Hsp-90-mediated folding of the lymphoid cell kinase P56(lck1)

Several lines of evidence suggest that members of the 90-kDa family of heat shock proteins (hsp90) may support the folding of various homologues of the src kinase family. In this work, we utilized pulse-chase analyses in rabbit reticulocyte lysate to demonstrate that hsp90-bound intermediates existed for the majority of newly synthesized p56(lck) molecules. The hsp90-binding drug geldanamycin disrupted the association of p56lck with hsp90, prevented the kinase from demonstrating a protease-resistant conformation, and caused decreases in kinase specific activity. Requirements for geldanamycin-inhibitable hsp90 function and physical interactions between hsp90 and p56(lck) persisted during chase periods. Consistent with the effects observed in rabbit reticulocyte lysate, application of geldanamycin to fibroblasts caused specific reversion of lck-mediated transformation concomitant with loss of p56(lck) activity and protein. However, geldanamycin had no direct effect on purified p56(lck). Also consistent with functional linkages between hsp90 and p56(lck), physical interactions between these proteins were detected in cytoplasmic, but not membrane, fractions of LSTRA. cells. Although hsp90 functions in both the initial de novo folding and the reiterative support of p56(lck) structure in rabbit reticulocyte lysate, the specific occurrence of complexes between hsp90 and p56(lck) in the cytoplasm of T cells suggests that hsp90 primarily folds nascent molecules of p56(lck) in vivo.