We propose to continue our studies on the tyrosine kinase Src (also called c-Src), the cellular homolog of the v-Src oncoprotein. Activated Src causes transformation of some cell types and cancer in animals, but can stimulate differentiation, migration, survival or apoptosis when expressed in other cell types. Because activated Src phosphorylates many different cell proteins, the specific mechanisms underlying these different cellular outcomes have been difficult to dissect. Moreover, the roles of the normal cellular Src protein have been unclear, in part due to overlapping functions between Src and other close relatives. Src activity is elevated in many human cancers, including approximately 70% of carcinomas. Understanding a novel aspect of Src biology is the general goal of this proposal. Over the past funding period, our study of a Src substrate, Dab1, led us to uncover a Src-dependent signaling pathway that regulates the migrations of neuronal precursors in the developing mammalian central nervous system. An extracellular protein, Reelin, acts via non-catalytic receptors to stimulate Src- dependent tyrosine phosphorylation of Dab1. Genetic evidence shows that tyrosine phosphorylation of Dab1 is both a cause and a consequence of activation of Src, and both events are required for downstream signaling. Reelin stimulates Dab1 binding to several identified signaling proteins, including the adaptor proteins Crk and CrkL. Reelin also stimulates Dab1 ubiquitylation and degradation, and Reelin receptor endocytosis. However, it is not yet clear whether Dab1 ubiquitylation regulates signaling or endocytbsis or is a feedback control, and it remains unclear how Dab1 degradation, Reelin endocytosis, Dab1-complex formation, and Src kinase activation link to each other and regulate neuron precursor movements in vivo. Wepropose to use genetic and biochemical approaches to uncover the mechanisms by which Src and Dab1 regulate cell migrations in vivo. We propose two broad aims: to uncover the importance of different Dab1 phosphorylation sites and signaling complexes and Dab1 degradation in Dab1 regulation and development;and to determine the specific roles of Crk, CrkL, their downstream mediator C3G, and endocytosis in signaling. By further study of this signaling process, we expect to identify principles of signal complex formation and regulation that may be relevant to other tyrosine kinase signaling pathways.
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