Chaperoning checkpoint kinase 1 (Chk1), an Hsp90 client, with purified chaperones.
Checkpoint kinase 1 (Chk1), a serine/threonine kinase that regulates DNA damage checkpoints, is destabilized when heat shock protein 90 (Hsp90) is inhibited, suggesting that Chk1 is an Hsp90 client. In the present work we examined the interplay between Chk1 and Hsp90 in intact cells, identified a source of unchaperoned Chk1, and report the in vitro chaperoning of Chk1 in reticulocyte lysates and with purified chaperones and co-chaperones. We find that bacterially expressed Chk1 is post-translationally chaperoned to an active kinase. This reaction minimally requires Hsp90, Hsp70, Hsp40, Cdc37, and the protein kinase CK2. The co-chaperone Hop, although not essential for the activation of Chk1 in vitro, enhanced the chaperoning process, whereas the co-chaperone p23 did not stimulate the chaperoning reaction. Additionally, we found that the C-terminal regulatory domain of Chk1 affects the association of Chk1 with Hsp90. Collectively these results provide new insights into Hsp90-dependent chaperoning of a client kinase and identify a novel, biochemically tractable model system that will be useful to further dissect the Hsp90-dependent chaperoning of this important and ubiquitous class of Hsp90 clients.
Arlander
Sonnet J H
SJ
Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Graduate School.
Felts
Sara J
SJ
Wagner
Jill M
JM
Stensgard
Bridget
B
Toft
David O
DO
Karnitz
Larry M
LM
D047390:Casein Kinase II; D002474:Cell-Free System; D000071877:Checkpoint Kinase 1; D018841:HSP90 Heat-Shock Proteins / Q000502:physiology; D006367:HeLa Cells; D006360:Heat-Shock Proteins / Q000502:physiology; D006801:Humans; D018832:Molecular Chaperones / Q000302:isolation & purification / Q000502:physiology*; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*
D018841:HSP90 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; C079373:STIP1 protein, human; D011494:Protein Kinases; C000605867:CHEK1 protein, human; D047390:Casein Kinase II; D000071877:Checkpoint Kinase 1