BACKGROUND
glycogen synthase kinase- <dig> , an insulin-inhibited protein kinase, has been linked to the control of many cellular processes  <cit> .
it was originally identified as the protein kinase that phosphorylated and inactivated glycogen synthase in rabbit muscle  <cit> .
two highly related forms of gsk <dig>  are expressed from distinct genes  <cit> .
they share greater than 95% identity in their kinase domains and appear to be ubiquitously expressed in mammals  <cit> .
in resting cells gsk <dig> activity is high and inhibition of the kinase is achieved by at least two mechanisms, firstly through phosphorylation of an n-terminal serine residue   <cit> , and secondly through protein protein interaction  <cit> .
insulin promotes the phosphorylation of ser-9/ <dig> of gsk <dig> by activation of protein kinase b   <cit> , while canonical wnt signalling inhibits gsk <dig> independently of this n-terminal phosphorylation  <cit> .
in liver, inhibition of gsk <dig> reduces expression of the phosphoenolpyruvate carboxykinase  and glucose- <dig> phosphatase  genes, two rate limiting enzymes in hepatic glucose production  <cit> , as well as the igf binding protein- <dig>  gene  <cit> .
these gene promoters share a related dna sequence termed the thymine-rich insulin response element  that plays an important role in their response to insulin  <cit> .
expression of these genes as well as gsk <dig> activity is abnormally high in insulin resistant states such as type  <dig> diabetes mellitus   <cit> .
meanwhile overexpression of gsk <dig> antagonises insulin regulation of igfbp <dig>  <cit> .
therefore inhibition of gsk <dig> is proposed as a potential target for the treatment of t2dm  <cit> .
indeed selective gsk <dig> inhibitors significantly reduce hepatic glucose output and blood glucose levels in several animal models of obesity and diabetes  <cit> .
however, gsk <dig> is reported to have over  <dig> substrates, and mutation of one of these, apc, is linked to the development of colonic cancer  <cit> .
in addition, genetic deletion of one of the isoforms  leads to liver cell death, possibly due to increased sensitivity to tnfα  <cit> .
hence great care and consideration is being given to the development of gsk <dig> inhibitors for use in humans.
with this in mind we have initiated a search for molecules that link gsk <dig> to the gluconeogenic gene promoters and in particular the tire, since inhibition of gsk <dig> is sufficient to repress the isolated dna element  <cit> .
identification of the gsk <dig> substrate that regulates igfbp <dig>  g6pase and pepck gene expression, may permit manipulation of this specific interaction without affecting other gsk <dig> targets, thereby reducing potential side effects.
gsk <dig> is an unusual kinase in that most substrates must be phosphorylated  on a ser/thr four or five residues c-terminal to the gsk <dig> target residue .
priming of the substrate is performed by a distinct protein kinase, therefore inhibition of the priming kinase indirectly inhibits gsk <dig> regulation of the substrate, but only those substrates primed by that particular priming kinase.
priming kinases are potential targets for more specific interference with gsk <dig> function.
the basic leucine zipper transcription factor, caat-enhancer binding protein alpha  is a potential link between gsk <dig> and the gluconeogenic genes based on the following information in the literature; 1) c/ebpα is phosphorylated on two residues  by gsk <dig>  although the functional effect of phosphorylation is not yet clear  <cit> , 2) c/ebpα is a key molecule in the regulation of the pepck and g6pase gene promoters by camp  <cit>  and t <dig>  <cit> , 3) c/ebpα is regulated by wnt  <cit>  and insulin  <cit>  in brown adipocytes, and the insulin regulation involves pkb   <cit> , 4) pepck and g6pase expression, as well as glucose and glycogen metabolism is abnormal in animals lacking c/ebpα  <cit>  and 5) binding of a tire-interacting protein is blocked using an oligonucleotide representing a known c/ebpα binding sequence  <cit> .
therefore, we attempted to determine whether c/ebpα could modulate the activity of the isolated tire, or the gene promoters of interest, in a gsk <dig> regulated manner.
in addition, although t222/t <dig> of c/ebpα were identified as targets for gsk <dig> it has not previously been established whether s <dig> was a target for a 'priming' kinase, so we have also investigated whether mutation of s <dig> influences c/ebpα regulation.
RESULTS
c/ebpα regulates the tire independently of t222/ <dig> phosphorylation
we have previously shown that insulin, or pharmacological inhibition of gsk <dig>  reduced the expression of endogenous pepck, g6pase and igfbp <dig> genes and also the activity of the isolated thymine-rich insulin response element  found in these gene promoters  <cit> .
in an attempt to further characterise the gsk <dig> regulation of pepck, g6pase and igfbp <dig> we assessed whether a target for gsk <dig>  namely c/ebpα, could regulate tire activity.
in the hepatocyte c/ebpα activity is linked to camp induction of genes, so insulin inhibition of c/ebpα could explain repression of these genes by insulin.
in this model, phosphorylation of c/ebpα by gsk <dig> would have to induce c/ebpα activity toward the tire.
interestingly, c/ebpα overexpression had a repressive effect on tire activity in the rat hepatoma h4iie cells , and the repression was lost when the tire sequence was mutated at two residues to produce an insulin insensitive sequence .
hence c/ebpα regulated the tire in a sequence specific and insulin-like manner.
however, mutation of the gsk3-regulated residues on c/ebpα  to alanines  did not alter the ability of c/ebpα to repress tire activity .
one possibility is that the dephosphorylated form of c/ebpα repressed the tire, and that the overexpressed c/ebpα was not substantially phosphorylated by gsk <dig>  consistent with this hypothesis, co-expression of an active form of gsk <dig> reduced the effect of wild-type c/ebpα on the tire .
unfortunately, increased gsk <dig> activity also blocked the repressive action of the c/ebpα-aaa  clearly demonstrating that phosphorylation of t222/t226/s <dig> was not required for the action of gsk <dig> on the isolated tire in h4iie cells.
this data questioned the functional importance of t222/t <dig> phosphorylation in the regulation of c/ebpα activity.
gsk <dig> phosphorylates c/ebpα in intact cells
it was therefore important to establish whether t <dig> and t <dig> were actually phosphorylated by gsk <dig> in intact cells.
cells were transfected with wild-type and mutant c/ebpα and total and phosphorylated c/ebpα visualised by western blotting.
t222/ <dig> phosphorylation, measured with this antibody, was clearly present in intact cells .
incubation with  <dig> or  <dig> μm of the gsk <dig> inhibitor chir <dig>  <cit>  for  <dig> or  <dig> h significantly reduced the phosphorylation of t222/ <dig> of wild-type c/ebpα , confirming that gsk <dig> was a t222/ <dig> kinase in these cells.
at shorter incubations,  <dig> μm chir <dig> was sufficient to give maximal reduction in t222/ <dig> phosphorylation, while  <dig> μm had a greater effect at  <dig> h incubation.
this is similar to the effect of these concentrations on β-catenin activation in the h4iie cells  <cit> .
meanwhile, co-expression of active gsk3α  only produced a marginal induction of phosphorylation  suggesting that the expressed c/ebpα was either almost fully phosphorylated at t222/ <dig> or that it was not a particularly good substrate for gsk <dig>  mutation of t222/ <dig> to alanines made the mutant c/ebpα unresponsive to the phosphospecific antibody .
interestingly, the dephosphorylation of c/ebpα in h4iie cells treated with chir <dig> was rapid, but not complete .
however, there was no significant reduction in t222/ <dig> phosphorylation following up to  <dig> h incubation with insulin , even though insulin promoted phosphorylation of ser9/ser <dig> of gsk3β and gsk3α respectively.
insulin reduced gsk <dig> activity around 50% in the h4iie cell line but this is clearly not sufficient to reduce t222/ <dig> phosphorylation.
therefore it appears that insulin does not regulate t222/ <dig> phosphorylation in these cells through inhibition of gsk <dig>  however, the data suggested that some level of t222/ <dig> phosphorylation by gsk <dig> occurs on the overexpressed protein .
c/ebpα is a relatively poor substrate for gsk <dig> in vitro
we next characterised c/ebpα phosphorylation by gsk <dig> in vitro .
incubation of c/ebpα with gsk3β for up to one hour resulted in a slow incorporation of less than  <dig>  mol phosphate per mol of protein .
in comparison, p42map kinase phosphorylation of c/ebpα increased over  <dig> min peaking around  <dig>  mol per mol .
phosphorylation of c/ebpα by p42map kinase did not occur at t <dig>  t <dig> or s <dig>  and is more likely to be on s <dig>  <cit> .
interestingly western blot analysis of c/ebpα following incubation with gsk3β gave little indication of this slow increase in phosphorylation, and a very strong signal was obtained with the phosphospecific antibody even at low stoichiometry of phosphorylation .
these data demonstrated that c/ebpα was a relatively poor substrate for gsk <dig> in vitro ,but that the phospho-t222/ <dig> antibody was highly sensitive.
it is difficult to directly compare phosphorylation of c/ebpα with other substrates of gsk <dig> as most are poorly phosphorylated prior to priming.
for example crmp phosphorylation by gsk <dig>  incorporates around  <dig>  mol/mol phosphate within  <dig> h of incubation  <cit> .
however, this increases more than 5-fold if the substrate is first primed.
of course we can never rule out the possibility that an intrinsic factor improves the gsk3-c/ebpα interaction in vivo ,either promoting priming at an unidentified residue or allowing greater phosphorylation by gsk <dig>
gsk <dig> phosphorylation of t222/ <dig> in cells does not require s <dig> phosphorylation
many substrates of gsk <dig> are primed for kinase recognition by prior phosphorylation at a s/t residue four amino acids c-terminal to the target site  <cit> .
s <dig> lies within this 'consensus' gsk <dig> targeting sequence .
therefore s <dig> phosphorylation could prime for t <dig> phosphorylation by gsk <dig>  permitting subsequent phosphorylation of t <dig> by gsk <dig>  however, mutation of s <dig> to alanine had no effect on t222/ <dig> phosphorylation in intact cells .
this argued that s <dig> phosphorylation did not prime for t <dig> phosphorylation by gsk <dig> in resting cells, or that the wild-type c/ebpα is not being efficiently phosphorylated at s <dig>  since the amino acid immediately following s <dig> is a proline , we hypothesised that priming may only occur after induction of a proline-directed kinase such as a member of the map kinase family.
therefore we attempted to induce s <dig> phosphorylation by incubation of cells with serum, which stimulates the classical map kinase and several other proline-directed kinases such as cyclin-dependent kinases.
however, t222/ <dig> phosphorylation of wild-type or s230a c/ebpα was not altered in serum treated cells , despite activation of the p90rsk downstream of the p42/p44map kinase .
this demonstrated we could not alter s <dig> phosphorylation by incubating cells with or without serum.
interestingly, serum treatment induced phosphorylation of s21/ <dig> of gsk3α/β , suggesting some inhibition of gsk <dig> occurs in the serum treated cells.
this was not sufficient to reduce c/ebpα phosphorylation but did partially reduce glycogen synthase phosphorylation .
similarly, treatment of the cells with chir <dig> did not completely inhibit t222/ <dig> phosphorylation , despite a complete loss of glycogen synthase phosphorylation .
therefore t222/ <dig> must be relatively resistant to dephosphorylation or a distinct t222/ <dig> protein kinase must be present in these cells.
again, potential substrates for gsk <dig> have been reported that have their priming at a residue more distant from the gsk <dig> target site, and we cannot discount that possibility.
however, our data strongly argues against priming through s <dig> phosphorylation.
prephosphorylation of cebpα by p <dig> map kinase did not prime for subsequent phosphorylation by gsk <dig> in vitro , confirming that s <dig> is not targeted by p <dig> map kinase.
similarly, incubation of cells with either a p42map kinase inhibitor  or a p38map kinase inhibitor  had no effect on phosphorylation of cebpα at t222/ <dig> .
c/ebpα induced a consensus binding element while repressing the tire in h4iie cells
in contrast to the repressive action on tire activity, overexpression of c/ebpα in h4iie cells induced the activity of a consensus c/ebp binding element  .
we were therefore able to investigate whether the gsk <dig> regulation of t222/t <dig> was dependent on the dna sequence analysed.
mutation of t222/226/s <dig> to alanines  made no difference to the induction of the cbe by c/ebpα .
similarly, inhibition of gsk <dig> using chir <dig> or insulin had no effect on cbe activity .
therefore gsk <dig> phosphorylation of t222/ <dig> does not regulate either induction of the cbe or repression of the tire in the h4iie cells.
interestingly cbe activity was induced in the presence of the synthetic glucocorticoid dexamethasone to a similar level as that seen with c/ebpα overexpression .
c/ebpα overexpression does not regulate endogenous tire-containing genes
in order to establish whether changes in c/ebpα phosphorylation could alter expression of the insulin regulated genes igfbp <dig> and g6pase, we overexpressed c/ebpα in h4iie cells using an adenoviral vector to produce almost 100% transfection efficiency .
we observed no effect of c/ebpα expression on igfbp <dig>  or g6pase  gene expression, and no alteration in regulation of either gene by dexamethasone, insulin or chir <dig>  however, there was a significant repression of the co-expressed bp1-tire by c/ebpα when compared with its effect on the insulin resistant tire  .
this suggests that c/ebpα overexpression represses the isolated element but has no effect on this element when it is in the context of the intact gene promoters.
this is an example of the dangers of studying isolated promoter elements and extrapolating to intact gene promoters without examining endogenous regulation.
interestingly, a postnatal knockout of c/ebpα resulted in reduced basal expression of pepck and g6pase but did not alter regulation of these genes by camp  <cit> .
again, this would argue against a repressive effect of this factor on the endogenous genes.
c/ebpα regulation of the cbe in preadipocytes is greater when t222/ <dig> are mutated
c/ebpα has an important role in adipocyte differentiation  <cit> , therefore we transfected 3t3-l <dig> preadipocytes with c/ebpα and measured cbe activity .
in contrast to the h4iie cells, mutation of t222/ <dig> to alanines resulted in a significant increase in transactivation potential compared to wild-type c/ebpα .
this suggested that phosphorylation of these residues was inhibiting c/ebpα activity in these cells.
consistent with this hypothesis, inhibition of gsk <dig> using chir <dig> or insulin induced wild-type c/ebpα activity .
in addition, the c/ebpα s230a mutant had similar transactivation potential to the aa mutant , strongly suggesting that s <dig> was a priming site for t222/t <dig> phosphorylation in these cells.
however we could find no evidence that phosphorylation of t222/ <dig> was reduced in the s230a mutant .
although there was a great deal of inter-experiment variation in the phosphorylation of t222/t <dig> in the wt and the s230a mutant there was not a significant difference between the two, and in  <dig> out of the three experiments the s230a mutant exhibited higher phosphorylation than the wt protein .
therefore, changes in phosphorylation of these two sites cannot account for the difference in c/ebpα activity when the s <dig> site is mutated to alanine.
ideally it would be beneficial to confirm these data on endogenous gene expression in the adipocyte cell line, however these cells do not transfect efficiently with the adenoviral constructs making it impossible to examine their effect on endogenous genes.
CONCLUSIONS
in summary, c/ebpα is not the link between gsk <dig> and the hepatic tire-containing gene promoters.
however, c/ebpα can regulate the tire, at least when overexpressed, and the residues t222/ <dig> and s <dig> all influence c/ebpα function in preadipocytes.
interestingly we demonstrate opposite effects of c/ebpα overexpression on tire and cbe activity, and a tissue specific role for the t222/ <dig> motif in this activity.
our data also provides further evidence of the differences in transcription factor activity when measured using isolated promoter elements as opposed to intact gene promoters.
during the course of this study a regulation of c/ebpβ by gsk <dig> in adipocytes was reported  <cit> .
therefore it will be of interest to confirm whether this c/ebp isoform is the link between gsk <dig> inhibition and tire regulation in hepatocytes.

 glycogen synthase kinase-, an insulin-inhibited protein kinase, has been linked to the control of many cellular processes. it was originally identified as the protein kinase that phosphorylated and inactivated glycogen synthase in rabbit muscle. two highly related forms of gsk are expressed from distinct genes. they share greater than 95% identity in their kinase domains and appear to be ubiquitously expressed in mammals. in resting cells gsk activity is high and inhibition of the kinase is achieved by at least two mechanisms, firstly through phosphorylation of an n-terminal serine residue, and secondly through protein protein interaction. insulin promotes the phosphorylation of ser-9/ of gsk by activation of protein kinase b, while canonical wnt signalling inhibits gsk independently of this n-terminal phosphorylation. in liver, inhibition of gsk reduces expression of the phosphoenolpyruvate carboxykinase and glucose- phosphatase genes, two rate limiting enzymes in hepatic glucose production, as well as the igf binding protein- gene. these gene promoters share a related dna sequence termed the thymine-rich insulin response element that plays an important role in their response to insulin. expression of these genes as well as gsk activity is abnormally high in insulin resistant states such as type diabetes mellitus. meanwhile overexpression of gsk antagonises insulin regulation of igfbp. therefore inhibition of gsk is proposed as a potential target for the treatment of t2dm. indeed selective gsk inhibitors significantly reduce hepatic glucose output and blood glucose levels in several animal models of obesity and diabetes. however, gsk is reported to have over substrates, and mutation of one of these, apc, is linked to the development of colonic cancer. in addition, genetic deletion of one of the isoforms leads to liver cell death, possibly due to increased sensitivity to tnfα. hence great care and consideration is being given to the development of gsk inhibitors for use in humans. with this in mind we have initiated a search for molecules that link gsk to the gluconeogenic gene promoters and in particular the tire, since inhibition of gsk is sufficient to repress the isolated dna element. identification of the gsk substrate that regulates igfbp g6pase and pepck gene expression, may permit manipulation of this specific interaction without affecting other gsk targets, thereby reducing potential side effects. gsk is an unusual kinase in that most substrates must be phosphorylated on a ser/thr four or five residues c-terminal to the gsk target residue. priming of the substrate is performed by a distinct protein kinase, therefore inhibition of the priming kinase indirectly inhibits gsk regulation of the substrate, but only those substrates primed by that particular priming kinase. priming kinases are potential targets for more specific interference with gsk function. the basic leucine zipper transcription factor, caat-enhancer binding protein alpha is a potential link between gsk and the gluconeogenic genes based on the following information in the literature; 1) c/ebpα is phosphorylated on two residues by gsk although the functional effect of phosphorylation is not yet clear, 2) c/ebpα is a key molecule in the regulation of the pepck and g6pase gene promoters by camp and t, 3) c/ebpα is regulated by wnt and insulin in brown adipocytes, and the insulin regulation involves pkb, 4) pepck and g6pase expression, as well as glucose and glycogen metabolism is abnormal in animals lacking c/ebpα and 5) binding of a tire-interacting protein is blocked using an oligonucleotide representing a known c/ebpα binding sequence. therefore, we attempted to determine whether c/ebpα could modulate the activity of the isolated tire, or the gene promoters of interest, in a gsk regulated manner. in addition, although t222/t of c/ebpα were identified as targets for gsk it has not previously been established whether s was a target for a'priming' kinase, so we have also investigated whether mutation of s influences c/ebpα regulation.  c/ebpα regulates the tire independently of t222/ phosphorylation we have previously shown that insulin, or pharmacological inhibition of gsk reduced the expression of endogenous pepck, g6pase and igfbp genes and also the activity of the isolated thymine-rich insulin response element found in these gene promoters. in an attempt to further characterise the gsk regulation of pepck, g6pase and igfbp we assessed whether a target for gsk namely c/ebpα, could regulate tire activity. in the hepatocyte c/ebpα activity is linked to camp induction of genes, so insulin inhibition of c/ebpα could explain repression of these genes by insulin. in this model, phosphorylation of c/ebpα by gsk would have to induce c/ebpα activity toward the tire. interestingly, c/ebpα overexpression had a repressive effect on tire activity in the rat hepatoma h4iie cells, and the repression was lost when the tire sequence was mutated at two residues to produce an insulin insensitive sequence. hence c/ebpα regulated the tire in a sequence specific and insulin-like manner. however, mutation of the gsk3-regulated residues on c/ebpα to alanines did not alter the ability of c/ebpα to repress tire activity. one possibility is that the dephosphorylated form of c/ebpα repressed the tire, and that the overexpressed c/ebpα was not substantially phosphorylated by gsk consistent with this hypothesis, co-expression of an active form of gsk reduced the effect of wild-type c/ebpα on the tire. unfortunately, increased gsk activity also blocked the repressive action of the c/ebpα-aaa clearly demonstrating that phosphorylation of t222/t226/s was not required for the action of gsk on the isolated tire in h4iie cells. this data questioned the functional importance of t222/t phosphorylation in the regulation of c/ebpα activity. gsk phosphorylates c/ebpα in intact cells it was therefore important to establish whether t and t were actually phosphorylated by gsk in intact cells. cells were transfected with wild-type and mutant c/ebpα and total and phosphorylated c/ebpα visualised by western blotting. t222/ phosphorylation, measured with this antibody, was clearly present in intact cells. incubation with or μm of the gsk inhibitor chir for or h significantly reduced the phosphorylation of t222/ of wild-type c/ebpα, confirming that gsk was a t222/ kinase in these cells. at shorter incubations, μm chir was sufficient to give maximal reduction in t222/ phosphorylation, while μm had a greater effect at h incubation. this is similar to the effect of these concentrations on β-catenin activation in the h4iie cells. meanwhile, co-expression of active gsk3α only produced a marginal induction of phosphorylation suggesting that the expressed c/ebpα was either almost fully phosphorylated at t222/ or that it was not a particularly good substrate for gsk mutation of t222/ to alanines made the mutant c/ebpα unresponsive to the phosphospecific antibody. interestingly, the dephosphorylation of c/ebpα in h4iie cells treated with chir was rapid, but not complete. however, there was no significant reduction in t222/ phosphorylation following up to h incubation with insulin, even though insulin promoted phosphorylation of ser9/ser of gsk3β and gsk3α respectively. insulin reduced gsk activity around 50% in the h4iie cell line but this is clearly not sufficient to reduce t222/ phosphorylation. therefore it appears that insulin does not regulate t222/ phosphorylation in these cells through inhibition of gsk however, the data suggested that some level of t222/ phosphorylation by gsk occurs on the overexpressed protein. c/ebpα is a relatively poor substrate for gsk in vitro we next characterised c/ebpα phosphorylation by gsk in vitro. incubation of c/ebpα with gsk3β for up to one hour resulted in a slow incorporation of less than mol phosphate per mol of protein. in comparison, p42map kinase phosphorylation of c/ebpα increased over min peaking around mol per mol. phosphorylation of c/ebpα by p42map kinase did not occur at t t or s and is more likely to be on s. interestingly western blot analysis of c/ebpα following incubation with gsk3β gave little indication of this slow increase in phosphorylation, and a very strong signal was obtained with the phosphospecific antibody even at low stoichiometry of phosphorylation. these data demonstrated that c/ebpα was a relatively poor substrate for gsk in vitro,but that the phospho-t222/ antibody was highly sensitive. it is difficult to directly compare phosphorylation of c/ebpα with other substrates of gsk as most are poorly phosphorylated prior to priming. for example crmp phosphorylation by gsk incorporates around mol/mol phosphate within h of incubation. however, this increases more than 5-fold if the substrate is first primed. of course we can never rule out the possibility that an intrinsic factor improves the gsk3-c/ebpα interaction in vivo,either promoting priming at an unidentified residue or allowing greater phosphorylation by gsk gsk phosphorylation of t222/ in cells does not require s phosphorylation many substrates of gsk are primed for kinase recognition by prior phosphorylation at a s/t residue four amino acids c-terminal to the target site. s lies within this'consensus' gsk targeting sequence. therefore s phosphorylation could prime for t phosphorylation by gsk permitting subsequent phosphorylation of t by gsk however, mutation of s to alanine had no effect on t222/ phosphorylation in intact cells. this argued that s phosphorylation did not prime for t phosphorylation by gsk in resting cells, or that the wild-type c/ebpα is not being efficiently phosphorylated at s since the amino acid immediately following s is a proline, we hypothesised that priming may only occur after induction of a proline-directed kinase such as a member of the map kinase family. therefore we attempted to induce s phosphorylation by incubation of cells with serum, which stimulates the classical map kinase and several other proline-directed kinases such as cyclin-dependent kinases. however, t222/ phosphorylation of wild-type or s230a c/ebpα was not altered in serum treated cells, despite activation of the p90rsk downstream of the p42/p44map kinase. this demonstrated we could not alter s phosphorylation by incubating cells with or without serum. interestingly, serum treatment induced phosphorylation of s21/ of gsk3α/β, suggesting some inhibition of gsk occurs in the serum treated cells. this was not sufficient to reduce c/ebpα phosphorylation but did partially reduce glycogen synthase phosphorylation. similarly, treatment of the cells with chir did not completely inhibit t222/ phosphorylation, despite a complete loss of glycogen synthase phosphorylation. therefore t222/ must be relatively resistant to dephosphorylation or a distinct t222/ protein kinase must be present in these cells. again, potential substrates for gsk have been reported that have their priming at a residue more distant from the gsk target site, and we cannot discount that possibility. however, our data strongly argues against priming through s phosphorylation. prephosphorylation of cebpα by p map kinase did not prime for subsequent phosphorylation by gsk in vitro, confirming that s is not targeted by p map kinase. similarly, incubation of cells with either a p42map kinase inhibitor or a p38map kinase inhibitor had no effect on phosphorylation of cebpα at t222/. c/ebpα induced a consensus binding element while repressing the tire in h4iie cells in contrast to the repressive action on tire activity, overexpression of c/ebpα in h4iie cells induced the activity of a consensus c/ebp binding element. we were therefore able to investigate whether the gsk regulation of t222/t was dependent on the dna sequence analysed. mutation of t222/226/s to alanines made no difference to the induction of the cbe by c/ebpα. similarly, inhibition of gsk using chir or insulin had no effect on cbe activity. therefore gsk phosphorylation of t222/ does not regulate either induction of the cbe or repression of the tire in the h4iie cells. interestingly cbe activity was induced in the presence of the synthetic glucocorticoid dexamethasone to a similar level as that seen with c/ebpα overexpression. c/ebpα overexpression does not regulate endogenous tire-containing genes in order to establish whether changes in c/ebpα phosphorylation could alter expression of the insulin regulated genes igfbp and g6pase, we overexpressed c/ebpα in h4iie cells using an adenoviral vector to produce almost 100% transfection efficiency. we observed no effect of c/ebpα expression on igfbp or g6pase gene expression, and no alteration in regulation of either gene by dexamethasone, insulin or chir however, there was a significant repression of the co-expressed bp1-tire by c/ebpα when compared with its effect on the insulin resistant tire. this suggests that c/ebpα overexpression represses the isolated element but has no effect on this element when it is in the context of the intact gene promoters. this is an example of the dangers of studying isolated promoter elements and extrapolating to intact gene promoters without examining endogenous regulation. interestingly, a postnatal knockout of c/ebpα resulted in reduced basal expression of pepck and g6pase but did not alter regulation of these genes by camp. again, this would argue against a repressive effect of this factor on the endogenous genes. c/ebpα regulation of the cbe in preadipocytes is greater when t222/ are mutated c/ebpα has an important role in adipocyte differentiation, therefore we transfected 3t3-l preadipocytes with c/ebpα and measured cbe activity. in contrast to the h4iie cells, mutation of t222/ to alanines resulted in a significant increase in transactivation potential compared to wild-type c/ebpα. this suggested that phosphorylation of these residues was inhibiting c/ebpα activity in these cells. consistent with this hypothesis, inhibition of gsk using chir or insulin induced wild-type c/ebpα activity. in addition, the c/ebpα s230a mutant had similar transactivation potential to the aa mutant, strongly suggesting that s was a priming site for t222/t phosphorylation in these cells. however we could find no evidence that phosphorylation of t222/ was reduced in the s230a mutant. although there was a great deal of inter-experiment variation in the phosphorylation of t222/t in the wt and the s230a mutant there was not a significant difference between the two, and in out of the three experiments the s230a mutant exhibited higher phosphorylation than the wt protein. therefore, changes in phosphorylation of these two sites cannot account for the difference in c/ebpα activity when the s site is mutated to alanine. ideally it would be beneficial to confirm these data on endogenous gene expression in the adipocyte cell line, however these cells do not transfect efficiently with the adenoviral constructs making it impossible to examine their effect on endogenous genes.  in summary, c/ebpα is not the link between gsk and the hepatic tire-containing gene promoters. however, c/ebpα can regulate the tire, at least when overexpressed, and the residues t222/ and s all influence c/ebpα function in preadipocytes. interestingly we demonstrate opposite effects of c/ebpα overexpression on tire and cbe activity, and a tissue specific role for the t222/ motif in this activity. our data also provides further evidence of the differences in transcription factor activity when measured using isolated promoter elements as opposed to intact gene promoters. during the course of this study a regulation of c/ebpβ by gsk in adipocytes was reported. therefore it will be of interest to confirm whether this c/ebp isoform is the link between gsk inhibition and tire regulation in hepatocytes. 
the work demonstrates an important role for this domain in the regulation of c/ebpα activity in adipocytes but not hepatocytes, however gsk phosphorylation of these residues does not mediate regulation of this c/ebp activity. pharmacological inhibition of gsk mimics the effect of insulin on phosphoenolpyruvate carboxykinase, glucose- phosphatase and igf binding protein- gene expression. finally, we demonstrate that c/ebpα is a very poor substrate for gsk in vitro and in cells. caat/enhancer binding protein alpha regulates these gene promoters in liver and is phosphorylated on two residues by gsk although the functional outcome of the phosphorylation has not been established. we aimed to establish whether cebpα is a link between gsk and these gene promoters. phosphorylation of t222/t is decreased by gsk inhibition, suggesting gsk does phosphorylate t222/ in intact cells. c/ebpα represses the igfbp thymine-rich insulin response element, but mutation of t or t of c/ebpα to non-phosphorylatable alanines has no effect on c/ebpα activity in liver cells. meanwhile c/ebpα activity in 3t l preadipocytes was enhanced by mutation of t222/t and/or s to alanine residues. in short, we find no evidence that c/ebpα activity is regulated by direct phosphorylation by gsk 
 glycogen synthase kinase- activity is repressed following insulin treatment of cells. pharmacological inhibition of gsk mimics the effect of insulin on phosphoenolpyruvate carboxykinase, glucose- phosphatase and igf binding protein- gene expression. caat/enhancer binding protein alpha regulates these gene promoters in liver and is phosphorylated on two residues by gsk although the functional outcome of the phosphorylation has not been established. we aimed to establish whether cebpα is a link between gsk and these gene promoters.  c/ebpα represses the igfbp thymine-rich insulin response element, but mutation of t or t of c/ebpα to non-phosphorylatable alanines has no effect on c/ebpα activity in liver cells. phosphorylation of t222/t is decreased by gsk inhibition, suggesting gsk does phosphorylate t222/ in intact cells. however, phosphorylation was not altered by treatment of liver cells with insulin. meanwhile c/ebpα activity in 3t l preadipocytes was enhanced by mutation of t222/t and/or s to alanine residues. finally, we demonstrate that c/ebpα is a very poor substrate for gsk in vitro and in cells.  the work demonstrates an important role for this domain in the regulation of c/ebpα activity in adipocytes but not hepatocytes, however gsk phosphorylation of these residues does not mediate regulation of this c/ebp activity. in short, we find no evidence that c/ebpα activity is regulated by direct phosphorylation by gsk 