Substantial evidence exists supporting direct roles for ErbB-2/neu and src tyrosine kinase activation in breast cancer. CHK (Csk Homologous Kinase) is a cytoplasmic protein tyrosine kinase that phosphorylates and negatively regulates src kinase activity. In this proposed study, we aim to elucidate the role of CHK in breast cancer cells. CHK is highly restricted in its expression and normally found in brain and hematopoietic cells. Our recent studies reveal 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 and src in these primary specimens (6/6). Furthermore, we observed that CHK participates in signaling in breast cancer cells by associating, via its SH2 domain, with ErbB-2 following heregulin stimulation. This association appears to be receptor specific (ErbB-2) and ligand specific (heregulin). Site-directed mutagenesis and phosphopeptide inhibition experiments indicated that CHK-SH2 binds to Tyr1253 of the rodent ErbB-2 (neu) or to Tyr1248 of the human ErbB-2. 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 results strongly suggest that CHK is a potential novel negative growth regulator in human breast cancer, and lead us to hypothesize that: (i) CHK expression is upregulated in breast cancer cells; (ii) CHK is able to antagonize the growth-promoting signals that are mediated by src and ErbB-2 tyrosine kinases; (iii) overexpression of CHK inhibits the oncogenicity of ErbB-2; (iv) CHK interacts with specific substrate(s) in breast tumor cells. In order to test these hypotheses, we propose to focus on the following specific aims: (1) To further assess the expression of CHK, relative to src and ErbB-2 tyrosine kinases, in human breast tumors resected at various stages of malignant progression; (2) To determine the molecular and functional analyses of CHK-ErbB-2 and CHK pp60src interactions in breast cancer cells; (3) To characterize the biological role(s) of CHK in breast cancer cellular responses and cell growth; and (4) To identify and characterize signaling molecules that associate with CHK in breast cancer cells. New information gained from these studies on the role of CHK as a putative negative growth regulator in breast cancer may provide a basis for utilizing this novel tyrosine kinase to oppose the malignant process.
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