Class IA phosphatidylinositol 3 -kinase involvement in cytoskeletal regulation

Abstract

Phosphoinositide 3-kinases (PI 3-kinases) phosphorylate the D3 position on the inositol ring of phosphatidylinositol. PI 3-kinases products act as second messengers in many different signaling events. The best-characterized PI 3-kinases are the Class IA enzymes, which are heterodimers consisting of a catalytic subunit (p110) and a regulatory subunit (p85). p85/p110 PI 3-kinases are activated by tyrosine kinases, and in intact cells produce primarily PI 3,4,5-trisphosphate (PIP3).;My thesis work examines the role Class IA PI 3-kinases play in EGF-stimulated lamellipod extension and cytoskeleton regulation in MTLn3 cells. MTLn3 cells are rat adenocarcinoma cells that undergo a chemotactic response to EGF. In this response, MTLn3 cells produce an actin-dependent lamellipod extension and membrane ruffling. Treatment of MTLn3 cells with wortmannin, a specific inhibitor of PI 3-kinase, inhibits EGF-stimulated lamellipod extension. This would suggest a participation of PI 3-kinases.;I examined whether Class IA PI 3-kinase isoforms were involved in EGF-stimulated lamellipod extension. In summary, data shows that p110alpha and not p110beta is necessary for EGF-stimulated lamellipodia extension and new barbed end formation, whereas both p110alpha and p110beta are necessary for optimal DNA synthesis.;The N-terminal half of the Class IA regulatory subunit, p85, contains several protein-protein interaction domains, including a SH3 domain, a GTPase-binding domain (BCR), and two proline-rich (PR) domains. The PR and BCR domains of p85 specifically couple PI 3-kinase to EGF-stimulated lamellipod extension.;In order to understand which signaling proteins may be involved in this isoform-specific activity, I generated MTLn3 cells that stably overexpress one isoform. The cells that overexpress p110alpha extend lamellipods during EGF-stimulation, whereas the cells that overexpress p110beta do not. In summary, there seems to be a difference in the regulation of signaling proteins involved in cytoskeleton regulation, more specifically cofilin, among the p110beta and p110alpha overexpressing cells. These data lay the foundation for further analysis of signaling differences that may be involved in Class IA PI 3-kinase isoform-specific signaling. (Abstract shortened by UMI.).