11 months ago

2004 Scientific Report

Laboratory of Signal

Laboratory of Signal Regulation and Cancer Sara A. Courtneidge, Ph.D. Dr. Courtneidge completed her Ph.D. at the National Institute for Medical Research in London. She began her career in the basic sciences in 1978 as a postdoctoral fellow in the laboratory of J. Michael Bishop at the University of California School of Medicine. She later joined her alma mater as a member of the scientific staff. In 1985 Dr. Courtneidge joined the European Molecular Biology Laboratory as group leader and in 1991 was appointed a senior scientist with tenure. She joined Sugen in 1994 as Vice President of Research, later becoming Senior Vice President of Research and then Chief Scientist. Dr. Courtneidge joined VARI in January 2001 as a Distinguished Scientific Investigator. Laboratory Members Staff Eduardo Azucena, Ph.D. Paul Bromann, Ph.D. Hasan Korkaya, Ph.D. Ian Pass, Ph.D. Darren Seals, Ph.D. Rebecca Uzarski, Ph.D. Rebecca Cruz, M.S. Daniel Salinsky, M.S. Students Erik Freiter Lia Tesfay Research Interests Our laboratory wants to understand at the molecular level how proliferation is controlled in normal cells and by what mechanisms these controls are subverted in tumor cells. We largely focus on a family of oncogenic tyrosine kinases, the Src family. The prototype of the family, vSrc, originally discovered as the transforming protein of Rous sarcoma virus, is a mutated and activated version of a normal cellular gene product, cSrc. The activity of all members of the Src family is normally under strict control; however, the enzymes are frequently activated or overexpressed, or both, in human tumors. In normal cells, Src family kinases have been implicated in signaling from many types of receptors, including receptor tyrosine kinases, integrin receptors, and G protein–coupled receptors. Signals generated by Src family kinases are thought to play a role in cell cycle entry, cytoskeletal rearrangements, cell migration, and cell division. In tumor cells, Src may play a role in growth factor–independent proliferation or in invasiveness. Furthermore, some evidence points to a role for Src family kinases in angiogenesis. Some of the current projects in the laboratory are outlined below. The role of the Src substrate Fish in tumorigenesis Fish is an adaptor protein which has five SH3 domains and a phox homology (PX) domain. Fish is tyrosine phosphorylated in Srctransformed 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. The PX domain of Fish associates with phosphatidylinositol 3-phosphate and phosphatidylinositol 3,4-bisphosphate, and it is required to target Fish to podosomes. 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. We have begun to dissect the role of Fish in Src transformation. Our preliminary data (using RNA interference to reduce the level of Fish in cells by approximately 80%) suggest that Fish is required for 18

efficient formation of podosomes and for extracellular matrix degradation. Current experiments seek to define other Fish binding proteins, as well as to determine whether the ADAMs family plays a role in podosome formation and/or function. We have also found that the Fish protein is overexpressed in invasive breast cancer cell lines relative to noninvasive cells. This observation has prompted us to begin an analysis of Fish expression in human breast cancer tissues, with the aim of determining if it is a marker for invasive breast cancer. Characterization of the Src-like kinase Frk We are particularly interested in the human kinase Frk. This kinase has a domain structure typical of Src family kinases. 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 determined that Frk is negatively regulated by its tail tyrosine, but Csk is unlikely to be the responsible enzyme. Like other members of the Src superfamily, active forms of Frk can activate the MAP kinase pathway, but Frk is unusual in that it appears to act downstream of Ras. Furthermore, Frk is not found on cellular membranes, but rather is found in both the cytoplasm and the nucleus. We have now begun to characterize the transforming ability of Frk in epithelial cells. The role of Src family kinases in mitogenic signaling pathways We have previously shown that Src family kinases are required for both Myc induction and DNA synthesis in response to PDGF stimulation of NIH3T3 fibroblasts. We have also previously identified and characterized a small molecule inhibitor of Src family kinases called SU6656. We recently compared PDGF-stimulated gene expression in untreated and SU6656-treated cells. We determined that a discrete subset of PDGF-responsive genes requires Src family kinases. By using other inhibitors, we could show that these genes are for the most part distinct from the genes requiring MAP kinase or PI3-kinase activities for stimulation. We are currently determining which of these Src-specific events are required for the induction of Myc. 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 fully established. We have begun our studies by characterizing the role of Src in estrogen-stimulated signal transduction pathways in breast cancer cell lines. We have shown that Src family kinase activity is required for estrogen to stimulate mitogenesis in MCF7 cells. Furthermore, inhibition of Src prevents both estrogen stimulation of Myc and MAP kinase activity. We are currently dissecting which Src signaling pathways are necessary for estrogen-stimulated growth, as well as how Src activity results in the activation of MAP kinase and in the production of Myc. Recent Publications Abram, Clare L., Darren F. Seals, Ian Pass, Daniel Salinsky, Lisa Maurer, Therese M. Roth, and Sara A. Courtneidge. 2003. The adaptor protein Fish associates with members of the ADAMs family and localizes to podosomes of Src-transformed cells. Journal of Biological Chemistry 278(19): 16844–16851. Courtneidge, S.A. 2003. Isolation of novel Src substrates. Biochemical Society Transactions 31(Pt. 1): 25–28. Scaife, Robin M., Sara A. Courtneidge, and Wallace Y. Langdon. 2003. The multi-adaptor protooncoprotein Cbl is a key regulator of Rac and actin assembly. Journal of Cell Science 116(Pt. 3): 463–473. 19

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