Rapid activation of c-Src kinase by dioxin is mediated by the Cdc37-HSP90 complex as part of Ah receptor signaling in MCF10A cells.

We investigated the mechanism by which activation of the Ah receptor by dioxin (TCDD) was accompanied by rapid activation of c-Src kinase activity. A Western blotting analysis showed that such action of TCDD in MCF10A cells could effectively be suppressed by treatment with a specific inhibitor of Src family kinase, PP-2, as judged by Western blot detection of the active form of Src protein, indicating that Src kinase is directly activated by TCDD. Such an event, occurring within 10-30 min of the addition of TCDD, is also accompanied by simultaneous translocation of both Src and cdc37 proteins from cytosol into the 100,000 x g membrane fraction containing the plasma membrane. By dissociating the cytosolic Src-cdc37-HSP90 complex with 17 nM geldanamycin, an optimum concentration for affecting this cytosolic cdc37 complex, but not the cytosolic Ah receptor complex, we could show that the action of TCDD in activating c-Src and cdc37 was abolished, but not its action on CYP1A1. The important role of cdc37 in the action of TCDD-induced activation of c-Src was also confirmed by blocking cdc37 gene translation with the antisense oligonucleotide treatment as well as the siRNA preparation designed to silence cdc37 expression. To understand the functional meaning of the disruption of the Src-cdc37-HSP90 complex by 17 nM geldanamycin at the cellular level, we investigated its effect on TCDD-induced anti-apoptotic action. The results showed that geldanamycin at this concentration could also abolish this cellular effect of TCDD. Interestingly, such a role of cdc37 in mediating the action of TCDD appears to be limited to activation of c-Src kinase, but not kinases associated with activation of NFkB, C/EBPalpha, or ERK. Together, these observations support the hypothesis that there is a specific coordination between the activation of the cytosolic Ah receptor and the c-Src- and cdc37-containing HSP90 complex.