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2002 Scientific Report

  • Text
  • Institute
  • Report
  • Tumors
  • Protein
  • Signaling
  • Michigan
  • Molecular
  • Proteins
  • Laboratory

homology (PX) domain.

homology (PX) domain. Fish is tyrosine phosphorylated in Src-transformed fibroblasts (suggesting that it is a target of Src in vivo) and in normal cells after treatment with several growth factors. We have recently found that in Src-transformed cells, Fish is localized to specialized regions of the plasma membrane called invadopodia or podosomes. These actin-rich protrusions from the plasma membrane are sites of matrix invasion and locomotion. We have also determined that the PX domain of Fish associates with phosphatidylinositol 3-phosphate and that this domain targets Fish to the podosomes. Furthermore, the fifth SH3 domain of Fish mediates its association with members of the ADAMs family of membrane metalloproteases, which in Src-transformed cells are also localized to podosomes. Our current research aims to probe the role of Fish in invasion in both Src-transformed cells and in human tumor cell lines. Other novel proteins identified in the substrate screen have also been shown to be tyrosine phosphorylated in Src-transformed cells and are being characterized. Using a Src-selective inhibitor to probe its role in signaling pathways The use of small molecule inhibitors to study molecular components of cellular signal transduction pathways provides a complementary means of analysis to techniques such as antisense, dominant negative (interfering) mutants and constitutively activated mutants. We have recently identified and characterized a small molecule inhibitor, SU6656, which exhibits selectivity for Src and other members of the Src family. The use of SU6656 confirmed our previous findings that Src family kinases are required for both Myc induction and DNA synthesis in response to PDGF stimulation of NIH 3T3 fibroblasts. We are currently comparing both PDGF-stimulated gene expression and tyrosine phosphorylation events in untreated and SU6656- treated cells to define which events require Src family kinases. SU6656 should prove a useful tool for further dissecting the role of Src kinases in this and other signal transduction pathways. The connection between Src and p53 The tumor suppressor p53 is present at low levels in growing cells. Many DNA tumor viruses encode proteins that inactivate p53 by direct association or ubiquitination-mediated degradation, presumably to facilitate the entry of cells into cycle and therefore viral replication. We have recently shown that the product of one such DNA tumor virus, the SV40 large T antigen, bypasses the requirements for several signals emanating from growth factor receptors. In particular, cells that lack p53, or in which p53 has been inactivated by T antigen binding, no longer require Src family kinases for growth factor signaling. These findings suggest that Src kinases are required to overcome the inhibitory effects of p53. There are perhaps also implications for the use of signal transduction inhibitors in human cancers where negative regulators such as p53 are frequently mutated or absent. We are now investigating in more detail the way in which Src kinases impact p53 function. Breast cancer Increased Src activity can be demonstrated in the majority of breast cancers, both estrogendependent and estrogen-independent, yet the role of Src in breast tumorigenesis has not been established. We have begun to characterize the role of Src in both EGF-stimulated and estrogen-stimulated signal transduction pathways in breast cancer cell lines. Novel members of the Src family We are particularly interested in the human kinase Frk. This kinase has a domain structure typical of Src family kinases and is probably regulated in a similar manner. However Frk lacks the amino-terminal myristylation sequences and instead has a nuclear localization sequence in its SH2 domain. Interestingly, Frk is predominantly expressed in epithelial cells and is overexpressed in a high proportion of human tumors and tumor cells lines, particularly those deriving from lung. We have begun an extensive characterization of Frk, including its substrate specificity, regulation, transforming ability, and function. 19

Publications Courtneidge, S.A. 2002. The role of Src in signal transduction pathways. Biochemical Society Transactions 30(2): 11–17. From left to right, back row: Cruz, Mauer, Courtneidge middle row: Korkaya, Freiter, Seals front row: Salinsky, Azucena, Uzarski, Roth 20

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