Molecular bases of growth control and cell cycle regulation

Our goal is to understand the molecular basis of cell growth control and cell cycle regulation. Many growth factor receptors are protein-tyrosine kinases (PTK), which are activated by ligand-induced dimerization. Mitogenic signalling by such PTKs requires tyrosine phosphorylation of critical target proteins. In our search for PTK targets we have identified phospholipase C-gamma and more recently the SH2/SH3 adaptor protein, Nck, and shown that the c-Src nonreceptor PTK is essential for mitogenic signalling by the PDGF receptor PTK. We are currently investigating what signals lie downstream of Nck and c-Src. We are also studying how the integrin receptors for extracellular matrix proteins, such as fibronectin, signal when cells adhere. We have shown that integrin occupancy activates the focal adhesion kinase (FAK) PTK, which elicits binding of c-Src. Bound c-Src phosphorylates FAK and thus creates a binding site for the SH2/SH3 adaptor protein Grb2 leading to activation of the Ras/ERK MAP kinase pathway. To complement our work on PTKs, we are also investigating the functions of protein-tyrosine phosphatases (PTPs) in cell signalling. We have found that the receptor-like PTP, RPTP-alpha, which has no known ligand, is constitutively phoshorylated on Tyr789, and that Grb2 is bound to P.Tyr789. The crystal structure of the membrane proximal catalytic domain of RPTP-alpha shows that it exists as an inactive dimer, suggesting that ligand-induced dimerization of RPTP-alpha, and other RPTPs, may negatively regulate their activity. This principle of regulation would be the exact opposite of that observed for receptor PTKs. Our studies on the role of phosphorylation in mammalian cell cycle regulation are focused on the cyclins, cyclin-dependent kinases (Cdk), and their inhibitors, and on the NimA protein-serine kinase. We have identified p27, a novel cyclin/Cdk inhibitor (CDI), and we are investigating the role of p27 and other CDIs in cell cycle regulation. We have identified two potential novel nuclear substrates for phosphorylation by cyclin/Cdk, whose functions are being investigated. We have found that the G2/M transition in mammalian cells can be regulated by the Aspergillus NimA protein-serine kinase, suggesting that mammalian cells have a NimA-like mitotic pathway. In studying this pathway we have found that Aspergillus NimA interacts with Pin1, a small human protein that contains a WW domain and a prolyl isomerase domain. Pin1 is the functional homologue of yeast Ess1p, a protein essential for progression through mitosis. We are currently looking for Pin1 targets and trying to find a vertebrate NimA homologue.