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...

The E1 protein of bovine papillomavirus type 1 (BPV-1) is a phosphoprotein which specifically binds and unwinds the virus replication origin by ATPdependent helicase activity. The E1 protein has been shown to be multiply phosphorylated in vivo, although the sites of modification are incompletely mapped. Examination of the predicted amino acid sequence of all available E1 proteins revealed

The E1 protein of bovine papillomavirus type 1 (BPV-1) is the origin recognition protein and is essential for the initiation of viral DNA replication. We reported previously that there is a conserved motif between residues 25 and 60 of all papillomavirus E1 proteins that resembles a casein kinase II (CKII) phosphorylation site. The corresponding serine in BPV-1, serine-48, is an efficient substrate for CKII in vitro. To examine the functional role of this potential phosphorylation site, three amino acid substitutions were constructed at serine-48. Conversion of serine-48 to a glycine (S48G) resulted in a BPV-1 genome that was unable to replicate and had reduced transformation capacity. The S48G E1 protein also failed to support replication of a BPV-1 origin-containing plasmid when expressed from a heterologous vector rather than the viral genome, indicating a direct replication defect. In contrast, conversion of serine-48 to acidic residues (S48D or S48E), which mimic the charge and structure of phosphoserine, maintained the wild-type replication phenotype. These mutational results are consistent with a replication requirement for a negative charge at serine-48, presumably supplied by in vivo phosphorylation. The mechanistic basis for the negative charge requirement was examined by testing several activities of the S48G mutant E1 protein in vivo using yeast one-and two-hybrid systems. No gross defect was observed for stability, origin binding or interaction with E2 or for E1-E1 interaction, although subtle defects in these activities would not likely be detected. Overall, the results suggest that important phosphoregulatory control of E1 replication function is mediated through the N-terminal region of this protein.

A functional replication origin was reconstituted using oligonucleotide cassettes corresponding to three sequence subelements within the Bovine Papillomavirus Type 1 (BPV-1) replication origin: the 23-bp AT-rich region (ATR), the 18-bp binding site for the viral replication initiator protein E1 (E1BS), and a binding site for the viral transcriptional transactivator and replication enhancer protein E2 (E2BS). Replication of the reconstituted origin depended on heterologous expression of both the E1 and E2 proteins and on the presence of both the E1BS and E2BS, indicating that it is functionally analogous to the authentic BPV-1 origin. In addition, pairwise testing of subelement combinations revealed that the ATR was also essential and that a functional origin required at least one copy of all three subelements. While the E1BS and E2BS are sequence-specific elements, the function of the BPV-1 ATR could be at least partially substituted with heterologous AT-rich sequences, suggesting that the role of this element is primarily AT content-dependent rather than sequence-dependent. A stringent requirement for the ATR was also observed in the context of an authentic minimal origin sequence confirming that it is an intrinsic property of the BPV-1 origin and not simply an artifact of the reconstitution system. This study indicates that the minimal functional BPV-1 origin shares the tripartite modular organization characteristic of other simple eukaryotic replication origins. The reconstitution system described now provides a convenient approach to define the physical and functional interrelationships between the three subelements in a systematic fashion.