The Src family of tyrosine kinases is required for cells to progress from the G2 phase of the cell cycle into mitosis. Following up on our previous studies, which uncovered the mechanism of activation of Src at G2/M and identified a primary mitotic target, the RNA binding protein Sam68, we will study the key signalling proteins acting upstream and downstream of Src at mitosis. We have found that the prtotein kinase activity of the Raf-1 proto-oncoprotein is stimulated at mitosis.
In Specific Aim 1 we will explore the possibility that Raf-1 is a primary target of Src at G2/M. We will also determine whether Ras or other signalling proteins play a role in Raf-1 activation at G2/M and whether Raf-1 activation leads to activation of MAPK pathways at G2/M. Finall y we will determine whether Raf-1 and downstream effector pathways are required for cells to enter mitosis and whether Raf-1 plays a role in celular decisions at G2/M to divide or undergo apoptosis.
In Specific Aim 2 we will elucidate the cellular function of Sam68 by identifying the biologically relevant RNA targets with which Sam68 interacts. We will also investigate mechanisms by which Src and cell cycle regulators might regulate Sam68 function and will exploit the documented interaction between Sam68 and components of the poliovirus RNA replication machinery to gain insight into normal Sam68 function.
In Specific Aim 3 we will investigate the regulation of the best candidate for an upstream activator of Src family kinases at G2/M, the protein tyrosine phosphatase PTP alpha. We will determine the mechanisms underlying the increased expression of PTP alpha at G2/M and whether the accompanying increase in PTP alpha phosphatase activity is responsible for G2/M activation of Src. These studies are important for understanding the molecular basis of human cancer. Constitutive upregulation of Src, Raf-1 and PTP alpha activities by mutation or overexpression leads to cellular transformation. Furthermore, the activities of Src and Raf-1 are elevated and PTP alpha RNA levels are increased in some human cancers. In addition, all three proteins have links, either upstream or downstream, to Ras, perhaps the most frequently mutated proto-oncogene in human cancer. The aberrant mitoses of transformed cells and the increased sensitivity of cancer cells to G2/M-targetted drugs such as taxol mandate understanding proto- oncoprotein signalling pathways at G2/M to form the basis of rational drug design for new types of chemotherapeutic intervention.
Showing the most recent 10 out of 44 publications
