Hsp90 and its co-chaperone Cdc37 are required for the activity Of numerous eukaryotic protein kinases. c-Jun N-terminal kinases (JNKs) appear to be Hsp90-independent kinases, as their activity is unaffected by Hsp90 inhibition. It is Currently Unknown why some protein kinases are Hsp90- and Cdc37-dependent for their function, while others are not. Therefore, we investigated what structural motifs within JNKs confer or defer Hsp90 and Cdc37 interaction. Both Hsp90 and Cdc37 recognized structural features that were exposed or destabilized upon deletion of JNK1 alpha I's N-terminal non-catalytic structural motif, while only Hsp90 bound JNK when its C-terminal non-catalytic structural motif was deleted. Mutations in JNK's activation loop that are known to constitutively activate or inactivate its kinase activity had no effect on JNK's kick of interaction with Hsp90 and Cdc37. Our findings suggest a model in which Hsp90 and Cdc37 each recognize distinct features within the catalytic domains of kinases. (c) 2005 Elsevier Inc. All rights reserved.