Lymphoid cell kinase (lck) is a src-family nonreceptor protein tyrosine kinase mediating both thymocyte development and T-cell signaling. Aberrant expression of the lck gene has been implicated in several lymphomas. Changes in expression or regulation of lck are correlated with the progression of AIDS. The goal of the proposed research is to elucidate the pathway for posttranslational maturation of lck by testing three independent hypotheses: (1) that the 90-kDa heat shock protein (hsp90) and cohorts of hsp90 chaperone the folding of lck during its maturation; (2) that the pathways for lck folding and regulation overlap; (3) that the posttranslational maturation of lck can be modulated pharmacologically. The primary methodologies proposed are: (1) equilibrium and pulse-chase radiolabeling of LSTRA T-cells and coimmunoadsorption of lack with hsp90 and hsp90-cohorts to determine if nascent lck matures through a hsp90-bound intermediate; (2) expression of lck in hsp90-deficient yeast strains to determine if hsp90 is required for lck maturation; (3) assays to determine the functional status of the SH3, SH2 and kinase domains of lck; (4) structural characterization of lck by protease-nicking assays to assess changes in conformation occurring during lck maturation; (5) subcellular fractionation of cell lysates to determine the localization of lck changes. Possible pharmacological targets essential to lck maturation or function will be identified by: (1) identifying cohorts of hsp90 that occur in complexes with lck; (2) characterizing the effects of hsp90 inhibitors and immunophilins on lck biochemistry in LSTRA cells. The function of hsp90 will be modeled in the rabbit-reticulocyte lysate (and heterologous folding systems, e.g., wheat germ) to assess the structure and function of specific protein domains, roles of specific hsp90 cohorts, and effect of hsp90-inhibitors on lck folding. To determine if folding intermediates can serve as substrates for regulatory phosphorylations, the phosphorylation status of nascent lck will be characterized in an in vitro model system and in vivo. Specific regions of lck available for interactions with other proteins will be determined by coimmunoadsorption of hsp90 by a select battery of anti-lck antibodies. This research will enhance understanding of the immune response, facilitate development of pharmacological approaches to suppress lck-dependent immune functions and dysfunctions. Principles will extrapolate to processes regulated by other src-family kinases.

R01GM051608

1997-02-01

2002-01-31