Hsp90-mediated folding of the lymphoid cell kinase p56lck.
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 p56lck 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 p56lck 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 p56lck activity and protein. However, geldanamycin had no direct effect on purified p56lck. Also consistent with functional linkages between hsp90 and p56lck, 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 p56lck structure in rabbit reticulocyte lysate, the specific occurrence of complexes between hsp90 and p56lck in the cytoplasm of T cells suggests that hsp90 primarily folds nascent molecules of p56lck in vivo.
Hartson
S D
SD
Department of Biochemistry and Molecular Biology, Oklahoma State University, Stillwater 74078-0454, USA. shartson@bmb-fs1.bioechem.okstate.edu
Barrett
D J
DJ
Burn
P
P
Matts
R L
RL
D000818:Animals; D002461:Cell Line, Transformed; D002474:Cell-Free System; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism*; D066298:In Vitro Techniques; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D008214:Lymphocytes / Q000201:enzymology*; D017510:Protein Folding; D011817:Rabbits; D051381:Rats; D012156:Reticulocytes / Q000378:metabolism; D019061:src-Family Kinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D018841:HSP90 Heat-Shock Proteins; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D019061:src-Family Kinases