Elevated protein kinase C betaII is an early promotive event in colon carcinogenesis.

Protein kinase C (PKC) has been implicated in colon carcinogenesis in humans and in rodent models. However, little is known about the specific role of individual PKC isozymes in this process. We recently demonstrated that elevated expression of PKC betaII in the colonic epithelium induces hyperproliferation in vivo (N. R. Murray et al., J. Cell Biol., 145: 699-711, 1999). Because hyperproliferation is a major risk factor for colon cancer, we assessed whether specific alterations in PKC betaII expression occur during azoxymethane-induced colon carcinogenesis in mice. An increase in PKC betaII expression was observed in preneoplastic lesions (aberrant crypt foci, 3.7-fold) compared with saline-treated animals, and in colon tumors (7.8-fold; P = 0.011) compared with uninvolved colonic epithelium. In contrast, PKC alpha and PKC betaI (a splicing variant of PKC betaII) expression was slightly decreased in aberrant crypt foci and dramatically reduced in colon tumors. Quantitative reverse transcription-PCR analysis revealed that PKC mRNA levels do not directly correlate with PKC protein levels, indicating that PKC isozyme expression is likely regulated at the posttranscriptional/translational level. Finally, transgenic mice expressing elevated PKC betaII in the colonic epithelium exhibit a trend toward increased colon tumor formation after exposure to azoxymethane. Taken together, our results demonstrate that elevated expression of PKC betaII is an important early, promotive event that plays a role in colon cancer development.

Mapping of a molecular determinant for protein kinase C betaII isozyme function.

In human erythroleukemia (K562) cells, the highly related protein kinase C (PKC) alpha and PKC betaII isozymes serve distinct functions in cellular differentiation and proliferation, respectively. Previous studies using two domain switch PKC chimera revealed that the catalytic domains of PKC alpha and betaII contain molecular determinants important for isozyme-specific function (Walker, S. D., Murray, N. R., Burns, D. J., and Fields, A. P. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 9156-9160). We have now analyzed a panel of PKC chimeras to determine the specific region within the catalytic domain important for PKC betaII function. A cellular assay for PKC betaII function was devised based on the finding that PKC betaII selectively translocates to the nucleus and phosphorylates nuclear lamin B in response to the PKC activator bryostatin. This response is strictly dependent upon expression of PKC betaII or a PKC chimera that functions like PKC betaII. We demonstrate that a PKC alpha/betaII chimera containing only the carboxyl-terminal 13 amino acids from PKC betaII (betaII V5) is capable of nuclear translocation and lamin B phosphorylation. These results are consistent with our recent observation that the PKC betaII V5 region binds to phosphatidylglycerol (PG), a potent and selective PKC betaII activator present in the nuclear membrane (Murray, N. R., and Fields, A. P. (1998) J. Biol. Chem. 273, 11514-11520). Soluble betaII V5 peptide selectively inhibits PG-stimulated PKC betaII activity in a dose-dependent fashion, indicating that PG-mediated activation of PKC betaII involves interactions with the betaII V5 region of the enzyme. We conclude that betaII V5 is a major determinant for PKC betaII nuclear function and suggest a model in which binding of PG to the betaII V5 region stimulates nuclear PKC betaII activity during G2 phase of the cell cycle.

Wortmannin converts insulin but not oxytocin from an antilipolytic to a lipolytic agent in the presence of forskolin.

Insulin is an important regulator of glucose transport and lipolysis in adipocytes. The present studies compared the effects of insulin in rat adipocytes with the effects of oxytocin and peroxovanadate, which mimic some effects of insulin. The antilipolytic effects of peroxovanadate and oxytocin were unaffected by 500 nmol/L wortmannin, which blocked the antilipolytic action of insulin. However, wortmannin, which is a relatively specific inhibitor of phosphatidylinositol 3-kinase, did block most of the stimulation of glucose metabolism by peroxovanadate while having little effect on that due to oxytocin. Under appropriate conditions, it was also possible to demonstrate a lipolytic action of insulin, especially with low (0.1 to 1 nmol/L) concentrations of insulin after exposure of adipocytes to 50 nmol/L wortmannin. The data provide additional support for the hypothesis that oxytocin and peroxovanadate affect adipose tissue metabolism by mechanisms distinctly different from those involved in insulin action.

Enhanced desensitization and phosphorylation of the beta 1-adrenergic receptor in rat adipocytes by peroxovanadate.

Peroxovanadate (PVN) is an insulin-like agent that inhibits the dephosphorylation of the insulin receptor kinase. PVN inhibited the lipolytic action of 0.1 microM isoproterenol by 88%, which is a relatively specific beta 1 catecholamine agonist at this concentration, but was largely ineffective against beta 3 agonists or forskolin. To determine whether PVN-mediated desensitization of the beta 1 AR was associated with enhanced phosphorylation, we immunoprecipitated the beta 1 AR from rat adipocytes that were metabolically labeled with 32PO4. Isoproterenol enhanced the net phosphorylation of the beta 1 AR by 8 +/- 2-fold over control. PVN increased the net phosphorylation of the beta 1 AR by 5 +/- 0.5-fold, and together with isoproterenol, they enhanced the phosphorylation of the beta 1 AR by 2-fold over isoproterenol alone. Phosphoamino acid analysis of the phosphorylated receptor revealed phosphate incorporation into serine that was proportional to the radioactivity incorporated into the immunoprecipitated receptor. PVN inhibited the serine/threonine phosphatase calcineurin, suggesting that inhibition of receptor dephosphorylation may play a role in the actions of PVN. Cyanogen bromide cleavage of the phosphorylated beta 1 AR generated a phosphoprotein with a molecular mass consistent with carboxyl-terminal phosphorylation. Furthermore, the magnitude of receptor phosphorylation by isoproterenol was 3-fold larger than that due to forskolin, suggesting that beta 1 AR is a substrate for the beta AR kinase that phosphorylates carboxyl-terminal residues in the beta(2) AR. Our findings suggest that PVN may be a powerful new tool with which to study the phosphorylation of other G protein-coupled receptors.

Inhibition by NMDA of carbachol-stimulated inositol tetrakisphosphate accumulation in rat brain cortical slices.

The present studies examined the effect of NMDA on carbachol-stimulated accumulation of inositol polyphosphates, with emphasis on the accumulation of inositol 1,3,4,5-tetrakisphosphate (Ins 1,3,4,5-P4), at short time periods in rat brain cortical slices. There was a stimulatory effect of NMDA on accumulation of labeled inositol mono-, bis- and trisphosphates but not on labeled inositol tetrakisphosphates. In the presence of carbachol Ins 1,3,4,5-P4 accumulation was preferentially inhibited by NMDA at early time periods (within 30 seconds after NMDA addition). Subsequently, total phosphoinositide breakdown was inhibited by NMDA. NMDA did not stimulate accumulation of total Ins 1,3,4,5-P4 but immediately inhibited carbachol stimulated accumulation of Ins 1,3,4,5-P4. The inhibitory effect of NMDA (1 mM) was not mimicked by increasing K+ in the medium from 10 to 30 mM. However 30 mM K+ reversed the inhibitory effect of 1 mM NMDA on carbachol-stimulated Ins 1,3,4,5-P4. Parallel experiments with veratridine (a sodium channel activator) suggest that the early inhibitory effects of NMDA on Ins 1,3,4,5-P4 accumulation are not due to decreases in ATP availability or elevations in intracellular Na+. These data indicate that NMDA increases inositol mono-, bis- and trisphosphate accumulation while blocking muscarinic cholinergic stimulated accumulation of Ins 1,3,4,5-P4.

Insulin sensitizes beta-agonist and forskolin-stimulated lipolysis to inhibition by 2',5'-dideoxyadenosine.

In isolated rat adipocytes incubated in the absence of insulin, 2',5'-dideoxyadenosine blocked the increase in total adenosine 3',5'-cyclic monophosphate (cAMP) accumulation due to beta 1- or beta 3-catecholamine agonists and forskolin without affecting their stimulation of lipolysis. The inhibition of cAMP accumulation by 2',5'-dideoxyadenosine was not reflected in the total cytosolic cAMP-dependent protein kinase A activity, suggesting that the inhibition of cAMP occurred in cellular compartments distinct from those involved in the regulation of bulk protein kinase A activity. However, there was a good correlation between effects of lipolytic agents on cytosolic protein kinase A activity in fat cell extracts and lipolysis. Furthermore, it was possible to see an inhibition of the increase due to beta-agonists in cAMP accumulation, protein kinase A activity, and lipolysis by 2',5'-dideoxyadenosine in the presence of insulin. These data suggest that the readily measurable accumulation of cAMP seen with catecholamines in the absence of insulin is in a compartment separate from that involved in protein kinase A activation.

Inhibition by veratridine of carbachol-stimulated inositol tetrakisphosphate accumulation in rat brain cortical slices.

The present studies examined the inhibitory effect of veratridine (a Na+ channel activator) on carbachol (a cholinergic agonist) stimulated inositol 1,3,4,5-tetrakisphosphate accumulation in rat brain cortical slices. Veratridine inhibited carbachol stimulation of inositol 1,3,4,5-tetrakisphosphate formation (after a delay of about 30 seconds) at 60 or 120 seconds when there was little inhibition of inositol 1,4,5 trisphosphate accumulation. The inhibitory effect of veratridine on carbachol stimulated inositol 1,3,4,5-tetrakisphosphate accumulation was abolished in the presence of ouabain or tetrodotoxin but was unaffected in low calcium conditions. Veratridine reduced the total ATP content and this effect was abolished by tetrodotoxin. The inhibitory effect of 10 but not 30 microM veratridine on inositol 1,3,4,5-tetrakisphosphate accumulation in the presence of carbachol was reversed by the presence of exogenous 8-bromo cyclic AMP or forskolin which activates adenylyl cyclase. However, the decrease in brain slice ATP seen in the presence of veratridine was unaffected by forskolin. Our results are compatible with the hypothesis that veratridine inhibition of carbachol-stimulated inositol 1,3,4,5-tetrakisphosphate formation is due to depletion of ATP at the site of Ins 1,3,4,5-P4 formation from Ins 1,4,5-P3.