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 colocalization 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 CHK SH2 domain. Phosphopeptide inhibition of the in vitro interaction of CHK SH2 or native CHK with ErbB-2/neu, as well as site-directed mutagenesis of ErbB-2/neu, indicated that CHK SH2 binds to Tyr 1253 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 Tyr 1253 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.Breast cancer is the second leading cause of cancer death among women in the United States and is the leading cause of death among women aged 30 to 70 (1-3). The majority of breast carcinomas appear to be sporadic and have a complex accumulation of molecular and cellular abnormalities that constitute the malignant phenotype (4 -5). In many cases, random onset of breast cancer has been correlated with increased ErbB-2/neu receptor expression and Src tyrosine kinase activity (6 -12). Substantial evidence indicates that the c-Src proto-oncogene and ErbB-2/neu play important roles in breast cancer (7, 13). Src kinase activity is elevated in ErbB-2/neu (Neu) induced mammary tumors, and this elevated activity correlates with its capacity to physically associate with ErbB-2/neu (14 -15). A common pathway linking the activation mechanisms in ErbB-2/neu amplification in breast cancer is increased tyrosine kinase activity, which leads to cellular transformation (16).Four members of the ErbB (HER) family are presently known: p170ErbB-1 (epidermal growth factor receptor (EGF-R) 1 ), p185 ErbB-2 , p180 ErbB-3 , and p180 17,18). In particular, the overexpression of the p185ErbB-2 correlates with a poor clinical prognosis in breast cancer (9). ErbB-2/neu undergoes autophosphorylation on five tyrosine residues that are located on its non-catalytic C terminus (19,20). The autophosphorylated tyrosine re...

A genetic screen for modifiers of the lats tumor suppressor gene identifies C-terminal Src kinase as a regulator of cell proliferation in Drosophila

“…dCSK is more closely related to the human and mouse CSK proteins than any other SFKs ( Figure 2d). Furthermore, as a member of the CSK family, dCSK lacks an N-terminal myristylation site and the C-terminal tyrosine present in other SFKs Okada et al, 1991;Zrihan-Licht et al, 1998). Finally, dCSK specifically phosphorylates the C-terminal tyrosine residue of the Drosophila Src42 protein (Figure 6b).…”

“…There are two members of the CSK family in mammals, CSK and CHK (CSK homologous kinase), which are related to the Src family of tyrosine kinases (SFKs) (Brown and Cooper, 1996; Thomas and Brugge, 1997;Zrihan-Licht et al, 1998;Stamenovic and Coughlin, 1999). Database searches revealed that dCSK is the only CSK homolog in the Drosophila genome.…”

Functional Diversity of Csk, Chk, and Src SH2 Domains due to a SingleResidueVariation

“…In normal and cancerous breast cells, Csk is highly expressed and fully active (19). In contrast, Chk is not expressed in normal breast cells, but expression of Chk is induced in breast cancer cells (14). The increased expression level of Chk correlates to suppression of Src, although constitutively expressed Csk fails to suppress Src in these cells (19).…”

“…To determine whether the similarity in the Chk and Src SH2 domains in binding to Flu-GpYEEI would extend to a physiological phosphopeptide, we synthesized and tested a fluoresceinated phosphopeptide mimicking pTyr 1248 of ErbB2, Flu-NEPEpYLGLDV (FlupY1248) (14,19). This phosphorylation site is responsible for ErbB2 binding to the Chk SH2 domain (14).…”

“…Csk SH2 domain-binding proteins include the Csk-binding protein (CBP) (5), protein tyrosine phosphatase-HSCF (6), ␤-dystroglycan (7), an SHP2-interacting transmembrane adaptor protein (8), insulin-like growth factor-1 receptor (9), T-cell receptor and ⑀ (10), and GTPase-activating protien complex (11,12). Chk SH2 domain-binding proteins include ErbB2 (13,14), c-Kit receptor tyrosine kinase (15), paxillin (16), TrkA receptor (17), and RAFTK/Pyk2 (18). This comparison raises the possibility that the Csk and Chk SH2 domains may have different binding preferences.…”