Recent work on this project has provided new insights into the role of the proline-directed kinase, Cdk5, in regulating cell-cell and cell-matrix adhesion. Previous work from this laboratory had shown that the Cdk5 activiting protein, p39, is able to bind both myosin essential light chain and the Kelch-domain protein, muskelin. In an extension of this finding, we have now shown that p39 in fact links muskelin to myosin in stress fibers and that muskelin, p39, and Cdk5 are all essential for Rho-dependent contraction of stress fibers. Our previous studies had indicated that two distinct mechanisms of action are involved in Cdk5-dependent regulation of adhesion and migration: one involving Cdk5 kinase activity and a second, which is independent of kinase activity, but requires an intact phosphorylation site at Cdk5(Y15). Recent evidence has linked both of these mechanisms to Cdk5-dependent regulation of Src. We have found that cSrc is phosphorylated by Cdk5 in normal lens epithelial cells at a site in the unique region of the cSrc N-terminus. Phosphorylation at this site then targets cSrc for ubiquitylation by the E3 ubiquitin ligase, Cullin5. Loss of Cullin5, Cdk5 kinase activity, or site-specific mutagenesis of the phosphorylation site in cSrc, inhibits ubiquitin-dependent degradation of the active form of cSrc, allowing it to accumulate 1.5 to 2.0 fold. While Cdk5 kinase activity regulates Src by targeting the active Src for degradation, other work in this laboratory has shown that the adaptor function of Cdk5 is involved in yet another aspect of Src regulation: activation of cSrc at nascent focal adhesions. Suppressing Cdk5 expression with siRNA or overexpressing mutated Cdk5 proteins that can not be phosphorylated on Y15 prevents the activation of cSrc normally seen immediately after plating. Cdk5 siRNA does not interfere with cell attachment to extracellular matrix via integrins or with binding of focal adhesion kinase (FAK) to the integrin cytoplasmic tail;but binding of cSrc to the integrin-FAK complex is blocked, preventing focal adhesion maturation. These findings suggest that the adaptor function of Cdk5 is required either to transport Src to sites of focal adhesion formation or to facilitate its binding to autophosphorylated FAK at these sites. Our work has also implicated Cdk5 in regulating cell-cell adhesion. We had previously found that pharmacological inhibitors of Cdk5 reduced cell-cell adhesion and promoted degradation of E-cadherin. To extend our study of Cdk5s role in cell-cell adhesion, we generated a stable human corneal epithelial cell line with very low levels of endogenous Cdk5 using lentiviral mediated gene transfer to suppress Cdk5 expression with small hairpin RNA (shRNA). Analysis of this line (shHCLE) has confirmed that lack of Cdk5 expressionlike inhibition of Cdk5 activity-- impairs junctional stability, leading to internalization and degradation of E-cadherin. Remarkably, this finding is opposite to that obtained in neurons and lens epithelial cells, both of which express primarily N-cadherin. In these cells, inhibition of Cdk5 activity increases junctional stability. By transfecting either E-cadherin or N-cadherin into a cell line which otherwise expresses no cadherins, we found that the differing effects of Cdk5 are inherent in the cadherin. Measurements of Rac and Rho activity demonstrated that suppressing Cdk5 or inhibiting its activity inhibited Rac activity and increased Rho activity in corneal epithelial cells. This increase in Rho activity was required for the destabilization of E-cadherin seen when Cdk5 was blocked. In contrast, inhibiting Cdk5 activity in cells that express N-cadherin promoted Rho activation and stabilized the junctions. In view of the opposite effects of Cdk5 on E-cadherin and N-cadherin, we examined the expression of N-cadherin following inhibition of Cdk5. The results showed that the total N-cadherin expression and the amount of N-cadherin based junction formation increased when Cdk5 was blocked. Thus, when Cdk5 is inhibited or suppressed E-cadherin dependent cell-cell junctions are gradually degraded and replaced by new junctions containing N-cadherin. Since our previous in vitro studies of Notch signaling in the lens had implicated Notch 2 in lens differentiation, we extended our study of this pathway by generating conditional knockouts of the Notch 2 gene in the lens, corneal epithelium, and other ectodermally-derived eye tissues using the Le-Cre line for Cre recombinase expression. Conditional knockouts (cKOs) show a severe lens phenotype, with developmental abnormalities of the iris, ciliary body, and anterior chamber. Analysis of a variety of epithelial cell and fiber cell markers, carried out in collaboration with Dr. Nadean Brown, indicates a relative lack of cells expressing epithelial cell markers in Notch2 cKOs at early developmental ages, as also seen in cKOs of Rbpj and Jag1. In contrast to results obtained with these other Notch pathway components, however, Notch2 cKOs show no change in the proportion of proliferating cells, indicating that Notch1 is sufficient to maintain proliferation. Microarray analysis of E19.5 Notch2 cKOs identified a number of differentially expressed genes, including several Wnt pathway genes. Additional studies are in progress to identify the target genes that may contribute to the phenotype of the Notch2 cKO lenses. A collaborative project with the Brown laboratory has also succeeded in generating mice with conditional deletions of both Notch1 and Notch2, (double cKOs) to determine the extent to which these receptors have overlapping and distinct functions in the eye. Notch1,2 double cKOs have a severe lens phenotype which closely resembles that of the Rbpj and Jag1 cKOs, suggesting that Notch1 and Notch2 are the major Notch receptors involved in lens development. Further analysis of the double cKOs, including expression of epithelial cell and fiber cell markers, microarray analysis, and BrdU labeling, is in progress. In addition to the developmentally important Notch pathway, we have undertaken a novel study of the role of the CasZ1 gene in eye development by developing mice with a floxed allele of CasZ1. CasZ1 is the mammalian homolog of the drosophila gene, Castor, a transcription factor with a role in fate determination. In situ hybridizations performed in collaboration with the laboratory of Dr. Anand Swaroop (NEI) have shown that CasZ1 is upregulated in cells at the lens equator, which are in the early stages of fiber cell differentiation. Mice homozygous for the floxed allele are currently being bred with mice expressing the Le-Cre promoter to assess the role of CasZ1 in eye development.
