James E. Watson

Molecular and genetic studies imply Akt-mediated signaling promotes protein kinase CbetaII alternative splicing via phosphorylation of serine/arginine-rich splicing factor SRp40.

Insulin regulates alternative splicing of PKCβII mRNA by phosphorylation of SRp40 via a phosphatidylinositol 3-kinase pathway (Patel, N. A., Chalfant, C. E., Watson, J. E., Wyatt, J. R., Dean, N. M., Eichler, D. C., and Cooper, D. C. (2001) J. Biol. Chem. 276, 22648–22654). Transient transfection of constitutively active Akt2 kinase promotes PKCβII exon inclusion. Serine/arginine-rich (SR) RNA-binding proteins regulating the selection of alternatively spliced exons are potential substrates of Akt kinase because many of them contain RXRXX(S/T) motifs. Here we show that Akt2 kinase phosphorylated SRp40 in vivo and in vitro. Mutation of Ser86 on SRp40 blocked in vitro phosphorylation. In control Akt2(+/+) fibroblasts, insulin treatment increased the phosphorylation of endogenous SR proteins, but their phosphorylation state remained unaltered by insulin in fibroblasts from Akt2(-/-) mice. Levels of PKCβII protein were up-regulated by insulin in Akt2(+/+) cells; however, only very low levels of PKCβII were detected in Akt2(-/-) cells and did not change following insulin treatment. Endogenous PKCβI and -βII mRNA levels in Akt2(+/+) and Akt2(-/-) gastrocnemius muscle tissues were compared using quantitative real time PCR. The results indicated a 54% decrease in the expression of PKCβII levels in Akt(-/-), whereas PKCβI levels remained unchanged in both samples. Further, transfection of Akt2(-/-) cells with a PKCβII splicing minigene revealed defective βII exon inclusion. Co-transfection of the mutated SRp40 attenuated βII exon inclusion. This study provides in vitro and in vivo evidence showing Akt2 kinase directly phosphorylated SRp40, thereby connecting the insulin, PI 3-kinase/Akt pathway with phosphorylation of a site on a nuclear splicing protein promoting exon inclusion. This model is upheld in Akt2-deficient mice with insulin resistance leading to diabetes mellitus.

Regulation of the human fas promoter by YB-1, Purα and AP-1 transcription factors

Fas (CD95/Apo-1) gene expression is dysregulated in a number of diseased states. Towards understanding the regulation of fas gene expression, we previously identified activator and repressor elements within the human fas promoter. Using a combination of expression screening and reporter gene assays, we have identified transcription factors which bind to these elements and thereby regulate transcription of the fas promoter. These are three single-stranded DNA binding proteins, YB-1, Puralpha and Purbeta and two components of the AP-1 complex, c-Fos and c-Jun. c-Jun is a potent transcriptional activator of fas and stimulated expression levels up to 184-fold in reporter gene assays. Co-expression with c-Fos abrogated c-Jun-mediated activation. YB-1 and Puralpha are transcriptional repressors of fas and decreased basal transcription by 60-fold in reporter gene assays. Purbeta was predominantly an antagonist of YB-1/Puralpha-mediated repression. Overexpression of YB-1 and Puralpha in Jurkat cells was shown to reduce the level of cell surface Fas staining, providing further evidence that these proteins regulate the fas promoter. It has been suggested that YB-1 plays a role in cell proliferation as an activator of growth-associated gene expression. We have shown that YB-1 is a repressor of a cell death-associated gene fas. These results suggest that YB-1 may play an important role in controlling cell survival by co-ordinately regulating the expression of cell growth-associated and death-associated genes.

Glucose-induced synthesis of diacylglycerol de novo is associated with translocation (activation) of protein kinase C in rat adipocytes

Addition of glucose (5–20 mM) to rat adipocytes provoked dose‐related increases in diacylglycerol, without increasing production of [3H]inositol phosphates. Cytosolic protein kinase C enzyme activity and immunoreactivity decreased within 1–5 min of 5 mM glucose addition, and further over 20 min. Membrane protein kinase C increased stoichiometrically during the first 5 min and then decreased. Higher concentrations (10 and 20 mM) of glucose provoked greater and more rapid decreases of cytosolic and membrane protein kinase C. Our findings suggest that glucose stimulates diacylglycerol production by providing substrate for phosphatidic acid synthesis de novo, and this is associated with translocative activation of protein kinase C.

Insulin Regulates Protein Kinase CβII Expression through Enhanced Exon Inclusion in L6 Skeletal Muscle Cells A NOVEL MECHANISM OF INSULIN- AND INSULIN-LIKE GROWTH FACTOR-I-INDUCED 5′ SPLICE SITE SELECTION

The protein kinase Cβ (PKCβ) gene encodes two isoforms, PKCβI and PKCβII, as a result of alternative splicing. The unique mechanism that underlies insulin-induced alternative splicing of PKCβ pre-mRNA was examined in L6 myotubes. Mature PKCβII mRNA and protein rapidly increased >3-fold following acute insulin treatment, while PKCβI mRNA and protein levels remained unchanged. Mature PKCβII mRNA resulted from inclusion of the PKCβII-specific exon rather than from selection of an alternative polyadenylation site. Increased PKCβII expression was also not likely accounted for by transcriptional activation of the gene or increased stabilization of the PKCβII mRNA, and suggest that PKCβII expression is regulated primarily at the level of alternative splicing. Insulin effects on exon inclusion were observed as early as 15 min after insulin treatment; by 20 min, a new 5′-splice site variant of PKCβII was also observed. After 30 min, the longer 5′-splice site variant became the predominate species through activation of a downstream 5′ splice site. Similar results were obtained using IGF-I. Although the role of this new PKCβII mRNA species is presently unknown, inclusion of either PKCβII-specific exon results in the same PKCβII protein.

Insulin Promotes Neuronal Survival via the Alternatively Spliced Protein Kinase CδII Isoform

Background: Alternatively spliced PKCδII is a pro-survival protein. Results: Insulin regulates alternative splicing of PKCδII pre-mRNA, which promotes Bcl2 and bcl-xL expression. Conclusion: PKCδII is a key regulator of insulin-mediated neuronal survival. Significance: Elucidation of the molecular mechanisms by which insulin promotes survival in neuronal cells is necessary to understand how intranasal insulin improves cognition. Insulin signaling pathways in the brain regulate food uptake and memory and learning. Insulin and protein kinase C (PKC) pathways are integrated and function closely together. PKC activation in the brain is essential for learning and neuronal repair. Intranasal delivery of insulin to the central nervous system (CNS) has been shown to improve memory, reduce cerebral atrophy, and reverse neurodegeneration. However, the neuronal molecular mechanisms of these effects have not been studied in depth. PKCδ plays a central role in cell survival. Its splice variants, PKCδI and PKCδII, are switches that determine cell survival and fate. PKCδI promotes apoptosis, whereas PKCδII promotes survival. Here, we demonstrate that insulin promotes alternative splicing of PKCδII isoform in HT22 cells. The expression of PKCδI splice variant remains unchanged. Insulin increases PKCδII alternative splicing via the PI3K pathway. We further demonstrate that Akt kinase mediates phosphorylation of the splicing factor SC35 to promote PKCδII alternative splicing. Using overexpression and knockdown assays, we demonstrate that insulin increases expression of Bcl2 and bcl-xL via PKCδII. We demonstrate increased cell proliferation and increased BrdU incorporation in insulin-treated cells as well as in HT22 cells overexpressing PKCδII. Finally, we demonstrate in vivo that intranasal insulin promotes cognitive function in mice with concomitant increases in PKCδII expression in the hippocampus. This is the first report of insulin, generally considered a growth or metabolic hormone, regulating the alternative isoform expression of a key signaling kinase in neuronal cells such that it results in increased neuronal survival.

Acute hyperglycemia regulates transcription and posttranscriptional stability of PKCβII mRNA in vascular smooth muscle cells

Acute hyperglycemia may contribute to the progression of atherosclerosis by regulating protein kinase C (PKC) isozymes and by accelerating vascular smooth muscle cell (VSMC) proliferation. We investigated acute glucose regulation of PKCβ gene expression in A10 cells, a rat aortic smooth muscle cell line. Western blot analysis showed that PKCβII protein levels decreased with high glucose (25 mM) compared to normal glucose (5.5 mM), whereas PKCβI levels were unaltered. PKCβ mRNA levels were depleted by 60–75% in hyperglycemic conditions. To elucidate whether high glucose regulated PKCβ expression via the common promoter for PKCβI and PKCβII, deletion constructs of the PKCβ promoter ligated to CAT as reporter gene were transfected into A10 cells. Construct D (−411 to + 179CAT) showed quenching in high glucose (25 mM) and suggested the involvement of a carbohydrate response element in the 5′ promoter region of the PKCβ gene. In actinomycin D‐treated A10 cells, a 60% decrease in PKCβ mRNA with high glucose treatment indicated that posttranscriptional destabilization by glucose was also occurring. We have demonstrated that glucose‐induced posttranscriptional destabilization of PKCβII message is mediated via a nuclease activity present in the cytosol. The specificity of glucose to post‐transcriptionally destabilize PKCβII mRNA, but not the PKCβI mRNA, was confirmed in both A10 cells and primary cultures from human aorta.—Patel, N. A., Chalfant, C. E., Yamamoto, M., Watson, J. E., Eichler, D. C., Cooper, D. R. Acute hyperglycemia regulates transcription and posttranscriptional stability of PKCβII mRNA in vascular smooth muscle cells. FASEB J. 13, 103–113 (1999)

Sulfonylurea-Stimulated Glucose Transport Association With Diacylglycerollike Activation of Protein Kinase C in BC3H1 Myocytes

The extrapancreatic effects of sulfonylurea drugs include increased glucose uptake by certain peripheral tissues. To study this effect, we used BC3H1 myocytes, which are reported to respond to these drugs. Within 30 min, tolbutamide and glyburide increased [3H]-2-deoxyglucose uptake in a dose-dependent manner. The inactive analogue carboxytolbutamide had no effect on glucose transport. Because increases in glucose transport may be mediated by activation of the diacylglycerol-protein kinase C signaling system, we examined the effects of these drugs on lipid metabolism and protein kinase C activity. Unlike insulin, tolbutamide and glyburide failed to increase [3H]glycerol labeling of diacylglycerol or labeling of phospholipids by 32P. After 30 min of treatment with tolbutamide or glyburide, however, membraneassociated and cytosolic protein kinase C activity were each increased. When cells were treated with 12-O- tetradecanoylphorbol-13-acetate (TPA) for 48 h to deplete certain isoforms of protein kinase C, glyburide, tolbutamide, and acute TPA treatment failed to increase glucose uptake, suggesting that TPA and sulfonylureas operate through activation of a common pathway. The effect of glyburide was additive to TPA in stimulating glucose uptake at low but not high TPA concentrations. As with insulin and TPA, extracellular Ca2+ was not essential for sulfonylurea-stimulated glucose uptake. Staurosporine, a protein kinase C inhibitor, blocked glyburide-, tolbutamide-, and insulinstimulated glucose uptake. In intact cells, glyburide stimulated the phosphorylation of both 80,000-Mr and 40,000-Mr proteins, which are markers for protein kinase C activation. Addition of sulfonylureas directly to the protein kinase C assay system in vitro provoked dioleinlike effects, in that sensitivity of the enzyme to Ca2+ was increased. Our findings suggest that tolbutamide and glyburide increase glucose uptake in BC3H1 myocytes by a postreceptor mechanism, which may involve direct activation of protein kinase C.

Direct evidence for protein kinase c involvement in insulin-stimulated hexose uptake

Insulin has been reported to translocate protein kinase C (PKC) in rat adipocytes, and activation of PKC by phorbol esters is known to increase hexose uptake in these cells (1.2). To test the hypothesis that PKC may participate in insulin-stimulated hexose uptake, adipocytes were partially depleted of protein kinase C by overnight phorbol ester treatment, thereby impairing insulin effects on hexose uptake. Purified PKC was then introduced into these PKC-depleted adipocytes by electropermeabilization, and this fully restored insulin-stimulated hexose uptake. These findings provide direct evidence that PKC is required for insulin-stimulated hexose uptake.

Insulin translocates PKC-ϵ and phorbol esters induce and persistently translocate PKC-β2 in BC3H-1 myocytes

Abstract Initial studies suggested that insulin increases diacylglycerol and activates protein kinase C (PKC) in BC3H-1 myocytes. In these earlier studies, insulin was found to translocate PKC-β, but the presence of PKC-ϵ was not appreciated. More recently, the presence of PKC-ϵ was documented, but PKC-β was not detected, and it was questioned whether insulin activates PKC in BC3H-1 myocytes [Stumpo, D.J., Haupt, D.M. and Blackshear, P.J. (1994)J. Biol. Chem. 269:21184–21190]. We questioned whether insulin translocates PKC-ϵ in BC3H-1 myocytes, and re-evaluated the question of whether myocytes truly contain a PKC-β isoform whose existence can be verified by its response to phorbol ester treatment. We found that PKC-ϵ was acutely translocated by insulin and phorbol esters from the cytosol to the membrane fraction in BC3H-1 myocytes; in addition, PKC-ϵ, like PKC-α, was depleted by chronic phorbol ester treatment. We also found that BC3H-I myocytes containing a 76,000 Mr PKC-β isoform that is acutely translocated and subsequently depleted by phorbol esters. Moreover, chronic phorbol ester treatment induced an 84,000 Mr PKC-β2 isoform that appeared to be persistently translocated and activated, as suggested by studies of myristoylated arginine-rich C kinase substrate (MARCKS) phosphorylation. We conclude that: (1) insulin acutely translocates PKC-ϵ, as well as PKC-β, in BC3H-1 myocytes; and (2) PKC-β is not truly downregulated by phorbol esters in BC3H-1 myocytes.