Evaluation of the roles of protein serine/threonine kinases in the metabolic actions of insulin
Creators: name: Zhande, Rachel
Publisher: name: University of British Columbia
Year: 2009

description: Protein phosphorylation plays a central role in the biological effects of insulin. A number of proteins display increases in serine/threonine (Ser/Thr) phosphorylation in response to the hormone and include acetyl-CoA carboxylase (ACC) and the ribosomal protein S6. These phosphorylations are mediated by a discrete number of Ser/Thr protein kinases which are stimulated by the hormone. While the S6 kinases have been purified and are well characterized, very little is known about the insulin stimulated ACC-kinase. This study evaluated the roles of three different classes of the mitogen-activated protein (MAP) kinase family (ERKs, JNKs and p38) and that of protein kinase B in the metabolic responses of insulin in rat white adipose tissue. Initial evidence led to the hypothesis that an insulin-stimulated myelin basic protein (MBP) kinase may be an important ACC-kinase. A MBP kinase was highly purified from rat adipose tissue and displayed a number of properties indicating it was a member of the family of MAP kinases. However, the adipose tissue MBP kinase did not phosphorylate the insulin-directed site on ACC and did not induce insulin-like activation of ACC, indicating that it was not an ACC-kinase. This result therefore, argues strongly against the initial hypothesis that MBP/MAP kinases were significant insulin-stimulated ACC-kinases. Further characterization of the fat cell MBP kinase revealed its close association with the actin cytoskeleton. It is proposed that the cytoskeleton may act as an interface to nucleate signaling elements. This would provide efficiency and fidelity in signaling. Because extracellular osmolarity (and consequent regulation of cell volume) affects ! the three classes of MAP kinases and also influences liver carbohydrate metabolism, the effects of insulin and osmolarity on fatty acid biosynthesis and the activation state of the three classes of MAP kinases were therefore, investigated in adipose tissue. Changes in extracellular osmolarity alone, over the range from hypo-osmotic (228 mOsM) to hyperosmotic (404 mOsM) did not stimulate fatty acid biosynthesis in adipose tissue. Insulin stimulated the pathway to an equivalent extent under iso-osmotic (316 mOsM), hypo- and hyper-osmotic conditions. The activation of the ERKs differed from that of fatty acid synthesis, since hypo-osmotic medium alone activated the ERKs and because the activation of the ERKs was blocked in hyper-osmotic medium. Unlike fatty acid synthesis, JNKs were activated by both hypo-osmotic and hyper-osmotic medium in the absence of insulin. The activation state of p38 judged by tyrosine phosphorylation of p38, by associated MBP kinase activity or by the activity of a downstream heat-shock protein kinase was not affected by changes in osmolarity (200-400 mOsM) or by insulin. In conclusion, within a moderate physiological range, extracellular osmolarity did not lead to changes in the rates of de novo fatty acid synthesis in rat white adipose tissue (in contrast to previous studies with isolated rat hepatocytes). Therefore, activations of ERK1 and ERK2 are neither necessary nor sufficient for the activation of fatty acid biosynthesis. The p38 pathway was insensitive to insulin and activation of JNKs did not correlate with the stimulation of fatty acid biosynthesis. A newly-discovered protein kinase B (PKB), is insulin-activated and appears to play a significant role in regulating glycogen synthase. I therefore, examined the role of PKB in ACC regulation. Insulin provoked a rapid and sustained activation of PKB in rat white adipose. The activation of PKB was associated with a retardation of the mobility of the PKB protein on SDS-PAGE gels and was especially prominent in membrane fractions of rat adipose tissue. PKB immunoprecipitates strongly phosphorylated ACC and showed a striking preference for the 265-kDa ACC isoform. The major tryptic phosphopeptide from ACC phosphorylated by PKB immunoprecipitates co-migrated on 2-dimensional thin layer and high pressure chromatography with that obtained from ACC following phosphorylation with AMP-activated protein kinase (AMP-PK). Unlike AMP-PK, however, PKB immunoprecipitates did not phosphorylate the optimum (SAMS) peptide substrate. Furthermore, unlike AMP-PK, phosphorylation of ACC with PKB immunoprecipitates did not affect the enzymatic activity of ACC. Because specific PKB inhibitors are not yet available, we examined the effects of vanadium to shed light on the potential significance of PKB activation. Vanadium activated PKB, glycogen synthase and fatty acid biosynthesis with very similar dose dependencies. The maximal effects of vanadium were similar to the maximal effects of insulin and were not additive. PKB may therefore be required in the activation of glycogen synthase and fatty acid biosynthesis but further studies will be required to confirm that (ideally with PKB antagonists). In contrast, the anti-lipolytic effects of vanadium were evident at concentrations substantially below that required for PKB activation. Therefore, not all metabolic responses of vanadium are dependent on PKB activation.