Affinity isolation of heat-shock and other calmodulin-binding proteins following hyperthermia.
The interaction of calmodulin (CaM) with heat-shock and other binding proteins was studied in rat adenocarcinoma cells. Cells were equilibrium-labeled for 48 h prior to heating for 1 h at 43 degrees C, or pulse-labeled for 2 h at 37 degrees C after heating, to monitor the effect of heat on the affinity of CaM-binding proteins synthesized under these conditions. A CaM antagonist shown to sensitize to heat killing, W-7 [N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide], was used in competition assays to help monitor any changes in affinity. We found that heating tended to reduce the CaM-binding of proteins synthesized before heating relative to their 37 degrees C controls and proteins synthesized after heating tended to have increased binding relative to their respective controls. Members of the heat-shock protein (hsp) 90-, 70-, and 26-kDa families were among the proteins that bound to CaM and were eluted by W-7. The peak elution fractions for the hsp's and other cellular proteins varied, but hsp-70 eluted in the early fractions. The hsp-70 family was also found to be among a number of W-7-binding proteins. We conclude that the assumption that CaM antagonists potentiate killing of heated cells solely by competing nonspecifically for CaM-binding protein sites on CaM does not explain the process completely. These antagonists could also act by competing for CaM-binding sites with specific proteins whose interaction with CaM is important for survival following heating, or by directly binding to other proteins whose function is important for survival and inhibiting their activity. We do not have sufficient data to discern the predominant mechanism among these possibilities, but we believe all are likely to occur in heated cells and speculate that inhibition of the functions of the hsp-70 family is important in several of these antagonist actions.
Evans
D P
DP
Department of Tumor Biology, University of Texas M. D. Anderson Cancer Center, Houston 77030.
Tomasovic
S P
SP
D000230:Adenocarcinoma / Q000473:pathology; D000818:Animals; D002147:Calmodulin / Q000037:antagonists & inhibitors; D002148:Calmodulin-Binding Proteins / Q000302:isolation & purification*; D002470:Cell Survival / Q000187:drug effects; D002846:Chromatography, Affinity; D006360:Heat-Shock Proteins / Q000302:isolation & purification*; D006358:Hot Temperature; D066298:In Vitro Techniques; D008325:Mammary Neoplasms, Experimental / Q000473:pathology; D051381:Rats; D013449:Sulfonamides / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D006360:Heat-Shock Proteins; D013449:Sulfonamides; C017967:W 7