PTK6 [protein tyrosine kinase 6; also known as Brk (breast tumour kinase)] is a non-receptor tyrosine kinase, closely related to Src, but evolutionarily distinct, that is up-regulated in various cancers, including breast cancer. Hsp90 (heat-shock protein 90) was identified as a PTK6-interacting protein in HEK (human embryonic kidney)-293 cells overexpressing PTK6. Hsp90 interacted with the PTK6 tyrosine kinase catalytic domain, but catalytic activity was not required for the interaction. Geldanamycin, an Hsp90 inhibitor, significantly decreased the PTK6 protein level through proteasome-dependent degradation, but did not affect the level of Src. Geldanamycin treatment also decreased phosphorylation of PTK6 substrates due to reduced amounts of PTK6. Moreover, overexpression of CHIP [C-terminus of Hsc70 (heat-shock cognate 70)-interacting protein], a chaperone-dependent E3 ligase, enhanced proteosomal degradation of PTK6. Geldanamycin increased the interaction of PTK6 with CHIP, but decreased the interaction of PTK6 with Hsp90. We also found that endogenous PTK6 associated with Hsp90 and geldanamycin decreased expression of endogenous PTK6 in breast carcinoma cells. Finally, we report that silencing endogenous CHIP expression in breast carcinoma cells inhibited geldanamycin-induced PTK6 reduction. These results demonstrate that Hsp90 plays an essential role in regulating PTK6 stability and suggest that Hsp90 inhibitors may be useful as therapeutic drugs for PTK6-positive cancers, including breast cancer.

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