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Activation of Protein Kinase Cζ Induces Serine Phosphorylation of VAMP2 in the GLUT4 Compartment and Increases Glucose Transport in Skeletal Muscle

ABSTRACT Insulin stimulates glucose uptake into skeletal muscle tissue mainly through the translocation of glucose transporter 4 (GLUT4) to the plasma membrane. The precise mechanism involved in this process is presently unknown. In the cascade of events leading to insulin-induced glucose transport, insulin activates specific protein kinase C (PKC) isoforms. In this study we investigated the roles of PKCζ in insulin-stimulated glucose uptake and GLUT4 translocation in primary cultures of rat skeletal muscle. We found that insulin initially caused PKCζ to associate specifically with the GLUT4 compartments and that PKCζ together with the GLUT4 compartments were then translocated to the plasma membrane as a complex. PKCζ and GLUT4 recycled independently of one another. To further establish the importance of PKCζ in glucose transport, we used adenovirus constructs containing wild-type or kinase-inactive, dominant-negative PKCζ (DNPKCζ) cDNA to overexpress this isoform in skeletal muscle myotube cultures. We found that overexpression of PKCζ was associated with a marked increase in the activity of this isoform. The overexpressed, active PKCζ coprecipitated with the GLUT4 compartments. Moreover, overexpression of PKCζ caused GLUT4 translocation to the plasma membrane and increased glucose uptake in the absence of insulin. Finally, either insulin or overexpression of PKCζ induced serine phosphorylation of the GLUT4-compartment-associated vesicle-associated membrane protein 2. Furthermore, DNPKCζ disrupted the GLUT4 compartment integrity and abrogated insulin-induced GLUT4 translocation and glucose uptake. These results demonstrate that PKCζ regulates insulin-stimulated GLUT4 translocation and glucose transport through the unique colocalization of this isoform with the GLUT4 compartments.

The role of protein kinase C δ activation and STAT3 Ser727 phosphorylation in insulin-induced keratinocyte proliferation

Activation of the STAT family of transcription factors is regulated by cytokines and growth factors. STAT tyrosine and serine phosphorylation are linked to the transcriptional activation and function of STAT. We have previously described a unique pathway inducing keratinocyte proliferation, which is mediated by insulin stimulation and depends on protein kinase C δ (PKCδ). In this study, we assessed STAT3 activation downstream of this pathway and characterized the role of PKCδ activation in STAT3 tyrosine and serine phosphorylation and keratinocyte proliferation. Following insulin stimulation, STAT3 interacted with PKCδ but not with any other PKC isoform expressed in skin. Activated forms of PKCδ and STAT3 were essential for insulin-induced PKCδ-STAT3 activation in keratinocyte proliferation. Abrogation of PKCδ activity inhibited insulin-induced STAT3 phosphorylation, PKCδ-STAT3 association and nuclear translocation. In addition, overexpression of STAT3 tyrosine mutant eliminated insulin-induced PKCδ activation and keratinocyte proliferation. Finally, overexpression of a STAT3 serine mutant abrogated insulin-induced STAT3 serine phosphorylation and STAT3-induced keratinocyte proliferation, whereas STAT3 tyrosine phosphorylation was induced and nuclear localization remained intact. This study indicates that PKCδ activation is a primary regulator of STAT3 serine phosphorylation and that PKCδ is essential in directing insulin-induced signaling in keratinocyte proliferation.