The insulin receptor is the defining member of a family of protein(tyrosine) kinases. This family includes receptors for insulin- like growth factor, and hepatocyte growth factor, as well as Drosophila sevenless, Ros, and the UR2 transforming oncogene. The defining molecular feature is a conserved cluster of three tyrosines within the catalytic core of the kinase. Autophosphorylation of these tyrosines leads to activation of substrate phosphorylation. This activation is essential to the biological function of these kinases. However, the kinase domain of each family member is distinguished by unique autophosphorylation sites that are related to their respective unique cellular functions. Signal transduction through this family of receptors depends on the cellular environment and stimulation by the growth factor or hormone. The former is permissive of the biological effects, and the latter is necessary to raise the kinase from a basal to an activated state. At least four specific features of the kinase domain are intrinsic to this process of activation and thus to signal transduction: 1. """"""""core"""""""" autophosphorylation that activates the kinase, 2. """"""""subdomain"""""""" autophosphorylation characteristic of each kinase, which separately or together lead to 3. recognition of adapter proteins, and 4. phosphorylation of substrates. Thus, the apex of signal transduction for these receptors is activation of the kinase domain. The broad objective is to understand the molecular differences between basal and activated states of these tyrosine kinases that are activated by autophosphorylation. The specific objectives of this proposal are to examine (1) the molecular mechanisms that link autophosphorylation of the core tyrosines to kinase activation, and (2) the mechanisms that regulate reaction of the unique autophosphorylation sites. Each mechanism encompasses a set of conserved regulatory motifs that can be understood at the molecular level through rationally designed mutagenesis. Kinetics and physical- chemical measurements will then reveal both the catalytic events and conformational changes that are necessary and sufficient for activation. The early stages of this work focus on the isolated cytoplasmic kinase domain of the insulin receptor, which is more accessible for the proposed kinetic and physical studies. The long-term goals are to reconstruct activation via kinase domain interactions between transmembrane beta-subunits, and ultimately to establish the molecular mechanism of activation by insulin through binding to the alpha-subunit of the intact receptor.
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