We found earlier that pl30Cas (Cas), a 130 kDa v-src substrate protein, becomes tyrosine-phosphorylated upon integrin ligand binding and may function as a molecular switch activating downstream signaling pathways via protein-protein interactions. Our recent data support a crucial role for Cas in coordinating cell growth, morphology and cell migration. Our findings demonstrate that this role of Cas is not limited to integrin signaling, but Cas also functions downstream of cytokines, growth factors and oncogenes. Most notably, complex formation between the tyrosine-phosphorylated Cas and the SH2/SH3-containing adaptor protein Crk was found to play a central role in activating one of the MAP kinase pathways, namely the JNK pathway, in response to several cellular stimuli; this finding is the first demonstration of a stimuli-induced biochemical signaling pathway downstream of the Cas-Crk complex. Taken into consideration the evidence showing an important role for JNK in a number of physiological and pathological processes, the first two Aims within this proposal focus on defining the biological significance of the Cas-Crk-JNK pathway.
In Specific Aim l, we will study whether the Cas-Crk-JNK pathway regulates such well-defined biological events as transcriptional activity and cell migration in response to integrin-mediated cell adhesion; evidence in other signaling systems suggests that activation of the Cas-Crk-JNK cascade may well have a previously unidentified crucial role in these important cellular functions. In the Specific Aim 2, we will study the possibility that inadvertent activation of the Cas-Crk-JNK pathway may aid malignant transformation. As detailed in the Research Plan, oncogenic Met receptor-expressing cells provide a highly plausible and biologically significant model system, and will be utilized in these-studies. Finally, we have observed a time-dependent cleavage of Cas in apoptotic cells, which correlates with changes in- subcellular localization of Cas.
In Specific Aim 3, we will study the functional significance of Cas cleavage in apoptosis; these studies include determining the exact cleavage sites in Cas and expression of recombinant fragments of Cas and Cas-constructs that are resistant to cleavage in mammalian cells. Once completed, these studies are expected to yield significant new information on the role of the Cas-Crk signaling complex in intracellular signaling events.
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