Integrin adhesion receptor signaling through the Abl and Arg nonreceptor tyrosine kinases activates several major cytoskeletal effectors to coordinate changes in cytoskeletal structure. Abl/Arg-mediated signaling events are essential for nervous and immune system development and function, while inappropriately elevated Abl/Arg signaling is associated with several cancers. Despite these critical physiological roles, the mechanisms by which integrins activate Abl and Arg are poorly understood. Based on extensive preliminary evidence, we hypothesize a multistage model for integrin 1 activation of Arg kinase activity that we will test in three Aims.
Our first aim i s to identify and characterize he interfaces that mediate initial Arg recruitment to the integrin 1 tail. We find that a sequence in the integrin 1 tail not known to interact with other integrin-binding proteins binds directly to te isolated Arg kinase domain and activates Arg kinase activity. We will identify the interfaces on integrin 1 and Arg that mediate these interactions and use mutagenesis and binding assays to determine the contributions of specific residues to this interaction. We have expressed, purified and obtained crystals of the Arg kinase domain. We will now use X-ray crystallography to determine the structure of the Arg kinase domain in complex with integrin 1 tail peptides.
Our second aim i s to investigate the dual roles that the Arg SH2 domain plays in both retainment and reinforcement of Arg kinase activation. Arg can phosphorylate integrin 1 tail directly, thereby creating a binding site for the Arg SH2 domain, which we hypothesize retains Arg and promotes optimal kinase activation. We will use biochemistry and X-ray crystallography to elucidate an atomic resolution understanding of how the Arg SH2 domain and kinase domains bind coordinately to the phosphorylated integrin 1 tail. As part of this work, we will test the hypothesis that coordinated binding to the phosphorylated integrin 1 tail promotes cyclin-like Arg SH2 domain binding to the Arg kinase domain N-terminal lobe to reinforce kinase activation.
Our third aim i s to investigate the role of the recruitment, retainment, and reinforcement model in regulating Arg signaling and its control of dendritic spine and dendrite stability in vivo. Interin signaling through Arg and its substrate p190RhoGAP negatively regulates the RhoA GTPase, a major antagonist of synapse, dendritic spine, and dendrite stability. Genetic studies suggest that integrin 1 interacts functionally with Arg to mediate dendritic spine and dendrite stabilization i the adolescent mouse brain. We will generate integrin 1 and Arg mutants deficient in interactions that mediate Arg kinase recruitment, retainment, or reinforcement, and reconstitute them into integrin 1- or Arg-deficient fibroblasts and neurons. We will employ biochemical, FRET, and cell-based assays to determine how these interfaces contribute to integrin 1: Arg interactions and Arg- mediated signaling in fibroblasts and neurons, and control of dendritic spine and dendrite stability in neurons.
The Abl and Arg nonreceptor tyrosine kinases regulate the development and function of the immune and nervous sytstems. Chromosomal translocations involving the Abl and Arg nonreceptor tyrosine kinases have long been known to cause leukemia in humans and increased activation of native Abl and Arg may be associated with cancer progression of solid tumors, but how these kinases are activated by upstream receptors is unclear. We have found that direct binding of the intracellular tail of the integrin receptor ?1 subunit can activate the Arg tyrosine kinase. We propose to determine the mechanism and structural basis for integrin ?1 activation of Arg. We anticipate these studies will lead to new approaches to target Abl and Arg signaling in the treatment of cancer and other diseases.
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