Csk Homologous Kinase, a Novel Signaling Molecule, Directly Associates with the Activated ErbB-2 Receptor in Breast Cancer Cells and Inhibits Their Proliferation

Abstract

Substantial evidence exists supporting direct roles for ErbB-2/neu and Src kinase activation in breast cancer. The Csk homologous kinase (CHK) is a recently identified tyrosine kinase which, like Csk, phosphorylates the C-terminal tyrosine of Src kinases, resulting in inactivation of these enzymes. Recently, we observed that CHK is associated with the ErbB-2/neu receptor upon heregulin stimulation of breast cancer cells. Here, we report that CHK expression was observed in 70 out of 80 primary breast cancer specimens but not in normal breast tissues (0/19). Confocal microscopy analysis revealed co-localization of CHK with ErbB-2 in these primary specimens (6/6). In addition, we observed that the cytoplasmic domain of the ErbB-2/neu receptor is sufficient for its interaction with the CHKSH2 domain. Phosphopeptide inhibition of the in vitro interaction of CHKSH2 or native CHK with ErbB-2/neu, as well as site-directed mutagenesis of ErbB-2/neu, indicated that CHKSH2 binds to Tyr1253 of ErbB-2/neu. Interestingly, autophosphorylation at this site confers oncogenicity to this receptor. Moreover, CHK was able to down-regulate ErbB-2/neu-activated Src kinases. Overexpression of CHK in MCF-7 breast cancer cells markedly inhibited cell growth and proliferative response to heregulin as well as decreased colony formation in soft agar. These studies indicate that CHK binds, via its SH2 domain, to Tyr1253 of the activated ErbB-2/neu and down-regulates the ErbB-2/neu-mediated activation of Src kinases, thereby inhibiting breast cancer cell growth. These data strongly suggest that CHK is a novel negative growth regulator in human breast cancer.