Involucrin is an important precursor of the keratinocyte cornified envelope. In epidermis, expression of involucrin protein and mRNA is keratinocyte-specific and differentiation-dependent (i.e., expression is confined to the suprabasal layers). Our transgenic mouse and cell culture studies show that the human involucrin (hINV) gene upstream regulatory region is sufficient for this expression and that specific DNA elements in this region bindAPI, C/EBP and Spl transcription factors to drive expression. During the past funding period we identified a novelPKC, Ras, MEKK1, MEK3 and p388-ERKl/2 signaling cascade that regulates the activity and level of API, Spl and C/EBP transcription factors to increase involucrin gene expression. Although identifying this cascade represents a major step forward in our understanding of differentiation associated gene regulation, our understanding of this signal transduction cascade is far from complete. Having established the importance of this pathway, we now propose experiments designed to extend these findings. Our studies show that novel PKC isoforms, including PKC5, are the major activators of hINV gene expression in normal keratinocytes and that differentiation agents trigger tyrosine phosphorylation of PKC8 to regulate gene expression. However, the importance of PKC8 tyrosine phosphorylation in keratinocytes is complicated, controversial, and has not been thoroughly studied. Our first major goal is to identify the role of tyrosine phosphorylation of PKC8 on PKC8 activity, subcellular localization, and ability to regulate downstream signaling events. An important observation is that signal transduction in normal human keratinocytes converges at the MAPK level on p388. Our preliminary studies suggest that differential interaction of MEK3 and MEK6 with p38S may function to regulate the balance between keratinocyte differentiation and apoptosis. The second major goal of this study is to characterize the role of these kinases in regulating the balance between differentiation and apoptosis. In particular, we will focus on the role of PKC8, MEK3 and p388 in regulating keratinocyte differentiation. The overall goal of this proposal is to expand our knowledge regarding the molecular mechanisms that drive differentiation in keratinocytes. This work is particularly important, as this mitogen-activated protein kinase (MAPK) signaling cascade has been implicated in regulating keratinocyte survival, differentiation, death, and transformation. It is hoped that a better understand of this cascade will lead to new and effective therapies for epidermal disease.