This fiscal year has seen significant progress in understanding the role of the proline-directed kinase, Cdk5, in regulating cell-cell and cell-matrix adhesion. Building upon previous observations we have demonstrated that Cdk5 regulates cell-matrix adhesion in two distinct ways: via its kinase activity and via an adaptor function, which requires Src-dependent phosphorylation of Cdk5(Y15), but is independent of Cdk5 kinase activity. The adaptor function of Cdk5 is strictly required for stress fiber assembly, while the kinase activity of Cdk5 regulates stress fiber organization. More recent studies have shown that adaptor function of Cdk5 is specifically needed for recruitment of cSrc to nascent focal adhesions. Experimental conditions that prevent Cdk5(Y15) phosphorylation allow cell attachment to extracellular matrix via integrins. Focal adhesion kinase also binds to integrins and undergoes autophosphorylation; however, cSrc fails to join the integrin-FAK complex. Since cSrc activity is essential for further maturation of the focal adhesion and its connection to the cytoskeleton, cells are only weakly attached to the substrate. Experimental conditions that block Cdk5 activity without affecting Cdk5(Y15) phosphorylation, do not affect the recruitment of cSrc to focal adhesion, indicating that only the adaptor function of Cdk5 is involved in this effect. In a separate line of investigation, we have found that Cdk5 activity is an important regulator of Rho-dependent phosphorylation of myosin regulatory light chain (MRLC) during stress fiber contraction. Since stress fiber contraction strengthens cellular attachment to the substratum, this function of Cdk5 provides a potential mechanism for the previously observed increase in cell migration produced by Cdk5 inhibitors such as olomoucine.? We have furthered our study of signaling pathways that regulate cell adhesion by analyzing the phenotype and protein expression pattern of mice deficient in PdLim2, a PDZ-Lim domain protein that is highly expressed in the corneal epithelium. Published studies have implicated this protein in cytoskeletal regulation and have shown that it has E3 ubiquitin ligase activity. The results of our proteomic analysis of PdLim2 knock-out mice have identified a number of proteins whose expression is elevated. These include cytoskeletal proteins, (such as destrin, tubulin, and stathmin), ox-redox enzymes, (such as superoxide dismutase and peroxiredoxin), and chaperone proteins (such as alphaA-crystallin and tubulin-specific chaperone protein). The elevated expression of these proteins is not reflected in their respective mRNAs, indicating that it is due to translational or post-translational controls. Ongoing studies will determine whether these proteins are targeted for ubiquitin-dependent degradation by PdLim2. In addition, we have demonstrated that PdLim2 localizes to the actin cytoskeleton in corneal epithelial cells via a binding site in the mid-domain of the protein, which contains multiple potential phosphorylation sites. Future studies will determine the cytoskeletal protein(s) recognized by this region and investigate the possible role of phosphorylation.? Studies of Notch signaling during lens differentiation have been very fruitful. Using explants of rat lens epithelia, which differentiate in vitro when exposed to FGF, we have been able to study the signaling events occurring in a cohort of epithelial cells differentiating in concert. We have found that FGF induces expression of the Notch ligand, Jag1, via Erk1,2 activation. FGF is unique in this regard, as activation of Erk1,2 by other growth factors fails to induce Jag1. Jag1 induction is accompanied by Notch2 signaling, with production of the Notch2 intracellular domain (ICD), and induction of the Notch effector, Hes5. Immunostaining of lens epithelia immediately after dissection confirmed strong expression of Jag1 and the Notch2 ICD in transitional cells at the lens equator, which are in the early stages of differentiation. Jag1/Notch2 ICD immunostaining was seen in all cells in this region and was never mosaic. This pattern of expression is consistent with inductive signaling, in which Jag1 expression in one cell promotes Jag1 expression in neighboring cells. To test this possibility, Jag1 signaling was blocked in cultured explants by adding Jag1 function-blocking antibody to the culture medium. Under these conditions, expression of Jag1, Hes5, and the Notch2 ICD was greatly diminished, indicating that Jag1 expression is subject to Notch-dependent inductive signaling and establishing a role for Notch signaling in fiber cell differentiation.
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