Regulatory roles for small G proteins in the pancreatic beta-cell: lessons from models of impaired insulin secretion.

Emerging evidence suggests that GTP-binding proteins (G proteins) play important regulatory roles in physiological insulin secretion from the islet beta-cell. Such conclusions were drawn primarily from experimental data derived through the use of specific inhibitors of G protein function. Data from gene depletion experiments appear to further substantiate key roles for these signaling proteins in the islet metabolism. The first part of this review will focus on findings supporting the hypothesis that activation of specific G proteins is essential for insulin secretion, including regulation of their function by posttranslational modifications at their COOH-terminal cysteines (e.g., isoprenylation). The second part will overview novel, non-receptor-dependent mechanism(s) whereby glucose might activate specific G proteins via protein histidine phosphorylation. The third section will review findings that appear to link abnormalities in the expression and/or functional activation of these key signaling proteins to impaired insulin secretion. It is hoped that this review will establish a basis for future research in this area of islet signal transduction, which presents a significant potential, not only in identifying key signaling proteins that are involved in physiological insulin secretion, but also in examining potential abnormalities in this signaling cascade that lead to islet dysfunction and onset of diabetes.

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title: GTP and Its Binding Proteins in the Regulation of Insulin Exocytosis
abstract: Despite considerable evidence for the involvement of the purine nucleotides GTP* and ATP in exocytosis, there is currently no consensus about their exact role(s). With regard to GTP (the focus of this chapter), our recent studies (1) have suggested that there is at least a permissive role for GTP in insulin release. Normal rat pancreatic islets were pretreated overnight with each of four structurally-dissimilar inhibitors of a rate-limiting step in the cytosolic production of GTP (inosine monophosphate dehydrogenase; IMPDH). When islet concentrations of GTP were inhibited by more than about 80% (to levels down to less than 0.6–0.7 pmol/islet), insulin release subsequently stimulated by nutrients (glucose or α-ketoisocaproate) or by aphorbol ester was reduced 40–60%. In contrast, insulin secretion induced by direct depolarization of β-cells (by 50 m M K+) was refractory to inhibition. These results were attributed to a depletion of islet GTP stores, since repletion of GTP by coprovision of guanine (but not the normalization of both ATP and UTP content by the provision of adenine) reversed the inhibitory effects of the drugs both on GTP content and on insulin release. In subsequent studies, we observed that lesser degrees of inhibition of GTP, or, conversely, a stimulation of islet GTP content (by provision of guanine alone) to levels about 25% above basal, did not have any discernible negative or positive modulatory effects on secretion. These findings suggested that islet GTP content is normally saturating, and therefore mild to moderate changes in these levels do not alter secretion. Since the average islet content of GTP approximates 750–1000 μM, it appears that a reduction in total islet GTP levels to less than 150–200 μM (or, possibly, a comparable relative reduction of free GTP levels, which may be one-fifth to one-twentieth of total levels; ref. 2) is needed to vitiate the effects of GTP on secretion. Such drastic changes are not likely to be induced by physiologic agonists (and in fact were not induced by glucose, which had relatively modest effects on total islet GTP content); therefore, we defined the effects of GTP as “permissive.”

title: Elusive Proximal Signals of β-Cells for Insulin Secretion
abstract: The β-cell is unique because its major agonists, i.e., insulin secretagogues, undergo metabolism instead of interacting with a receptor. This perspectives presents the hypothesis that the first part of a metabolic signal of a secretagogue is specific to the secretagogue and the β-cell and can be envisioned as proximal. The second part, which occurs after transduction to more universal signaling mechanisms, is viewed as distal. Distal signaling and exocytosis in the β-cell operate the same as in other cells. Aerobic glycolysis is required for glucose-induced insulin release. Because glyceraldehyde, which enters metabolism at the triose phosphates in the glycolytic pathway, is a potent insulin secretagogue but pyruvate, which is metabolized in the mitochondrion, is not an insulin secretagogue, the proximal signal for glucose-induced insulin release originates with an interaction between the central part of the glycolytic pathway and mitochondrial metabolism. The proximal message in leucine-induced insulin release originates with leucine allosterically activating glutamate dehydrogenase, which activates endogenous glutamate metabolism, and by the metabolism of leucine itself. The methyl ester of succinate is a potent experimental insulin secretogogue. It is puzzling why the glucose signal requires the interplay of glycolysis and mitochondrial metabolism, whereas the signals from leucine and succinate originate entirely from within the mitochondrion. Leucine-induced insulin release is suppressed and glucose-induced insulin release is activated in islets cultured at a high concentration of glucose. Conversely, leucine-induced insulin release is activated and glucose-induced insulin release is suppressed in islets cultured at low glucose. We have correlated suppression of the insulinotropism of leucine and glucose with decreased expression of the genes that encode the catalytic subunit of the first component of the branched-chain ketoacid dehydrogenase complex and the pyruvate dehydrogenase complex, respectively. This indicates that the proximal signal is specific to the secretagogue, whereas distal signals are more universal and are shared by many secretagogues. The proximal signaling mechanisms have yet to be elucidated, but many distal mechanisms are known.

title: Metabolic coupling factors in pancreatic beta-cell signal transduction.
abstract: This chapter focuses on the biochemical mechanisms that mediate glucose-stimulated insulin secretion (GSIS) from beta-cells of the islets of Langerhans and the potentiating role played by fatty acids. We summarize evidence supporting the idea that glucose metabolism is required for GSIS and that the GLUT-2 facilitated glucose transporter and the glucose phosphorylating enzyme glucokinase play important roles in measuring changes in extracellular glucose concentration. The idea that glucose metabolism is linked to insulin secretion through a sequence of events involving changes in ATP:ADP ratio, inhibition of ATP-sensitive K+ channels, and activation of voltage-gated Ca2+ channels is critically reviewed, and the relative importance of ATP generated from glycolytic versus mitochondrial metabolism is evaluated. We also present the growing concept that an important signal for insulin secretion may reside at the linkage between glucose and lipid metabolism, specifically the generation of the regulatory molecule malonyl CoA that promotes fatty acid esterification and inhibits oxidation. Finally, we show that in contrast to its short term potentiating effect on GSIS, long-term exposure of islets to high levels of fatty acids results in beta-cell dysfunction, suggesting that hyperlipidemia associated with obesity may play a causal role in the diminished GSIS characteristic of non insulin-dependent diabetes mellitus (NIDDM).

title: Nutrient augmentation of Ca2+-dependent and Ca2+-independent pathways in stimulus-coupling to insulin secretion can be distinguished by their guanosine triphosphate requirements: studies on rat pancreatic islets.
abstract: To delineate the underlying mechanisms by which glucose augments both Ca2+-dependent and Ca2+-independent insulin release, the latter induced by the simultaneous activation of protein kinases A and C, we examined the effects of GTP depletion by mycophenolic acid (MPA), an inhibitor of GTP synthesis, on the augmentation of insulin release from rat pancreatic islets. MPA treatment reduced GTP content by 30-40% and completely abolished glucose-induced augmentation of Ca2+-independent insulin release. Thus, this pathway is extremely sensitive to a decrease in cellular GTP content. Complete inhibition was also observed in islets treated with MPA plus adenine, to maintain ATP levels, under which conditions GTP is selectively depleted. Provision of guanine, which increases the activity of a salvage pathway for GTP synthesis and normalizes GTP content, completely reversed the inhibitory effect of MPA. Neither glucose utilization nor glucose oxidation was affected by MPA. The augmentation of Ca2+-independent insulin release by several other metabolizable nutrients including alpha-ketoisocaproic acid (KIC) was also inhibited by MPA. In sharp contrast, augmentation of Ca2+-dependent insulin release by KIC was resistant to GTP depletion, indicating that nutrient-induced augmentation of the Ca2+-dependent- and Ca2+-independent secretory pathways can be differentiated by GTP dependency. We interpret these data in accord with current knowledge concerning the two known stimuli for exocytosis, Ca2+ and GTP (independently of Ca2+). We propose that both Ca2+-dependent and Ca2+-independent augmentation occurs via one metabolic pathway acting upon Ca2+- and upon GTP-stimulated exocytosis. Activation of PKA and PKC stimulates the GTP-sensitive exocytosis.

title: Small elevations of glucose concentration redirect and amplify the synthesis of guanosine 5'-triphosphate in rat islets.
abstract: Recent studies suggest a permissive requirement for guanosine 5'-triphosphate (GTP) in insulin release, based on the use of GTP synthesis inhibitors (such as myocophenolic acid) acting at inosine monophosphate (IMP) dehydrogenase; herein, we examine the glucose dependency of GTP synthesis. Mycophenolic acid inhibited insulin secretion equally well after islet culture at 7.8 or 11.1 mM glucose (51% inhibition) but its effect was dramatically attenuated when provided at < or = 6.4 mM glucose (13% inhibition; P < 0.001). These observations were explicable by a stimulation of islet GTP synthesis derived from IMP since, at high glucose: (a) total GTP content was augmented; (b) a greater decrement in GTP (1.75 vs. 1.05 pmol/islet) was induced by mycophenolic acid; and (c) a smaller "pool" of residual GTP persisted after drug treatment. Glucose also accelerated GTP synthesis from exogenous guanine ("salvage" pathway) and increased content of a pyrimidine, uridine 5'-triphosphate (UTP), suggesting that glucose augments production of a common regulatory intermediate (probably 5-phosphoribosyl-1-pyrophosphate). Pathway-specific radiolabeling studies confirmed that glucose tripled both salvage and de novo synthesis of nucleotides. We conclude that steep changes in the biosynthesis of cytosolic pools of GTP occur at modest changes in glucose concentrations, a finding which may have relevance to the adaptive (patho) physiologic responses of islets to changes in ambient glucose levels.

title: G Proteins and Modulation of Insulin Secretion
abstract: Guanine nucleotide–binding proteins (G proteins) are critically important mediators of many signal-transduction systems. Several important sites regulating stimulus-secretion coupling and release of insulin from pancreatic β-cells are modulated by G proteins. Gs mediates increases in intracellular cAMP associated with hormone-induced stimulation of insulin secretion. GI, mediates decreases in intracellular cAMP caused by inhibitors of insulin secretion, e.g., epinephrine, somatostatin, prostaglandin E2, and galanin. G proteins also regulate ion channels, phospholipases, and distal sites in exocytosis. Cholera and pertussis toxins irreversibly ADP ribosylate G proteins and are important tools that can be used both to manipulate G-protein–dependent modulators of insulin secretion and detect and quantify G proteins by electrophoretic techniques. The stage is set to pursue these initial observations in greater depth and ascertain whether G-protein research will provide important new insights into normal and abnormal regulation of insulin secretion.

title: G proteins: transducers of receptor-generated signals.
abstract: PERSPECTIVES AND SUMMARY .............................................................. 615 HISTORY ............................................................................................... 616 INDIVIDUAL G PROTEINS: FUNCTIONS AND MOLECULAR ENTITIES .......... 617 Criteria .for Involvement of a G Protein ..................................................... 618 Functions Regulated by G Proteins ........................................................... 619 Molecular Entities and Structure .............................................................. 622 LIGAND-G PROTEIN TERACTIONS ........................................................ 631 LIGAND-REGULATED PROTEIN-PROTEIN TERACTIONS ........................... 634 Receptor-G Protein Interactions ............................................................... 634 G Protein-Effector Interactions ................................................................ 638 G PROTEII~I-MEMBRANE INTERACTIONS .................................................. 643 COVALENT MODIFICATION F G PROTEINS ............................................ 644

title: Small GTP-binding proteins.
abstract: Small GTP-binding proteins (G proteins) exist in eukaryotes from yeast to human and constitute a superfamily consisting of more than 100 members. This superfamily is structurally classified into at least five families: the Ras, Rho, Rab, Sar1/Arf, and Ran families. They regulate a wide variety of cell functions as biological timers (biotimers) that initiate and terminate specific cell functions and determine the periods of time for the continuation of the specific cell functions. They furthermore play key roles in not only temporal but also spatial determination of specific cell functions. The Ras family regulates gene expression, the Rho family regulates cytoskeletal reorganization and gene expression, the Rab and Sar1/Arf families regulate vesicle trafficking, and the Ran family regulates nucleocytoplasmic transport and microtubule organization. Many upstream regulators and downstream effectors of small G proteins have been isolated, and their modes of activation and action have gradually been elucidated. Cascades and cross-talks of small G proteins have also been clarified. In this review, functions of small G proteins and their modes of activation and action are described.

title: New insights into the mechanisms of protein palmitoylation.
abstract: Since its discovery more than 30 years ago, protein palmitoylation has been shown to have a role in protein-membrane interactions, protein trafficking, and enzyme activity. Until recently, however, the molecular machinery that carries out reversible palmitoylation of proteins has been elusive. In fact, both enzymatic and nonenzymatic S-acylation reaction mechanisms have been proposed. Recent reports of protein palmitoyltransferases in Saccharomyces cerevisiae and Drosophila provide the first glimpse of enzymes that carry out protein palmitoylation. Equally important is the mechanism of depalmitoylation. Two major classes of protein palmitoylthioesterases have been described. One family is lysosomal and is involved in protein degradation. The second is cytosolic and removes palmitoyl moieties preferentially from proteins associated with membranes. This review discusses recent advances in the understanding of mechanisms of addition of palmitate to proteins and removal of palmitate from proteins.

title: The Heterotrimeric G-protein Gi Is Localized to the Insulin Secretory Granules of β-Cells and Is Involved in Insulin Exocytosis (*)
abstract: Mastoparan, a tetradecapeptide found in wasp venom that stimulates G-proteins, increases insulin secretion from β-cells. In this study, we have examined the role of heterotrimeric G-proteins in mastoparan-induced insulin secretion from the insulin-secreting β-cell line β-TC3. Mastoparan stimulated insulin secretion in a dose-dependent manner from digitonin-permeabilized β-TC3 cells. Active mastoparan analogues mastoparan 7, mastoparan 8, and mastoparan X also stimulated secretion. Mastoparan 17, an inactive analogue of mastoparan, did not increase insulin secretion from permeabilized β-TC3 cells. Mastoparan-induced insulin secretion from permeabilized β-TC3 cells was inhibited by pretreatment of the cells with pertussis toxin, suggesting that mastoparan-induced insulin secretion is mediated through a pertussis toxin-sensitive G-protein present distally in exocytosis. Enriched insulin secretory granules (ISG) were prepared by sucrose/nycodenz ultracentrifugation. Western immunoblotting performed on β-TC3 homogenate and ISG demonstrated that Gαi was dramatically enriched in ISG. Levels of Gαo and Gαq were comparable in homogenate and ISG. Mastoparan stimulated ISG GTPase activity in a pertussis toxin-sensitive manner. Mastoparan 7 and mastoparan 8 also stimulated GTPase activity in the ISG, while the inactive analogue mastoparan 17 had no effect. Selective localization of Gαi to ISG was confirmed with electron microscopic immunocytochemistry in β-TC3 cells and β-cells from rat pancreas. In contrast to Gαo and Gαq, Gαi was clearly localized to the ISG. Together, these data suggest that mastoparan may act through the heterotrimeric G-protein Gαi located in the ISG of β-cells to stimulate insulin secretion.

title: Evidence that Rap1 carboxylmethylation is involved in regulated insulin secretion.
abstract: Protein carboxylmethylation is a reversible posttranslational modification that regulates protein function. We examined the carboxylmethylation of small GTP-binding proteins in a pancreatic beta-cell line (beta TC cells). In vitro assays showed that carboxylmethylation of a membrane 23-kDa protein was induced by guanine nucleotides, best demonstrated by the nonhydrolyzable GTP analog, guanosine 5'-(3-O-thio)triphosphate (GTP gamma S). GTP gamma S also induced translocation of this 23-kilodalton (kDa) protein from cytosol to particulate fractions. Immunoblotting with antiserum sc-65 raised against Rap1 identified the carboxyl-methylated 23-kDa protein as Rap1. 1) The 23-kDa carboxyl-methylated protein separated by two-dimensional electrophoresis overlapped with the 23-kDa protein detected by immunoblotting. 2) GTP gamma S, in the presence of cytosol, increased the amount of detectable membrane-associated Rap1. Studies in intact beta TC cells demonstrated the carboxylmethylation of the 23-kDa protein in response to glucose and depolarizing concentrations of potassium, an effect that was abolished by the calcium channel inhibitor, D600. Similarly, N-acetyl-S-trans,trans-farnesyl-L-cysteine, an inhibitor of in vivo carboxylmethylation at COOH-terminal S-farnesylcysteine by methyltransferase, inhibited carboxylmethylation of the 23-kDa protein in intact cells and reduced insulin secretion in response to glucose and potassium. These data establish a correlation between insulin secretion and carboxylmethylation of a 23-kDa protein that comigrates with Rap1.

title: The small GTP-binding proteins in the cytosol of insulin-secreting cells are complexed to GDP dissociation inhibitor proteins.
abstract: Ras-related small GTP-binding proteins (SMGs) exist in a cytosolic and a membrane-bound pool. The mechanism regulating the intracellular distribution of SMGs remains to be elucidated. We have, therefore, investigated the properties of SMGs expressed in cells of the insulin-secreting lines RINm5F and HIT-T15. Phase-partitioning analysis revealed that smg25A/rab3A as well as all the SMGs in the 23-27 kDa range, labeled by radioactive GTP after blotting, were hydrophobic, regardless of their subcellular distribution. In contrast, the cytosolic forms of ADP ribosylation factor, rho, and CDC42 were hydrophilic. The cytosolic pool of the 23-27-kDa group, including smg25A/rab3A, sedimented in a sucrose density gradient as complexes with an apparent M(r) of about 80,000, whereas rho and CDC42 were recovered in 45-kDa complexes. ARF, however, was uncomplexed (M(r) close to 20,000). The 80-kDa aggregates were likely to be formed by 1:1 complexes with the regulatory protein smg25/GDP dissociation inhibitor (smg25/GDI). In fact, pure smg25/GDI by sucrose gradient exhibited a molecular mass of 55 kDa, but cosedimented with the 80-kDa complexes in cytosolic extracts of insulin-secreting cells. Moreover, purified smg25/GDI was able to extract the SMGs of the 23-27-kDa group from the membranes. Similarly, in cytosolic extracts, rho/GDI cosedimented with the 45-kDa aggregates. Blocking the synthesis of isoprenoid groups with lovastatin resulted in the appearance in the cytosol of SMGs that were hydrophilic. These SMGs were found to sediment with an apparent M(r) close to 25,000 and to be unable to form complexes with smg25/GDI. Lovastatin treatment also caused the accumulation of the noncomplexed form of CDC42 but not of rho proteins. We propose that 1) except for ARF, all the SMGs detected in the cytosol of insulin-secreting cells are associated in 1:1 complexes with their regulatory proteins; 2) the different SMGs can be subdivided into functional groups according to the regulatory protein bound to them; 3) the formation of the 80-kDa complexes with smg25/GDI and of the CDC42 complexes with rho/GDI necessitate the correct carboxyl-terminal post-translational modification of the SMGs.

title: Modulation of insulin secretion from normal rat islets by inhibitors of the post-translational modifications of GTP-binding proteins.
abstract: Many GTP-binding proteins (GBPs) are modified by mevalonic acid (MVA)-dependent isoprenylation, carboxyl methylation or palmitoylation. The effects of inhibitors of these processes on insulin release were studied. Intact pancreatic islets were shown to synthesize and metabolize MVA and to prenylate several candidate proteins. Culture with lovastatin (to inhibit synthesis of endogenous MVA) caused the accumulation in the cytosol of low-M(r) GBPs (labelled by the [alpha-32P]GTP overlay technique), suggesting a disturbance of membrane association. Concomitantly, lovastatin pretreatment reduced glucose-induced insulin release by about 50%; co-provision of 100-200 microM MVA totally prevented this effect. Perillic acid, a purported inhibitor of the prenylation of small GBPs, also markedly reduced glucose-induced insulin secretion. Furthermore, both N-acetyl-S-trans,trans-farnesyl-L-cysteine (AFC), which inhibited the base-labile carboxyl methylation of GBPs in islets or in transformed beta-cells, and cerulenic acid, an inhibitor of protein palmitoylation, also reduced nutrient-induced secretion; an inactive analogue of AFC (which did not inhibit carboxyl methylation in islets) had no effect on secretion. In contrast with nutrients, the effects of agonists that induce secretion by directly activating distal components in signal transduction (such as a phorbol ester or mastoparan) were either unaffected or enhanced by lovastatin or AFC. These data are compatible with the hypothesis that post-translational modifications are required for one or more stimulatory GBPs to promote proximal step(s) in fuel-induced insulin secretion, whereas one or more inhibitory GBPs might reduce secretion at a more distal locus.

title: Inhibition of Glucose- and Calcium-Induced Insulin Secretion from βTC3 Cells by Novel Inhibitors of Protein Isoprenylation
abstract: The majority of low molecular weight G proteins undergoes a series of post-translational modification steps, e.g., isoprenylation, at their C-terminal cysteine, which seem to be critical for the transport of the modified proteins to the membrane sites for interaction with their respective effector proteins. Using lovastatin, an inhibitor of mevalonic acid, and hence, isoprenoid biosynthesis, we demonstrated previously that protein isoprenylation is critical for physiological insulin secretion from normal rat islets. Herein, we used more selective synthetic inhibitors of protein prenylation to examine their effects on glucose- and calcium-mediated insulin secretion from βTC3 cells. Both 3-allyl- and vinylfarnesols, which inhibit and/or modulate protein farnesyl transferases, significantly (80–95%) inhibited glucose- and KCl-stimulated insulin secretion from these cells. In a similar manner, the allyl and vinyl forms of geranylgeraniol, reagents targeted toward protein geranylation, attenuated insulin secretion elicited by glucose and KCl. Furthermore, manumycin A, a natural inhibitor of protein farnesylation, and geranylgeranyl transferase inhibitor-2147 (GGTI-2147), a peptidomimetic inhibitor of protein geranylgeranylation, also inhibited glucose- and KCl-induced insulin secretion to comparable degrees. Treatment of βTC3 cells with either 3-vinylfarnesol or 3-vinyl geranylgeraniol resulted in accumulation of unprenylated proteins in the cytosolic fraction. These data further support our original formulation that inhibition of isoprenylation of small molecular weight G proteins might impede their interaction with their putative effectors, which may be required for physiological insulin secretion.

title: Evidence for differential roles of the Rho subfamily of GTP-binding proteins in glucose- and calcium-induced insulin secretion from pancreatic beta cells.
abstract: We utilized clostridial toxins (with known specificities for inhibition of GTPases) to ascertain the contribution of candidate GTPases in physiologic insulin secretion from beta cells. Exposure of normal rat islets or isolated beta (HIT-T15) cells to Clostridium difficile toxins A and B catalyzed the glucosylation (and thereby the inactivation) of Rac, Cdc42, and Rho endogenous to beta cells; concomitantly, either toxin reduced glucose- or potassium-induced insulin secretion from rat islets and HIT cells. Treatment of beta cells with Clostridium sordellii lethal toxin (LT; which modified only Ras, Rap, and Rac) also reduced glucose- or potassium-induced secretion. However, clostridial toxin C3-exoenzyme (which ADP-ribosylates and inactivates only Rho) was without any effect on either glucose- or potassium-induced insulin secretion. These data suggest that Cdc42, Rac, Ras, and/or Rap (but not Rho) may be needed for glucose- or potassium-mediated secretion. The effects of these toxins appear to be specific on stimulus-secretion coupling, since no difference in metabolic viability (assessed colorimetrically by quantitating the conversion of the tetrazolium salt into a formazan in a reduction reaction driven by nutrient metabolism) was demonstrable between control and toxin (A or LT)-treated beta cells. Toxin (A or LT) treatment also did not alter glucose- or potassium-mediated rises in cytosolic free calcium concentrations ([Ca2+]i), suggesting that these GTPases are involved in steps distal to elevations in ) and Rac (present study) are regulated by glucose or potassium. Together, these findings provide direct evidence, for the first time, that the Rho subfamily of GTPases plays a key regulatory role(s) in insulin secretion, and they suggest that Cdc42 may be required for early steps in glucose stimulation of insulin release, whereas Rap and/or Rac may be required for a later step(s) in the stimulus-secretion coupling cascade (i.e. Ca2+-induced exocytosis of insulin).

title: Characterization of prenylcysteine methyltransferase in insulin-secreting cells.
abstract: Prenylcysteine carboxymethyltransferase, an enzyme involved in the post-translational modification of many signalling proteins, was characterized in insulin-secreting INS-1 cells and normal rat pancreatic islets. The activity of this enzyme was monitored by the methylation of an artificial substrate (a prenylated cysteine analogue) with S-adenosy1[methyl-3H]methionine as methyl donor. More than 95% of the methyltransferase activity was associated with the membranes, and high-salt treatment only partially extracted the enzyme from the membranes. The highest specific activity was in the insulin-granule-enriched 25000 g pellet obtained by differential centrifugation. However, a highly purified insulin-enriched fraction obtained by density centrifugation in Percoll did not exhibit methyltransferase activity. The analyses of marker enzymes for cellular organelles revealed that the methyltransferase was co-localized, with the plasma membrane and probably the endoplasmic reticulum, but not with the mitochondria or lysosomes. Guanosine 5'-[gamma-thio]-triphosphate failed to increase methyltransferase activity directly, although it promotes the methylation of GTP-binding proteins. Mastoparan, Ca2+, cAMP and the protein kinase C activator phorbol 12-myristate 13-acetate did not alter enzyme activity. In addition, methyltransferase activity was not stably modified by stimulation of intact cells using glucose or other agents. However, the carboxymethylation of certain low-molecular-mass G-proteins is increased by glucose stimulation; conversely, treatment of cells with N-acetyl-S-trans,trans-farnesyl-L-cysteine inhibited glucose- and forskolin-induced insulin secretion. These results suggest that the membrane-associated prenylcysteine carboxymethyltransferase may be constitutively active and that the methylation of target proteins in vivo is regulated by the access of these proteins to the methyltransferase, as well as by their active (GTP-liganded) configuration.

title: Evidence for the carboxyl methylation of nuclear lamin-B in the pancreatic beta cell.
abstract: Lamins are intermediate filament proteins that constitute the main components of the lamina underlying the inner-nuclear membrane and serve to organize chromatin. Lamins (e.g., lamin-B) undergo posttranslational modifications (e.g., isoprenylation and methylation) at their C-terminal cysteine. Such modifications are thought to render optimal association of lamins with the nuclear envelop. Herein, we examined whether nuclear lamin-B undergoes carboxyl methylation in islet beta cells. A 65- to 70-kDa protein was carboxyl methylated in intact rat islets and clonal beta (HIT or INS) cells or in homogenates which could be immunoprecipitated using lamin-B antiserum. Incubation of purified HIT cell-nuclear fraction with [(3)H]S-adenosyl methionine yielded a single carboxyl methylated protein peak (ca. 65-70 kDa); this protein was immunologically identified as lamin-B. Several methylation inhibitors, including acetyl farnesyl cysteine, a competitive inhibitor of protein prenyl cysteine methylation, inhibited the carboxyl methylation of lamin-B, indicating that the carboxyl-methylated amino acid is cysteine. These findings, together with our recent observations demonstrating that inhibition of protein isoprenylation causes apoptotic death of the pancreatic beta cell, raise an interesting possibility that inhibition of C-terminal cysteine modifications of lamin-B might result in disruption of nuclear assembly, leading to further propagation of apoptotic signals, including DNA fragmentation and chromatin condensation.

title: Carboxylmethylation of the catalytic subunit of protein phosphatase 2A in insulin-secreting cells: evidence for functional consequences on enzyme activity and insulin secretion.
abstract: We report the carboxylmethylation of a 36-kDa protein in intact normal rat islets and clonal beta (INS-1) cells. This protein was predominantly cytosolic. Its carboxylmethylation, as assessed by vapor phase equilibration assay, was resistant to inhibition by N-acetyl-S-trans, trans-farnesyl-L-cysteine, a competitive substrate for cysteine methyl transferases. These data suggest that the methylated C-terminal amino acid is not cysteine. The methylated protein was identified as the catalytic subunit of protein phosphatase 2A (PP2Ac) by immunoblotting. The carboxylmethylation of the PP2Ac increased its catalytic activity, suggesting a key role in the functional regulation of PP2A. Therefore, we studied okadaic acid, a selective inhibitor of PP2A that acts by an unknown mechanism. Okadaic acid (but not 1-nor-okadaone, its inactive analog) inhibited (Ki = 10 nM) the carboxylmethylation of PP2Ac and phosphatase activity in the cytosolic fraction (from normal rat islets and clonal beta-cells) as well as in intact rat islets. Furthermore, methylated PP2Ac underwent rapid demethylation (t 1/2 = 40 min) catalyzed by a methyl esterase localized in islet homogenates. Ebelactone, a purported inhibitor of methyl esterases, significantly delayed (> 200 min) the demethylation of PP2Ac. Furthermore, ebelactone reversibly inhibited glucose- and ketoisocaproate-induced insulin secretion from normal rat islets. These data identify, for the first time, a methylation-demethylation cycle for PP2Ac in the beta-cell and suggest a key functional relationship between PP2A activity and the carboxylmethylation of its catalytic subunit. These findings thus suggest a negative modulatory role for PP2A in nutrient-induced insulin exocytosis.

title: Lipid Modifications of Trimeric G Proteins (*)
abstract: G protein alpha subunits and beta gamma dimers are covalently modified by lipids. The emerging picture is one in which attached lipids provide more than just a nonspecific "glue" for sticking G proteins to membranes. We are only beginning to understand how different lipid modifications of different G protein subunits affect specific protein-protein interactions and localization to specific cellular sites. In addition, regulation of these modifications, particularly palmitoylation, can provide new ways to regulate signals transmitted by G proteins.

title: Inhibition of Protein Palmitoylation, Raft Localization, and T Cell Signaling by 2-Bromopalmitate and Polyunsaturated Fatty Acids*
abstract: The ability of the Src family kinases Fyn and Lck to participate in signaling through the T cell receptor is critically dependent on their dual fatty acylation with myristate and palmitate. Here we identify a palmitate analog, 2-bromopalmitate, that effectively blocks Fyn fatty acylation in general and palmitoylation in particular. Treatment of COS-1 cells with 2-bromopalmitate blocked myristoylation and palmitoylation of Fyn and inhibited membrane binding and localization of Fyn to detergent-resistant membranes (DRMs). In Jurkat T cells, 2-bromopalmitate blocked localization of the endogenous palmitoylated proteins Fyn, Lck, and LAT to DRMs. This resulted in impaired signaling through the T cell receptor as evidenced by reductions in tyrosine phosphorylation, calcium release, and activation of mitogen-activated protein kinase. We also examined the ability of long chain polyunsaturated fatty acids (PUFAs) to inhibit protein fatty acylation. PUFAs have been reported to inhibit T cell signaling by excluding Src family kinases from DRMs. Here we show that the PUFAs arachidonic acid and eicosapentaenoic acid inhibit Fyn palmitoylation and consequently block Fyn localization to DRMs. We propose that inhibition of protein palmitoylation represents a novel mechanism by which PUFAs exert their immunosuppressive effects.

title: Palmitoylation of the 25-kDa synaptosomal protein (SNAP-25) in vitro occurs in the absence of an enzyme, but is stimulated by binding to syntaxin.
abstract: The neuronal N-ethylmaleimide-sensitive-factor-attachment-protein receptor (SNARE) proteins 25-kDa synaptosomal protein (SNAP-25), syntaxin 1 and synaptobrevin 2 interact to form the intermembrane SNARE complex, which mediates docking and fusion of synaptic vesicles with the plasma membrane. Assembly of the SNARE complex is accompanied by conformational changes, especially in SNAP-25. SNAP-25 is palmitoylated in vivo at cysteine residues located in the middle of the molecule. Acylation is required for membrane binding or membrane targeting of this intrinsically hydrophilic protein. Palmitoylation of recombinant SNAP-25 was studied in vitro in the absence of an enzyme source with [(3)H]palmitoyl-CoA as the lipid donor.

title: Inhibition of Receptor Signaling to Phospholipase D by Clostridium difficile Toxin B
abstract: Rho proteins have been reported to activate phospholipase D (PLD) in in vitro preparations. To examine the role of Rho proteins in receptor signaling to PLD, we studied the effect of Clostridium difficile toxin B, which glucosylates Rho proteins, on the regulation of PLD activity in human embryonic kidney (HEK) cells stably expressing the m3 muscarinic acetylcholine receptor (mAChR). Toxin B treatment of HEK cells potently and efficiently blocked mAChR-stimulated PLD. In contrast, basal and phorbol ester-stimulated PLD activities were not or only slightly reduced. Cytochalasin B and Clostridium botulinum C2 toxin, mimicking the effect of toxin B on the actin cytoskeleton but without involving Rho proteins, had no effect on mAChR-stimulated PLD. Toxin B did not alter cell surface mAChR number and mAChR-stimulated binding of (guanosine 5′-O-(thio)triphosphate (GTPS)) to G proteins. In addition to mAChR-stimulated PLD, toxin B treatment also inhibited PLD activation by the direct G protein activators, AlF and GTPS, studied in intact and permeabilized cells, respectively. Finally, C. botulinum C3 exoenzyme, which ADP-ribosylates Rho proteins, mimicked the inhibitory effect of toxin B on GTPS-stimulated PLD activity. In conclusion, the data presented indicate that toxin B potently and selectively interferes with receptor coupling mechanisms to PLD, and furthermore suggest an essential role for Rho proteins in receptor signaling to PLD.

title: Regulation of Gi and G,, by Mastoparan, Related Amphiphilic Peptides, and Hydrophobic Amines
abstract: Mastoparan (MP), a cationic, amphiphilic tetradecapeptide, stimuIates guanine nucleotide exchange by GTP-binding regulatory proteins (G proteins) in a manner similar to that of G protein-coupled receptors. 1) MP stimulated exchange by isolated G protein a subunits and @y trimers. Relative stimuIation was greater with aj3~ trimers and fly subunits could increase net MP-stimuIated activity. 2) MP action was enhanced by reconstitution of trimeric G protein into phospholipid vesicles. Hill coefficients for activation were 24. The membrane-bound a-helical conformation of MP appeared to be the activating species. 3) MP blocked the ability of G0 to increase the affinity of muscarinic receptors for agonist ligands, suggesting that MP and the receptor may compete for a common binding site on GO. 4) MP stimulated steady state GTPase activity at cl pM Mg*+ and stimulated the dissociation of both GDP and guanosine 5’-O-(3-thiotriphosphate) at <l nM Mg2+. Millimolar Mg2+ blocked the stimulatory effect of MP. Both high and low affinity Mg2+ binding sites are on the a subunit. 5) Increasing the amphiphilicity or hydrophobicity of MP enhanced its regulatory activity more than 2-fold and lowered the EC60 more than lo-fold. Several natural amphiphilic peptides also displayed modest stimulatory activity. 6) Benzalkonium chIoride competitively antagonized the stimulation of Gi by MP but potentIy stimulated nucleotide exchange on GO. Because cationic, amphiphilic sequences on the cytoplasmic faces of receptors are required for G protein regulation, these findings suggest that nucleotide exchange on G proteins is regulated by the presentation of multiple cationic structures on the inner face of the plasma membrane.

title: Mastoparan, a peptide toxin from wasp venom, mimics receptors by activating GTP-binding regulatory proteins (G proteins).
abstract: Mastoparan, a peptide toxin from wasp venom, is a nonspecific secretagogue. We show here that mastoparan increases the GTPase activity and the rate of nucleotide binding of several purified GTP-binding regulatory proteins (G proteins) whose function is to couple cell-surface receptors to intracellular mediators. Mastoparan accelerated guanosine-5'-(3-O-thiotriphosphate binding and consequent G protein activation in part by promoting the dissociation of bound GDP, the mechanism by which receptors regulate G proteins. ADP-ribosylation by pertussis toxin, which uncouples receptors from G proteins, selectively inhibited mastoparan-stimulated activation. Like receptors, mastoparan was more potent if the G protein was reconstituted in phospholipid vesicles and was active at micromolar concentrations of Mg2+. The structure of mastoparan in a lipid bilayer is similar to that predicted for a cationic intracellular loop of G protein-coupled receptors. Mastoparan thus displays a novel mode of toxicity by acting directly on G proteins to mimic the role normally played by agonist-liganded receptors.

title: A defect late in stimulus-secretion coupling impairs insulin secretion in Goto-Kakizaki diabetic rats.
abstract: A widely accepted genetically determined rodent model for human type 2 diabetes is the Goto-Kakizaki (GK) rat; however, the lesion(s) in the pancreatic islets of these rats has not been identified. Herein, intact islets from GK rats (aged 8-14 weeks) were studied, both immediately after isolation and after 18 h in tissue culture. Despite intact contents of insulin and protein, GK islets had markedly deficient insulin release in response to glucose, as well as to pure mitochondrial fuels or a non-nutrient membrane-depolarizing stimulus (40 mmol/l K+). In contrast, mastoparan (which activates GTP-binding proteins [GBPs]) completely circumvented any secretory defect. Basal and stimulated levels of adenine and guanine nucleotides, the activation of phospholipase C by Ca2+ or glucose, the secretory response to pertussis toxin, and the activation of selected low-molecular weight GBPs were not impaired. Defects were found, however, in the autophosphorylation and catalytic activity of cytosolic nucleoside diphosphokinase (NDPK), which may provide compartmentalized GTP pools to activate G-proteins; a deficient content of phosphoinositides was also detected. These studies identify novel, heretofore unappreciated, defects late in signal transduction in the islets of our colony of GK rats, possibly occurring at the site of activation by NDPK of a mastoparan-sensitive G-protein-dependent step in exocytosis.

title: Protein kinases, protein phosphorylation, and the regulation of insulin secretion from pancreatic beta-cells.
abstract: Expression and Characteristics A. Ca2+/calmodulin-dependent protein kinases B. Cyclic nucleotide-dependent protein kinases C. Ca2+/phospholipid-dependent protein kinases D. Mitogen-activated protein kinases E. Protein tyrosine kinases III. Phosphorylation of Endogenous Proteins in β-Cells A. Methods for studying protein phosphorylation in β-cells B. Endogenous kinase substrates in β-cells IV. Protein Kinases and the Regulation of Insulin Secretion A. Ca2+/calmodulin-dependent protein kinases B. Cyclic nucleotide-dependent protein kinases C. Ca2+/phospholipid-dependent protein kinases D. Mitogen-activated protein kinases E. Protein tyrosine kinases V. Protein Phosphatases and Protein Dephosphorylation in β-Cells A. Serine/threonine protein phosphatases B. Protein phosphatases in β-cells VI. Protein Kinase-Independent Secretory Pathways VII.

title: Protein kinases and phosphatases that act on histidine, lysine, or arginine residues in eukaryotic proteins: a possible regulator of the mitogen-activated protein kinase cascade.
abstract: Phosphohistidine goes undetected in conventional studies of protein phosphorylation, although it may account for 6% of total protein phosphorylation in eukaryotes. Procedures for studying protein N- kinases are described. Genes whose products are putative protein histidine kinases occur in a yeast and a plant. In rat liver plasma membranes, activation of the small G-protein, Ras, causes protein histidine phosphorylation. Cellular phosphatases dephosphorylate phosphohistidine. One eukaryotic protein histidine kinase has been purified, and specific proteins phosphorylated on histidine have been observed. There is a protein arginine kinase in mouse and protein lysine kinases in rat. Protein phosphohistidine may regulate the mitogen-activated protein kinase cascade.

title: A novel regulatory mechanism for trimeric GTP-binding proteins in the membrane and secretory granule fractions of human and rodent beta cells.
abstract: Recently we described roles for heterotrimeric and low-molecular-mass GTP-binding proteins in insulin release from normal rat islets. During these studies, we observed that a protein with an apparent molecular mass (37 kDa) similar to that of the beta subunit of trimeric GTP-binding proteins underwent phosphorylation in each of five classes of insulin-secreting cells. Incubation of the beta cell total membrane fraction or the isolated secretory granule fraction (but not the cytosolic fraction) with [gamma-32P]ATP or [gamma-32P]GTP resulted in the phosphorylation of this protein, which was selectively immunoprecipitated by an anti-serum directed against the common beta subunit of trimeric G-proteins. Disruption of the alpha beta gamma trimer (by pretreatment with either fluoroaluminate or guanosine 5'(-)[gamma-thio]triphosphate) prevented beta subunit phosphorylation. Based on differential sensitivities to pH, heat and the histidine-selective reagent diethyl pyrocarbonate (and reversal of the latter by hydroxylamine), the phosphorylated amino acid was presumptively identified as histidine. Incubation of pure beta subunit alone or in combination with the exogenous purified alpha subunit of transducin did not result in the phosphorylation of the beta subunit, but addition of the islet cell membrane fraction did support this event, suggesting that membrane localization (or a membrane-associated factor) is required for beta subunit phosphorylation. Incubation of phosphorylated beta subunit with G alpha.GDP accelerated the dephosphorylation of the beta subunit, accompanied by the formation of G alpha-GTP. Immunoblotting detected multiple alpha subunits (of Gi, G(o) and Gq) and at least one beta subunit in the secretory granule fraction of normal rat islets and insulinoma cells. These data describe a potential alternative mechanism for the activation of GTP-binding proteins in beta cells which contrasts with the classical receptor-agonist mechanism: G beta undergoes transient phosphorylation at a histidine residue by a GTP-specific protein kinase; this phosphate, in turn, may be transferred via a classical Ping-Pong mechanism to G alpha.GDP (inactive), yielding the active configuration G alpha.GTP in secretory granules (a strategic location to modulate exocytosis).

title: A filter-based protein kinase assay selective for alkali-stable protein phosphorylation and suitable for acid-labile protein phosphorylation.
abstract: Alkali-stable phosphorylation of proteins, particularly phosphotyrosine and phosphohistidine, is an important phenomenon in cells. In the case of phosphohistidine and some other phosphoamino acids, the phosphorylation is acid-labile and in these cases studies have been severely limited by the absence of a rapid assay suitable for acid-labile phosphorylation. The assay presented here involves a conventional kinase assay reaction followed by mild alkaline hydrolysis and adsorption of the product to washed Nytran paper at high pH. After further washing, at pH 9, the radioactivity on the papers is determined by liquid scintillation counting. Hence, acid-labile phosphorylation is preserved. The assay is selective for alkali-stable phosphorylation but not fully specific, mainly due to the need to balance the severity of the partial alkaline hydrolysis with the stability of the protein-peptide bonds. The assay has been used for the purification and characterization of a protein histidine kinase from Saccharomyces cerevisiae.

title: Protein phosphatases 1, 2A, and 2C are protein histidine phosphatases.
abstract: Eukaryotic cellular proteins contain phosphohistidine. To search for protein histidine phosphatases, protein histidine kinase from Saccharomyces cerevisiae was used to phosphorylate histone H4 on histidine at position 75 in the H4 amino acid sequence. Incubation of the phosphorylated histone H4 with either protein phosphatase 1, 2A, or 2C resulted in extensive removal of phosphate from the phosphorylated histone. Thus, protein phosphatases 1, 2A, and 2C are histidine phosphatases as well as serine/threonine phosphatases. Calcium/calmodulin-regulated protein phosphatase (protein phosphatase 2B) did not remove phosphate from phosphohistidine. The histidine phosphatase reaction was tested for a magnesium requirement and effects of inhibitor-1 and okadaic acid. In all cases, the protein phosphatases behaved as they do in their serine/threonine phosphatase activity. Extracts of the yeast, S. cerevisiae, contain protein histidine phosphatase activity. Quantitative measurement of phosphatase activity shows that the activity against phosphohistidine is a major activity of protein phosphatases 1, 2A, and 2C.

title: Phosphorylation and dephosphorylation of histidine residues in proteins.
abstract: Protein phosphorylation is a key mechanism for intracellular signal transduction in both prokaryotic and eukaryotic cells. Vertebrate proteins are prevalently phosphorylated on side chains that contain a hydroxyl group, such as serine, threonine and tyrosine residues. In the past decade, however, an increasing number of examples of histidine phosphorylation has been described. Because acid treatment of phosphoproteins during purification and detection of phosphoamino acid analysis is routine, O-phosphomonoesters have been studied more often, and the existence of acid-labile phosphates has been largely overlooked. The latter class of N-phosphoamidates may well be more widespread than is generally believed, even though the O-phosphates remain the major class in terms of quantity and extent of distribution in proteins. Phosphohistidine currently is estimated to be 10- to 100-fold more abundant than phosphotyrosine, but less abundant than phosphoserine [Matthews, H.R. (1995) Pharmac. Ther. 67, 323-350.]. This minireview briefly summarizes the extensive knowledge of the key mechanisms and functions of phosphohistidine in bacteria. It also describes the still limited, yet increasing, data from homologs of the bacterial two-component system. Finally, novel mechanisms of phosphorylation and dephosphorylation of histidine residues not related to the two-component system are described.

title: Purification of a protein histidine kinase from the yeast Saccharomyces cerevisiae. The first member of this class of protein kinases.
abstract: An enzyme of molecular weight 32,000 comprising a single subunit has been isolated from whole cell extracts of the yeast Saccharomyces cerevisiae. In vitro, the enzyme transfers the gamma phosphate of ATP to a protein substrate, histone H4, to produce an alkali-stable phosphorylation. Modification of the substrate histidine with diethylpyrocarbonate prevented phosphorylation. Phosphoamino acid analysis of the phosphorylated substrate showed the presence of 1-phosphohistidine. Hence, the isolated enzyme is a protein histidine kinase. A novel assay for acid-labile alkali-stable protein phosphorylation was used in the purification of the kinase activity to a final specific activity of 2,700 nmol/15 min/mg. The purified enzyme phosphorylates specifically histidine 75 in histone H4 and does not phosphorylate histidine 18 nor histidine residues in any other core histone. Steady state kinetic data are consistent with an ordered sequential reaction with Km values for Mg-ATP and histone H4 of 60 and 17 microM, respectively. The protein histidine kinase requires a divalent cation such as Mg2+, Co2+, or Mn2+ but will not use Ca2+, Zn2+, Cu2+, Fe2+, spermine, or spermidine. This is the first purification of an enzyme that catalyzes N-linked phosphorylation in proteins.

title: ras proteins enhance the phosphorylation of a 38 kDa protein (p38) in rat liver plasma membrane
abstract: Phosphorylation of a 38 kDa protein (p38) present in rat liver plasma membrane has been shown for the first time to be enhanced by ras proteins. This increase in phosphorylation is about 3–16‐fold depending on the incubation time and the type of ras protein used. Acid treatment removes phosphate from this protein suggesting that the phosphorylation involves phosphoramidate derivatives of basic amino acids. Experiments carried out in the presence of diethylpyrocarbonate suggest that the phosphorylation occurs on (a) histidine residue(s). It is probable that the function of p38 in the cell is modulated by ras proteins through phosphorylation. The significance of phosphorylation of p38 in terms of malignant transformation is presently unknown.

title: Identification and characterization of a novel protein histidine kinase in the islet β cell: evidence for its regulation by mastoparan, an activator of G-proteins and insulin secretion
abstract: Using insulin-secreting cells, we previously demonstrated that specific proteins associated with the cytosolic, secretory granule, and mitochondrial fractions undergo a novel type of phosphorylation on their histidine residues. Subsequently, we identified these proteins as the nucleoside diphosphate kinase (NDPK) [Kowluru and Metz, Biochemistry 1994;33:12495–503], the β subunit of trimeric GTP-binding proteins [Kowluru et al ., ], and the α subunit of succinyl-CoA synthetase [Kowluru, Diabetologia 2001;44:89–94], respectively. Since several other enzymes of intermediary metabolism (e.g. ATP-citrate lyase and glucose-6-phosphatase) also undergo histidine phosphorylation, these initial findings may have a more generalized significance to β cells. Herein, we characterized a novel protein histidine kinase in pancreatic β cells, and determined it to be acid- and heat-labile as well as alkali-resistant in its phosphorylation of histone 4. Such an activity was detected in normal rat islets, human islets, and clonal β (HIT-T15 and INS-1) cells, and could utilize either ATP or GTP as a phosphoryl donor (with K m values in the range of 60–100 μM). On a size-exclusion column, its molecular mass was estimated to be in the range of 60–70 kDa. It was stimulated by divalent cations (Mg 2+ >Mn 2+ >control=Ca 2+ =Zn 2+ =Co 2+ ), but was resistant to polyamines. It was inactivated by known in vitro inhibitors of protein histidine phosphorylation (e.g. UDP or cromoglycate). Mastoparan, a global activator of G-proteins and insulin secretion from isolated β cells, but not mastoparan-17, its inactive analog, stimulated histidine kinase activity and histidine phosphorylation of G β subunit and insulin secretion from isolated rat islets. These studies identify, for the first time, a protein kinase activity in the pancreatic β cell that does not act on traditional -Ser, -Tyr, or -Thr residues. They also establish a possible link between histidine kinase activity and G β phosphorylation in isolated β cells.

title: Histidine phosphorylation of P-selectin upon stimulation of human platelets: A novel pathway for activation-dependent signal transduction
abstract: Transient phosphorylation of histidine characterizes the two-component systems in prokaryotes that control important physiological functions, but analogous events have not been implicated in signal transduction in mammalian cells. To explore histidine phosphorylation during activation of human cells, stimulated platelets were analyzed for the formation of protein phosphohistidine in a model system employing P-selectin. P-selectin, a leukocyte adhesion molecule, undergoes rapid phosphorylation and selective dephosphorylation of tyrosine, serine, and threonine. We now establish that phosphorylation following platelet activation with thrombin or collagen generates phosphohistidine at histidines on the cytoplasmic tail of P-selectin. With thrombin stimulation, the kinetics of phosphohistidine appearance and disappearance of P-selectin are very rapid. Platelets exhibit a novel ligand-induced signaling pathway to generate phosphohistidine. These results provide direct biochemical evidence for the induction of rapid and reversible histidine phosphorylation in mammalian cells upon cell activation and represent a novel paradigm for mammalian cell signaling.

title: Regulation of Cellular Functions by Nucleoside Diphosphate Kinases in Mammals
abstract: The role of nucleoside diphosphate (NDP) kinases in cell growth, differentiation, and tumormetastasis in relation to signal transduction was investigated. The essential role of NDP kinasein cell growth was validated by coupling between reduced NDP kinase levels, induced byantisense oligonucleotides, and the suppression of proliferative activity of a cultured cell line.In addition, because NDP kinase levels are often enhanced with development and differentiation,as has been demonstrated in postmitotic cells and tissues, such as the heart and brain, wefurther examined this possibility using the bone tissue (osteoblasts) and a cultured cell linePC12D. The enhanced NDP kinase accumulation was demonstrated in the matured osteoblastsin vivo and in vitro by immunohistochemistry. In PC12D cells neurite outgrowth took placein NDP kinase β-transfected clones without differentiation inducers, which was accompaniedby prolongation of doubling time. Neurite outgrowth, triggered by nerve growth factor and acyclic AMP analog, was down-regulated upon forced expression of inactive mutant NDPkinase by virtue of a dominant negative effect. NDP kinase α-transfected rat mammaryadenocarcinoma cells (MTLn3) and nm23-H2-transfected human oral squamous cell carcinomacells (LMF4) manifested reduced metastatic potential and were associated with an alteredsensitivity to environmental factors, such as motility and growth factors. NDP kinase α,compared to NDP kinase β, was involved in a wide variety of the cellular phenomena examined.Taken together, NDP kinase isoforms appear to elicit both their own respective and commoneffects. They may have an ability to lead cells to both proliferative and differentiated statesby modulating responsiveness to environmental factors, but their fate seems to depend on theirsurrounding milieu.

title: Detection and characterization of the phosphorylated form of microsomal glucose-6-phosphatase.
abstract: Incubation of rat liver microsomes with 32 P-glucose-6-phosphate results in rapid incorporation of 32 P into protein as N-3-phosphoryl histidine.

title: Localization and characterization of the mitochondrial isoform of the nucleoside diphosphate kinase in the pancreatic beta cell: evidence for its complexation with mitochondrial succinyl-CoA synthetase.
abstract: Nucleoside diphosphate kinase (NDPK) catalyzes the transfer of terminal phosphates from nucleoside triphosphates to nucleoside diphosphates to yield nucleotide triphosphates. The present study was undertaken to localize and characterize the mitochondrial isoform of NDPK (mNDPK) in the pancreatic beta cell since it could contribute to the generation of mitochondrial nucleotide triphosphates and, thereby, to the mitochondrial high-energy phosphate metabolism of the pancreatic beta cell. Mitochondrial fractions from the insulin-secreting beta cells were isolated by differential centrifugation. mNDPK activity was assayed as the amount of [(3)H]GTPgammaS formed from ATPgammaS and [(3)H]GDP. Incubation of isolated mitochondrial extracts with either [gamma-(32)P]ATP or GTP resulted in the formation [(32)P]NDPK, which could be immunoprecipitated by an anti-NDPK serum. mNDPK exhibited saturation kinetics with respect to its nucleoside diphosphate acceptors and nucleoside triphosphate donors and sensitivity to known inhibitors of NDPK (e.g., uridine diphosphate and cromoglycate). By Western blot analyses, at least three isoforms of NDPK were identified in various subcellular fractions of the beta cell. The nm23-H1 (NDPK-A) was predominantly soluble whereas nm23-H2 (NDPK-B) was associated with the soluble as well as membranous fractions. The mitochondrial isoform of NDPK, nm23-H4, was uniformly distributed in the beta cell mitochondrial subfractions. A significant amount of NDPK (as determined by the catalytic activity and immunological methods) was recovered in the immunoprecipitates of mitochondrial fraction precipitated with an antiserum directed against succinyl-CoA synthetase (SCS), suggesting that NDPK might remain complexed with SCS. We provide the first evidence for the localization of a mitochondrial isoform of the NDPK in the islet beta cell and thus offer a potential mechanism for the generation of intramitochondrial GTP which, unlike ATP, is not transported into mitochondria via the classical nucleotide translocase. Further work will be required to determine the importance of the NDPK/SCS complex to normal beta cell function in the secretion of insulin.

title: Histidine kinases and histidine phosphorylated proteins in mammalian cell biology, signal transduction and cancer.
abstract: Intensive investigation of protein tyrosine, serine and threonine phosphorylation has lead to advances in signal transduction research and cancer treatment. This feature summarizes research on mammalian proteins exhibiting histidine phosphorylation. Histidine kinases are well known in prokaryotic and lower eukaryotic systems where they form the 'two-component' signal transduction system. The relative invisibility of histidine phosphorylation in mammalian cells may result from technical obstacles such as its acid lability, which precludes detection in electrophoretic systems, amino acid sequencing, etc. Emerging data have identified mammalian histidine kinases for the kinase suppressor of ras, a scaffold molecule for the Map kinase pathway, as well as histone H4, aldolase C and the beta-subunit of heterotrimeric G proteins. Additional mammalian proteins of interest to signal transduction and cancer research exhibit histidine phosphorylation, including P-selectin, annexin I and the 20S proteasome. Other candidate histidine phosphorylated proteins are identified. These data suggest the existence of another series of phosphorylation patterns in signal transduction.

title: Ca2+-dependent membrane bound protein fraction from rabbit gastric mucosa contains a protein whose histidyl residue is phosphorylated.
abstract: We found an autophosphorylated protein with a molecular weight of 40 kDa (p40) in the crude annexin fraction of rabbit gastric mucosa, i.e., the materials released by EGTA from the membrane fraction obtained in the presence of Ca2+. This protein was enriched in chief cells in the gastric glands, and also found in the heart and the liver by Western blotting. The protein bound to phenyl-Sepharose in the presence of Ca2+ and showed extremely basic nature. The phosphorylation site of p40 was considered to be histidyl residue based on the stability to the various agents, the synthesizing activity of ATP from ADP, and the results of phosphoamino acid analysis. The autophosphorylation of p40 was augmented several tenth fold by GDP, Ras, myelin basic protein, or H1 histone at micromolar range. The phosphorylated form was rapidly dephosphorylated in the presence of cold ATP, succinate, and CoA, suggesting that p40 has succinyl-CoA synthetase activity. In fact, a peptide fragment from p40 showed a striking homology with the alpha subunits of succinyl-CoA synthetases from Escherichia coli, Dictyostelium discoideum, and rat liver. These results suggest that p40 is extramitochondrial alpha subunit of succinyl-CoA synthetase or its homologue.

title: Two-component kinase-like activity of nm23 correlates with its motility-suppressing activity.
abstract: Nm23 genes, which encode nucleoside diphosphate kinases, have been implicated in suppressing tumor metastasis. The motility of human breast carcinoma cells can be suppressed by transfection with wild-type nm23-H1, but not by transfections with two nm23-H1 mutants, nm23-H1(S12OG) and nm23-H1(P96S). Here we report that nm23-H1 can transfer a phosphate from its catalytic histidine to aspartate or glutamate residues on 43-kDa membrane proteins. One of the 43-kDa membrane proteins was not phosphorylated by either nm23-H1(P96S) or nm23-H1(S120G), and another was phosphorylated much more slowly by nm23-H1(P96S) and by nm23-H1(S120G) than by wild-type nm23-H1. Nm23-H1 also can transfer phosphate from its catalytic histidine to histidines on ATP-citrate lyase and succinic thiokinase. The rates of phosphorylation of ATP-citrate lyase by nm23-H1(S120G) and nm23-H1(P96S) were similar to that by wild-type nm23-H1. The rate of phosphorylation of succinic thiokinase by nm23-H1(S120) was similar to that by wild-type nm23-H1, and the rate of phosphorylation of succinic thiokinase by nm23-H1(P96S) was about half that by wild-type nm23-H1. Thus, the transfer of phosphate from nm23-H1 to aspartates or glutamates on other proteins appears to correlate better with the suppression of motility than does the transfer to histidines.

title: Phosphorylation of ATP-Citrate Lyase by Nucleoside Diphosphate Kinase (*)
abstract: Rat liver nucleoside diphosphate kinase (NDPK) and PC12 cell cytosol were used to determine whether NDPK could function as a protein kinase. NDPK was phosphorylated on its catalytic histidine using ATP, and the phosphorylated NDPK separated from [γ-32P]ATP. The addition of phosphorylated NDPK to dialyzed PC12 cell cytosol resulted in the phosphorylation of a protein with a subunit molecular mass of about 120 kDa. This phosphorylation appeared to occur by a direct transfer of a phosphoryl group from the catalytic histidine of NDPK to a histidine on the 120-kDa protein. The 120-kDa protein was partially purified and shown by peptide sequencing to be ATP-citrate lyase. ATP-citrate lyase is the primary source of cytosolic acetyl-CoA. NDPK phosphorylated the histidine at the catalytic site of ATP-citrate lyase. This histidine can also be phosphorylated by ATP, and its phosphorylation is the first step in the conversion of citrate and CoA to oxaloacetate and acetyl-CoA by ATP-citrate lyase. The level of phosphorylation of PC12 cell ATP-citrate lyase by phosphorylated NDPK was comparable with that by ATP. Thus, in addition to its nucleoside diphosphate kinase activity, NDPK can function as a protein kinase.

title: Characterization of nucleoside diphosphokinase activity in human and rodent pancreatic beta cells: evidence for its role in the formation of guanosine triphosphate, a permissive factor for nutrient-induced insulin secretion.
abstract: We have recently demonstrated a permissive role for GTP in nutrient-induced insulin secretion. One of the possible loci at which GTP might exert its regulatory effects include one (or more) of the GTP-binding proteins which we have identified in subcellular fractions (including secretory granules) of pancreatic islets. Herein, we characterize nucleoside diphosphokinase (NDP kinase) activity, which catalyzes the transphosphorylation of nucleotide diphosphate (e.g., GDP) to nucleotide triphosphates (e.g., GTP) in insulin-secreting cells. The presence of NDP kinase activity in normal rat and human islets, and pure beta (RIN and HIT) cells, was verified by three distinct approaches: first, its catalytic activity (formation of GTP or GTP gamma S from GDP and ATP or ATP gamma S); secondly, by immunologic detection; and third, by quantitating the phosphoenzyme intermediate of NDP kinase, which is involved in a ping-pong phosphotransfer mechanism. Subcellularly, NDP kinase is predominantly cytosolic (with a tetrameric molecular mass of 85-90 kDa) and requires divalent metal ions and thiols for its activity. UDP, which forms an abortive complex with the enzyme, inhibited its activity in a concentration-dependent manner (Ki = 2 mM). The phosphorylated intermediate of NDP kinase was differentially sensitive to heat, acidic pH, and a histidine-selective reagent, diethyl pyrocarbonate, suggesting that (one of) the phosphoamino acid(s) may be histidine. These data demonstrate that in beta cells NDP kinase undergoes transient phosphorylation and suggest that this phosphate, in turn, is transferred to GDP. If the GTP which is formed thereby is bound to, or channelled to, relevant GTP-binding proteins, it would facilitate the formation of active form of these proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

title: Stepwise phosphorylation mechanisms and signal transmission within a ligand–receptor–Gαβγ-protein-complex
abstract: An extended α-helical structural model of a G-protein-coupled receptor has been combined with a G-protein structure to assess the ternary complex's potential to function as a GTP synthase. The establishment of the structural constraints between the G-protein and the receptor model has been based on the likelihood of a proton signalling mechanism existing within the ternary complex which can conduct a proton from the interior of the receptor to a phosphorylated histidine residue on the Gβ-subunit. The candidate histidine residue is β183. The earlier development of the receptor's action as a ligand-activated trans-membrane proton transducer is concluded to be potentially more extensive in the extended receptor structure, the receptor's proton signalling mechanism being based on a Tyr–Arg–Tyr–Arg–Tyr proton shuttle pentad. Further proton shuttle mechanisms can exist in the Gα,Gβ subunit interface. The final Tyr–Arg–Tyr shuttle triad exhibits an oxygen–oxygen interatomic distance of some 12.0 A in the G-protein crystal structure consistent with the distance predicted previously within a receptor proton shuttle triad. The arrival of a proton on the acid labile histidine phosphoramidate would enable release of the phosphate group and, through an expected S N 2 reaction, allow transfer to the neighbouring arginine residue α201 (Gαt numbering) with the formation of a high energy phosphono-arginine intermediate. Subsequent transport of the phosphate group to the GDP phosphorylation site involving the known movement of the Gα α2-helix at the Gα–Gβ subunit interface would then complete the synthesis through this stepwise phosphorylation. In the general context of cell signalling under more active conditions, the direct phosphorylation mechanism is consistent with the cell requiring more energy input than can be obtained by a GDP/GTP exchange process, the explicit catalytic phosphorylation mechanism being faster than the generally accepted exchange mechanism.

title: Phosphotransfer reactions as a means of G protein activation
abstract: Heterotrimeric guanine nucleotide-binding regulatory proteins (G proteins) serve to transduce information from agonist-bound receptors to effector enzymes or ion channels. Current models of G protein activation-deactivation indicate that the oligomeric GDP-bound form must undergo release of GDP, bind GTP and undergo subunit dissociation, in order to be in active form (GTP bound α subunits and free βγ dimers) and to regulate effectors. The effect of receptor occupation by an agonist is generally accepted to be promotion of guanine nucleotide exchange thus allowing activation of the G protein. Recent studies indicate that transphosphorylation leading to the formation of GTP from GDP and ATP in the close vicinity, or even at the G protein, catalysed by membrane-associated nucleoside diphosphate kinase, may further activate G proteins. This activation is demonstrated by a decreased affinity of G protein-coupled receptors for agonists and an increased response of G protein coupled effectors. In addition, a phosphorylation of G protein β subunits and consequent phosphate transfer reaction resulting in G protein activation has also been demonstrated. Finally, endogenously formed GTP was preferentially effective in activating some G proteins compared to exogenous GTP. The aim of this report is to present an overview of the evidence to date for a transphosphorylation as a means of G protein activation (see also refs [1 and 2] for reviews).

title: Adenine and guanine nucleotide-specific succinyl-CoA synthetases in the clonal beta-cell mitochondria: implications in the beta-cell high-energy phosphate metabolism in relation to physiological insulin secretion
abstract: Succinyl-CoA synthetase catalyses the substrate level phosphorylation of ADP or GDP. It also supplies succinyl-CoA for heme synthesis. Recently, two distinct mitochondrial succinyl-CoA synthetase activities, one specific for ATP and the other for GTP, have been characterized in various tissues of pigeon. Because of the relative importance of mitochondrial high-energy phosphate metabolism in physiological insulin secretion and the few data available on mitochondrial succinyl-CoA synthetase in the beta cell, this study examined whether ATP-specific and GTP-specific succinyl-CoA synthetase activities are localized in the clonal beta-cell mitochondria.¶Methods. Using the mitochondrial extracts from clonal beta [INS-1 and HIT-T15] cells, we measured the formation of succinyl-CoA from succinate, CoA and ATP or GTP. To confirm the identity of these two enzymes, individual subunits of ATP-specific and GTP-specific to succinyl-CoA synthetase were identified by Western blot analysis.¶Results. Both ATP-and GTP activities of succinyl-CoA synthetase were observed in the mitochondrial fractions from these cells. The ratios of GTP to ATP activities of succinyl-CoA synthetase were near unity in both of the cell types studied. Using affinity-purified antisera directed specifically against individual (α and β) subunits of succinyl-CoA synthetase, we also identified both ATP-specific and GTP-specific forms of succinyl-CoA synthetase in HIT and INS cell mitochondria. Furthermore, using [γ-32P]ATP as a phosphoryl donor, we observed that the alpha subunit of succinyl-CoA synthetase undergoes autophosphorylation at a histidine residue; co-provision of exogenous succinate and CoA resulted in pronounced dephosphorylation of the phosphorylated alpha subunit of succinyl-CoA synthetase.¶Conclusion/interpretation. We provide evidence for the localization of two distinct activities of succinyl-CoA synthetase in the beta cell mitochondria. Whereas it is well established that ATP is critical for the beta cell mitochondrial metabolism, we propose that GTP generated by the activation of succinyl-CoA synthetase could promote key functional roles in the mitochondrial metabolism leading to insulin secretion.

title: Mastoparan stimulates exocytosis at a Ca(2+)-independent late site in stimulus-secretion coupling. Studies with the RINm5F beta-cell line.
abstract: Mastoparan, a tetradecapeptide from wasp venom, stimulated exocytosis in a concentration-dependent manner, which was enhanced by pertussis toxin pre-treatment, in the insulin secreting beta-cell line RINm5F. Mastoparan (3-20 microM) also elevated cytosolic free calcium concentration ([Ca2+]i), a rise that was not attenuated by nitrendipine. Divalent cation-free Krebs-Ringer bicarbonate (KRB) medium with 0.1 mM EGTA nullified the mastoparan-induced increase in [Ca2+]i, suggesting that the peptide increased Ca2+ influx but not through the L-type voltage-dependent Ca2+ channel. Depletion of the intracellular Ca2+ pool did not affect the mastoparan-induced elevation of [Ca2+]i. Remarkably, in divalent cation-free KRB medium with 0.1 mM EGTA and 2 microM thapsigargin in which mastoparan reduced [Ca2+]i, the mastoparan-stimulated insulin release was similar to that in normal Ca(2+)-containing KRB medium. Inhibitors of protein kinase C, such as bisindolylmaleimide, staurosporine, and 1-O-hexadecyl-2-O-methyl-rac-glycerol did not suppress the mastoparan-stimulated insulin release. Mastoparan at 10-20 microM did not increase cellular cAMP levels, nor did mastoparan at 5-10 microM affect [3H]arachidonic acid release. In conclusion, although mastoparan increased [Ca2+]i, this increase was not involved in the stimulation of insulin release. Rather, the data suggest that mastoparan directly stimulates exocytosis in a Ca(2+)-independent manner. As GTP-binding proteins (G proteins) are thought to be involved in the process of exocytosis and as mastoparan is known to exert at least some of its effects by activation of G proteins, an action of mastoparan to activate the putative stimulatory Ge (exocytosis) protein is likely.

title: Activation of heterotrimeric G proteins by a high energy phosphate transfer via nucleoside diphosphate kinase (NDPK) B and Gbeta subunits. Complex formation of NDPK B with Gbeta gamma dimers and phosphorylation of His-266 IN Gbeta.
abstract: G protein betagamma dimers can be phosphorylated in membranes from various tissues by GTP at a histidine residue in the beta subunit. The phosphate is high energetic and can be transferred onto GDP leading to formation of GTP. Purified Gbetagamma dimers do not display autophosphorylation, indicating the involvement of a separate protein kinase. We therefore enriched the Gbeta-phosphorylating activity present in preparations of the retinal G protein transducin and in partially purified G(i/o) proteins from bovine brain. Immunoblots, autophosphorylation, and enzymatic activity measurements demonstrated enriched nucleoside diphosphate kinase (NDPK) B in both preparations, together with residual Gbetagamma dimers. In the retinal NDPK B-enriched fractions, a Gbeta-specific antiserum co-precipitated phosphorylated NDPK B, and an antiserum against the human NDPK co-precipitated phosphorylated Gbetagamma. In addition, the NDPK-containing fractions from bovine brain reconstituted the phosphorylation of purified Gbetagamma. For identification of the phosphorylated histidine residue, bovine brain Gbetagamma and G(t)betagamma were thiophosphorylated with guanosine 5'-O-(3-[(35)S]thio)triphosphate, followed by digestion with endoproteinase Glu-C and trypsin, separation of the resulting peptides by gel electrophoresis and high pressure liquid chromatography, respectively, and sequencing of the radioactive peptides. The sequence information produced by both methods identified specific labeled fragments of bovine Gbeta(1) that overlapped in the heptapeptide, Leu-Met-Thr-Tyr-Ser-His-Asp (amino acids 261-267). We conclude that NDPK B forms complexes with Gbetagamma dimers and contributes to G protein activation by increasing the high energetic phosphate transfer onto GDP via intermediately phosphorylated His-266 in Gbeta(1) subunits.

title: Model of a quinary structure between Krebs TCA cycle enzymes: a model for the metabolon.
abstract: The enzymes which are responsible for catalyzing sequential reactions in several metabolic pathways have been proposed to be highly organized in supramolecular complexes termed metabolons. However, the in situ existence of these weak complexes is difficult to demonstrate because many of them are dissociated during isolation due to dilution effects. Consequently, the metabolon concept is subject to controversy. A model system consisting of genetically prepared bienzymatic fusion proteins has been used to immobilize sequential metabolic enzymes in close proximity and to demonstrate possible kinetic advantages of metabolons. These experiments use the sequential Krebs TCA cycle enzymes from yeast mitochondrial malate dehydrogenase (MDH), citrate synthase (CS), and aconitase (ACO). Using the porcine high-definition structures of these three enzymes, we have performed computer-modeling studies in order to understand how the molecules may interact. Among the thousands of docking orientations we have tried, one was found to respond to the structural and experimental constraints from the results obtained with the yeast fusion proteins. Interestingly, this quinary structure model shows substantial interacting surface areas with spatial and electrostatic complementarities which make the complex thermodynamically stable. This structure also contains an unbroken electrostatically favorable channel connecting the active sites of ACO and CS, as well as the one previously reported between CS and MDH active sites. Charged amino acids which could be involved in interactions stabilizing the complex have been identified. This model will be used as the basis for further experimental work on the structure of the Krebs TCA cycle metabolon.

title: ARF6-GTP recruits Nm23-H1 to facilitate dynamin-mediated endocytosis during adherens junctions disassembly
abstract: ARF6-regulated endocytosis of E-cadherin is essential during the disassembly of adherens junctions in epithelial cells. Here, we show that activation of ARF6 promotes clathrin-dependent internalization of E-cadherin and caveolae at the basolateral cell surface. Furthermore, we demonstrate that ARF6-GTP, a constitutively activate form of ARF6, interacts with and recruits Nm23-H1, a nucleoside diphosphate (NDP) kinase that provides a source of GTP for dynamin-dependent fission of coated vesicles during endocytosis. Finally, we show that ARF6-mediated recruitment of Nm-23-H1 to cell junctions is accompanied by a decrease in the cellular levels of Rac1-GTP, consistent with previous findings that Nm23-H1 down-regulates activation of Rac1. These studies provide a molecular basis for ARF6 function in polarized epithelia during adherens junction disassembly.

title: Interaction of the Ras-related protein associated with diabetes rad and the putative tumor metastasis suppressor NM23 provides a novel mechanism of GTPase regulation.
abstract: Rad is the prototypic member of a new class of Ras-related GTPases. Purification of the GTPase-activating protein (GAP) for Rad revealed nm23, a putative tumor metastasis suppressor and a development gene in Drosophila. Antibodies against nm23 depleted Rad-GAP activity from human skeletal muscle cytosol, and bacterially expressed nm23 reconstituted the activity. The GAP activity of nm23 was specific for Rad, was absent with the S105N putative dominant negative mutant of Rad, and was reduced with mutations of nm23. In the presence of ATP, GDP.Rad was also reconverted to GTP.Rad by the nucleoside diphosphate (NDP) kinase activity of nm23. Simultaneously, Rad regulated nm23 by enhancing its NDP kinase activity and decreasing its autophosphorylation. Melanoma cells transfected with wild-type Rad, but not the S105N-Rad, showed enhanced DNA synthesis in response to serum; this effect was lost with coexpression of nm23. Thus, the interaction of nm23 and Rad provides a potential novel mechanism for bidirectional, bimolecular regulation in which nm23 stimulates both GTP hydrolysis and GTP loading of Rad whereas Rad regulates activity of nm23. This interaction may play important roles in the effects of Rad on glucose metabolism and the effects of nm23 on tumor metastasis and developmental regulation.

title: Defective protein histidine phosphorylation in islets from the Goto-Kakizaki diabetic rat.
abstract: We recently described novel regulatory roles for protein histidine phosphorylation of key islet proteins (e.g., nucleoside diphosphate kinase and succinyl thiokinase) in insulin secretion from the islet beta-cell (Kowluru A. Diabetologia 44: 89-94, 2001; Kowluru A, Tannous M, and Chen HQ.  160-169, 2002). In this context, we also characterized a novel, ATP- and GTP-sensitive protein histidine kinase in isolated beta-cells that catalyzed the histidine phosphorylation of islet (endogenous) proteins as well as exogenously added histone 4, and we implicated this kinase in the activation of islet endogenous G proteins (Kowluru A. Biochem Pharmacol 63: 2091-2100, 2002). In the present study, we describe abnormalities in ATP- or GTP-mediated histidine phosphorylation of nucleoside diphosphate kinase in islets derived from the Goto-Kakizaki (GK) rat, a model for non-insulin-dependent diabetes. Furthermore, we provide evidence for a marked reduction in the activities of ATP- or GTP-sensitive histidine kinases in GK rat islets. On the basis of these observations, we propose that alterations in protein histidine phosphorylation could contribute toward insulin-secretory abnormalities demonstrable in the diabetic islet.

title: Gαolf identification by RT-PCR in purified normal pancreatic B cells and in islets from rat models of non-insulin-dependent diabetes
abstract: Recent reports using immunohistochemistry have shown that Galphaolf which shares 88% homology with Galphas was expressed in pancreatic islets. To test the specificity of the expression of this G protein isotype in rat islet cells, B and non-B cells were separated by flow cytometry. The expression of Galphaolf and adenylyl cyclases (AC) of types II, III, V, and VI was evaluated by reverse-transcriptase polymerase chain reaction (RT-PCR). Since alterations in the expression of AC III were recently reported in the GK rat (a model of non-insulin-dependent diabetes mellitus, NIDDM), we also have analyzed the mRNA expression of Galphaolf and AC isoforms in pancreatic islets from GK rats and from adult rats neonatally treated by streptozotocin (nSTZ rats), another model of NIDDM. Southern blots of amplicons generated with specific primers of Galphaolf revealed the presence of a 540-bp band only in B cells. AC of types II, III, V, and VI were expressed both in B and non-B cells. However, AC III mRNA was clearly more abundant in non-B than in B cells. Moreover, in B cells the expression of AC VI was higher than that of AC V, whereas equal expressions of AC V and AC VI were found in non-B cells. In GK rat islets, the mRNA expressions of Galphaolf, AC II, and AC III were clearly increased and no change in AC V and AC VI was found. In nSTZ rat islets, Galphaolf expression was barely detectable, but AC II and AC III mRNA levels were higher than those observed in controls. In conclusion, Galphaolf mRNA appeared specifically expressed in islet B cells and was increased in GK islets. The steady-state mRNA levels of AC II and AC III were clearly increased in the islets of the two rat models of NIDDM. Thus, alterations in the expression of G protein isotypes and AC isoforms could contribute to the diabetic phenotype.

title: Insulin Secretory Deficiency and Glucose Intolerance in Rab3A Null Mice*
abstract: Insulin secretory dysfunction of the pancreatic β-cell in type-2 diabetes is thought to be due to defective nutrient sensing and/or deficiencies in the mechanism of insulin exocytosis. Previous studies have indicated that the GTP-binding protein, Rab3A, plays a mechanistic role in insulin exocytosis. Here, we report that Rab3A−/− mice develop fasting hyperglycemia and upon a glucose challenge show significant glucose intolerance coupled to ablated first-phase insulin release and consequential insufficient insulin secretion in vivo, without insulin resistance. Thein vivo insulin secretory response to arginine was similar in Rab3A−/− mice as Rab3A+/+ control animals, indicating a phenotype reminiscent of insulin secretory dysfunction found in type-2 diabetes. However, when a second arginine dose was given 10 min after, there was a negligible insulin secretory response in Rab3A−/− mice, compared with that in Rab3A+/+ animals, that was markedly increased above that to the first arginine stimulus. There was no difference in β-cell mass or insulin production between Rab3A−/− and Rab3A+/+ mice. However, in isolated islets, secretagogue-induced insulin release (by glucose, GLP-1, glyburide, or fatty acid) was ∼60–70% lower in Rab3A−/− islets compared with Rab3A+/+ controls. Nonetheless, there was a similar rate of glucose oxidation and glucose-induced rise in cytosolic [Ca2+] i flux between Rab3A−/− and Rab3A+/+ islet β-cells, indicating the mechanistic role of Rab3A lies downstream of generating secondary signals that trigger insulin release, at the level of secretory granule transport and/or exocytosis. Thus, Rab3A plays an important in vivo role facilitating the efficiency of insulin exocytosis, most likely at the level of replenishing the ready releasable pool of β-granules. Also, this study indicates, for the first time, that the in vivoinsulin secretory dysfunction found in type-2 diabetes can lie solely at the level of defective insulin exocytosis.

title: Inosine monophosphate dehydrogenase: A molecular switch integrating pleiotropic GTP-dependent beta-cell functions.
abstract: Studies of pancreatic islet function in the pathogenesis of type 2 diabetes mellitus have tended to focus on the short-term control of insulin secretion. However, the long-term control of beta-cell mass is also relevant to diabetes, since this parameter is reduced substantially even in non-insulin-dependent diabetes in humans. In animal models of type 2 diabetes, the normal balance between beta-cell proliferation and programmed cell death is perturbed. We take the perspective in this overview that inosine monophosphate dehydrogenase (IMPDH; EC 1.1.1. 205) may represent a previously neglected molecular integrator or sensor that exerts both functional (secretory) and anatomical (proliferative) effects within beta-cells. These properties reflect the fact that IMPDH is a rate-limiting enzyme in the new synthesis of the purine guanosine triphosphate (GTP), which modulates both exocytotic insulin secretion and DNA synthesis, as well as a number of other critical cellular functions within the beta-cell. Alterations in the expression or activity of IMPDH may be central to beta-cell replication, cell cycle progression, differentiation, and maintenance of adequate islet mass, effects that are probably mediated both by GTP directly, and indirectly via low molecular mass GTPases. If GTP becomes depleted, a hierarchy of beta-cell functions becomes progressively paralyzed, until eventually the effete cell is removed via apoptosis.

title: Cytokines, nitric oxide and insulin secreting cells.
abstract: Cytokines are a group of regulatory and immunomodulatory proteins involved in a number of physiological processes. Various disease states are believed to involve alteration of normal cytokine activity, including insulin-dependent diabetes mellitus, an autoimmune disease in which insulin secreting beta cells within pancreatic islets of Langerhans are selectively destroyed. Glucose-induced insulin secretion is inhibited by the cytokines interleukin-1 beta (IL-1 beta), interleukin-6 and tumour necrosis factor alpha (TNF) when combined with IL-1 beta in cultured rat islets, by IL-1 beta, TNF and interferon gamma in mouse islets, and by combined treatment of IL-1 beta, TNF and interferon gamma in human islets. Continued cytokine treatment in many cases leads to destruction of some, if not all, islet cells. A key factor in the inhibitory effect of IL-1 beta and TNF in rat islets is the generation of nitric oxide which inactivates enzymes such as aconitase and ribonucleotide reductase by formation of iron-nitrosyl complexes. This in turn may lead to reduced oxidation of glucose and synthesis of ATP and DNA respectively. The causes of cytokine-induced beta cell death are less well defined, but important factors may be nitric oxide-mediated DNA damage, depletion of NAD levels and toxic effects of oxygen free radicals and eicosanoids generated in addition to nitric oxide. Potentially important defence and repair responses induced by IL-1 beta treatment of rat islets are formation of heat shock protein, haem oxygenase, and superoxide dismutase. Other protective responses may be induction of cytokines and cytokine receptor antagonists.(ABSTRACT TRUNCATED AT 250 WORDS)

title: The role of interleukin-1 in the pathogenesis of IDDM
abstract: "It is perhaps today, where a certain underevaluation well, even disrespect o f scientific research is growing useful again and again to refer to the fact o f how well suited the discovery o f insulin is to demonstrate that unprejudiced scientific research which may not appear to be o f immediate practical value, sooner or later will be fruitful also to clinical practice" Oskar Minkowski, speech at the celebration of the Society for Internal Medicine of Rhein-Westphalen, 18 November, 1929 .

title: Cytokines and Nitric Oxide in Islet Inflammation and Diabetes
abstract: Cytokines released by both T lymphocytes and activated macrophages, in particular interleukin-1 (IL-1), have been implicated as immunological effector molecules that both inhibit insulin secretion from the pancreatic β cell and induce β-cell destruction. Recent findings have demonstrated that production of the free radical nitric oxide (NO), resulting from the expression of the cytokine-inducible isoform of NO synthase (iNOS), mediates these deleterious effects. The cellular mechanism responsible for inhibition of β-cell function and destruction by NO involves, in part, inactivation of enzymes specifically localized to the β-cell mitochondria that contain iron-sulfur centers or clusters. Intraislet release of IL-1 also inhibits β-cell function by this same cellular mechanism involving the overproduction of NO. In addition, the cytokine, IL-1, induces the co-expression of both iNOS and the cytokine-inducible isoform of cyclooxygenase, COX-2. The expression of COX-2 results in the overproduction of the proinflammatory prostaglandins and thromboxanes. Furthermore, NO produced by iNOS directly stimulates the activities of both constitutive and inducible isoforms of COX, further augmenting the overproduction of these proinflammatory mediators, NO and prostaglandins, which may be important in initiating or maintaining the inflammatory response and destruction of the β cell associated with autoimmune diabetes.

title: GTP-binding proteins in cell survival and demise: the emerging picture in the pancreatic beta-cell.
abstract: It is widely believed that guanine nucleotide-binding regulatory proteins (G-proteins) play central roles as "molecular switches" in a variety of cellular processes ranging from signal transduction to protein and vesicle trafficking. To achieve these regulatory functions, G-proteins form complexes with a wide range of effector molecules whose activities are altered upon interaction with the G-protein. These effector molecules can be either soluble or membrane bound, and it is likely that some are localized to secretory granules where they direct the movement, docking, and fusion of granules during exocytosis. The effector molecules regulated by G-proteins are diverse and include phospholipases, protein kinases, protein phosphatases, ion channels, adenylate cyclases, cytoskeletal elements, as well as secretory vesicle and plasma membrane-associated fusion-proteins. The majority of studies performed in the pancreatic beta-cell have focused on the role of G-proteins in the regulation of insulin secretion, whereas very little attention has been focused on their potential involvement in other cellular processes. Such studies have identified and implicated both heterotrimeric (comprising alpha, beta, and gamma subunits) and monomeric (low molecular mass) G-proteins in the regulation of insulin secretion, but intriguing recent evidence has also begun to emerge which favors the view that they may be involved in the maintenance of beta-cell viability. In the present commentary, we will review this evidence and discuss the current understanding of the role of G-proteins in the life and death of the beta-cell.

title: Positive modulation by Ras of interleukin-1beta-mediated nitric oxide generation in insulin-secreting clonal beta (HIT-T15) cells.
abstract: In the present study, we have shown that exposure of insulin-secreting clonal beta (HIT-T15) cells to interleukin-1beta (IL-1beta) results in a time- and concentration-dependent increase in nitric oxide (NO) release. These effects by IL-1beta on NO release were mediated by induction of inducible nitric oxide synthase (iNOS) from the cells. Preincubation of HIT cells with Clostridium sordellii lethal toxin-82, which irreversibly glucosylates and inactivates small G-proteins, such as Ras, Rap, Ral, and Rac, but not Cdc42, completely abolished IL-1beta-induced NO release. Pre-exposure of HIT cells to C. sordellii lethal toxin-9048, which monoglucosylates and inhibits Ras, Cdc42, Rac, and Rap, but not Ral, also attenuated IL-1beta-mediated NO release. These data indicate that activation of Ras and/or Rac may be necessary for IL-1beta-mediated NO release. Preincubation of HIT cells with C. difficile toxin-B, which monoglucosylates Rac, Cdc42, and Rho, had no demonstrable effects on IL-mediated NO release, ruling out the possibility that Rac may be involved in this signaling step. Further, two structurally dissimilar inhibitors of Ras function, namely manumycin A and damnacanthal, inhibited, in a concentration-dependent manner, the IL-1beta-mediated NO release from these cells. Together, our data provide evidence, for the first time, that Ras activation is an obligatory step in IL-1beta-mediated NO release and, presumably, the subsequent dysfunction of the pancreatic beta cell. Our data also provide a basis for future investigations to understand the mechanism of cytokine-induced beta cell death leading to the onset of insulin-dependent diabetes mellitus.

title: Cell replacement therapy for type 1 diabetes.
abstract: Replacement of the insulin-producing pancreatic islet beta cells represents the ultimate treatment for type 1 diabetes. Recent advances in islet transplantation underscore the urgent need for developing alternatives to human tissue donors, which are scarce. Two possible approaches are the expansion of differentiated beta cells by reversible immortalization and the generation of insulin-producing cells from embryonic or adult stem cells. It is possible that new insights into endocrine pancreas development will ultimately lead to manipulation of progenitor-cell fate towards the beta-cell phenotype of insulin production, storage and regulated secretion. Both allogeneic and autologous surrogate beta cells are likely to require protection from recurring autoimmunity. This protection might take the form of tolerization, cell encapsulation, or cell engineering with immunoprotective genes. If successful, these approaches could lead to widespread cell replacement therapy for type 1 diabetes.

title: Engineered cell lines for insulin replacement in diabetes: current status and future prospects
abstract: The recently completed diabetes complications and control trial has highlighted the need for improvement of insulin delivery systems for treatment of insulin-dependent diabetes mellitus. Despite steady improvement in methods for islet and whole pancreas transplantation over the past three decades, the broad-scale applicability of these approaches remains uncertain due in part to the difficulty and expense associated with procurement of functional tissue. To address this concern, we and others have been using the tools of molecular biology to develop cell lines with regulated insulin secretion that might serve as a surrogate for primary islets or pancreas tissue in transplantation therapy. This article seeks to provide a brief summary of the current status of this growing field, with a particular emphasis on progress in producing cell lines with appropriate glucose-stimulated insulin secretion.