The aim of this proposal is to identify the function of c-Src which, in the osteoclast, is an absolute requirement for bone resorption (src- mutants develop osteopetrosis) and is unique to c-Src (this function cannot be compensated by other gene products within this cellular context). The hypothesis that c-src may play a unique role in osteoclast function stems from three observations. First, deletion of the gene encoding the proto-oncogene c-src by homologous recombination in the mouse leads to a phenotype of osteopetrosis without other apparent cellular defects. Second, in vivo and in vitro reconstitution experiments have demonstrated that the cellular defect induced by src gene deletion is cell autonomous. Third, the osteoclast expresses other members of the Src family of nonreceptor tyrosine kinases (c-Fyn, c-Lyn and c-Yes) implying that although present, they cannot compensate for src-deletion in osteoclast function. This research proposal includes both short-term strategies aimed at determining the various functions of c-Src in osteoclasts and long-term strategies aimed at identifying the unique function of c-Src in osteoclasts.
The specific aims are: (1) To further identify and analyze signal-transduction pathways in which c-src is involved and/or required in osteoclasts, searching for upstream elements (i.e. molecules that, directly or indirectly; activate c-Src or modulate its activity) both at the.plasma membrane (receptors) and in intracellular membranes); (2) To further identify and characterize substrates (i.e. downstream elements) which are associated with and/or phosphorylated by c-Src, in particular those found in functionally critical regions of the osteoclast; (3) To study the role of phosphorylation and dephosphorylation events in the regulation of csrc-kinase activity and determine the relationship between this kinase activity and bone resorption; and (4) To develop and implement strategies aimed at identifying osteoclast proteins which interact specifically and/or with high affinity with c-Src and determine which functional domain(s) of c- src are important in these interactions. The identification of the unique function played by c-src in osteoclasts would have a very significant impact, not only on the understanding of the molecular mechanisms of bone resorption and of the biological role of c-Src but also on the development of therapeutic means of intervention in diseases involving increased bone resorption (osteoporosis, periodontal disease, joint diseases).
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