Modular folding and evidence for phosphorylation-induced stabilization of an hsp90-dependent kinase.
The de novo folding of the individual domains of the src family kinase p56(lck) was examined within the context of full-length p56(lck) molecules produced in rabbit reticulocyte lysate containing active chaperone machinery. The catalytic domain required geldanamycin-inhibitable heat shock protein 90 (hsp90) function to achieve its active protease-resistant conformation, but the src homology 2 (SH2) domain acquired phosphopeptide-binding competence independently of hsp90 function. The SH2 domain of hsp90-bound p56(lck) was folded and functional. In addition to the facilitation by hsp90 of kinase biogenesis, a conditional role in maintenance folding could be demonstrated; although wild type p56(lck) molecules with a negative-regulatory C-terminal tyrosine matured to a nearly hsp90-independent state, p56(lck) molecules with a mutated C-terminal tyrosine continued to require hsp90-mediated maintenance. De novo folding could be distinguished from maintenance folding on the basis of proteolytic fingerprints and the effects of different temperatures on folding behavior. Results indicate that during p56(lck) biogenesis, the SH2 domain rapidly folds independently of hsp90 function, followed by the slower hsp90-dependent folding of the catalytic domain and suggest the final stabilization of p56(lck) structure by phosphorylation-mediated interdomain interactions.
Hartson
S D
SD
Department of Biochemistry and Molecular Biology, Oklahoma State University, Stillwater, Oklahoma 74078, USA. shartson@bmb-fs1.biochem.okstate.edu
Ottinger
E A
EA
Huang
W
W
Barany
G
G
Burn
P
P
Matts
R L
RL
D000818:Animals; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism*; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck) / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D017510:Protein Folding; D011817:Rabbits; D018909:src Homology Domains; D019061:src-Family Kinases / Q000737:chemistry / Q000378:metabolism
D018841:HSP90 Heat-Shock Proteins; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D019061:src-Family Kinases