Annual Report 2002 This project investigates signal transduction pathways that regulate proliferation, cell migration, and differentiation in the lens and corneal epithelium. Recent studies have focused on the signaling pathway linking EGF receptor activation with c-fos transcription in the lens and on the functions of Cdk5, a member of the cyclin dependent kinase family. Our previous studies of lens epithelial cells have shown that 12(S)HETE, a lipoxygenase metabolite of arachidonic acid, is required for EGF-dependent activation of the protein kinase C isoforms, PKCalpha and PKCbeta. Recent work has shown that 12(S)HETE and EGF have complementary effects on PKC. 12(S)HETE is required for membrane translocation of PKC, while EGF is required to activate the PtdIns3 kinase pathway to phosphorylate the PKC activation loop. In the absence of EGF, 12(S)HETE produces translocation of PKC, but not activation. In the absence of 12(S)HETE (either endogenously synthesized or exogenously supplied) EGF is unable to cause translocation, so PKC is not properly localized at the membrane and phosphorylation is ineffective. Thus, 12(S)HETE and EGF act in concert to activate PKC in lens epithelial cells. Our studies of Cdk5 suggest that this enzyme may regulate cell adhesion, migration, and survival in a variety of non-neuronal cells, including lens and corneal epithelial cells. To investigate the possible mechanism of these effects in the lens, a cDNA library has been constructed from E18 embryonic rat lenses for yeast two-hybrid screening using Cdk5, p35, and p39 as 'baits'. This screen has identified a specific interaction between p39 and the kelch-domain protein, muskelin. Overexpression of muskelin enables cells to attach and spread on thrombospondin, an extracellular matrix component associated with cell migration during embryogenesis and wound repair. Co-expression of p39 interferes with muskelin-dependent attachment to thrombospondin. The effects of p39 may be due to phosphorylation of muskelin by Cdk5/p39, since muskelin contains a potential site for phosphorylation by Cdk5 and immunoprecipitates containing Cdk5/p39 are able to phosphorylate muskelin in vitro. This finding provides the first example of a protein that binds preferentially to p39, as compared to p35, and suggests that these two activating proteins may serve to target Cdk5 to different sets of substrates in vivo. Further studies of Cdk5 functions in corneal epithelial cells have demonstrated that Cdk5 increases adhesion to fibronectin and decreases the rate of cell migration in vitro. Conversely, inhibiting endogenous Cdk5 function with a dominant negative mutation of Cdk5 increases the rate of cell migration, implying that endogenous Cdk5 activity is involved. Thus, our results indicate that Cdk5 may have both positive and negative effects on cell adhesion and migration, depending on the extracellular matrix material and the particular Cdk5 activating protein involved. Current studies are investigating the molecular mechanism of these effects.