Glycogen synthase kinase-3 (GSK-3) has attracted much scrutiny due to its plethora of cellular functions, novel mechanisms of regulation and its potential as a therapeutic target for several common diseases. In mammals, GSK-3 is encoded by two genes, termed GSK-3alpha and GSK-3beta, that yield related but distinct protein-serine kinases. GSK-3 is unusual in that its protein kinase activity tends to be high in resting cells and cellular stimuli, such as hormones and growth factors, result in its catalytic inactivation. Further, many of the substrate proteins of GSK-3 are functionally inhibited by phosphorylation. Thus, signals that inhibit GSK-3 often cause activation of its diverse array of target proteins. Regulation of GSK-3 is important for normal development, regulation of metabolism, neuronal growth and differentiation and modulation of cell death. Dysregulation of GSK-3 activity has been implicated in human pathologies such as neurodegenerative diseases and type-2 diabetes. In this introductory chapter we provide a primer on the modes of GSK-3 regulation and a description of the various signaling pathways and cellular processes in which GSK-3 is an active participant.

Gap junctions (GJs), composed of connexins, are intercellular channels ensuring electric and metabolic coupling between cardiomyocytes. We have shown previously that an endogenous mitogenic and cardioprotective protein, fibroblast growth factor-2 (FGF-2), decreases cardiomyocyte GJ permeability by stimulating phosphorylation of connexin-43 (Cx43). Identifying the kinase(s) phosphorylating cardiac Cx43 may thus provide a way of modulating cardiac intercellular communication. Because FGF-2 activates receptors linked to protein kinase C (PKC) and mitogen-activated protein kinase, we first investigated participation of these enzymatic systems in Cx43 phosphorylation. The inhibitor PD98059 blocked activation of mitogen-activated protein kinase, but it did not prevent the FGF-2 effects on GJs. In contrast, the PKC inhibitor chelerythrine blocked the effects of FGF-2 on Cx43 phosphorylation and permeability. Because the epsilon-isoform of PKC localizes to plasma membrane sites, we examined whether it is directly involved in the FGF-2-induced Cx43 phosphorylation. In nonstimulated myocytes, PKCepsilon displayed a discontinuous pattern of localization at intercellular contact sites and partial colocalization with Cx43. Treatment with FGF-2 or phorbol 12-myristate 13-acetate induced a more continuous pattern of PKCepsilon distribution, whereas the anti-Cx43 staining appeared to overlap extensively with that of PKCepsilon. In immunoprecipitation experiments using specific anti-Cx43 antibodies, PKCepsilon but not PKCalpha coprecipitated with Cx43. FGF-2 increased levels of coprecipitated PKCepsilon, suggesting increased association between PKCepsilon and Cx43 on stimulation. Transient gene transfer and overexpression of cDNAs coding for truncated or mutated dominant-negative forms of PKCepsilon decreased cardiomyocyte Cx43 phosphorylation significantly. We conclude that PKC mediates the FGF-2-induced effects on cardiac GJs and that PKCepsilon likely interacts with and phosphorylates cardiac Cx43 at sites of intercellular contact.

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