Department of Biochemistry, College of Life Science and Biotechnology, Yonsei University, Seoul 120-749, Korea.
J Biol Chem. 2010 Aug 20;285(34):26013-21. doi: 10.1074/jbc.M109.088971. Epub 2010 Jun 16.
PTK6 (also known as Brk) is a non-receptor-tyrosine kinase containing SH3, SH2, and catalytic domains, that is expressed in more than 60% of breast carcinomas but not in normal mammary tissues. To analyze PTK6-interacting proteins, we have expressed Flag-tagged PTK6 in HEK293 cells and performed co-immunoprecipitation assays with Flag antibody-conjugated agarose. A 164-kDa protein in the precipitated fraction was identified as ARAP1 (also known as centaurin delta-2) by MALDI-TOF mass analysis. ARAP1 associated with PTK6 in an EGF/EGF receptor (EGFR)-dependent manner. In addition, the SH2 domain of PTK6, particularly the Arg(105) residue that contacts the phosphate group of the tyrosine residue, was essential for the association. Moreover, PTK6 phosphorylated residue Tyr(231) in the N-terminal domain of ARAP1. Expression of ARAP1, but not of the Y231F mutant, inhibited the down-regulation of EGFR in HEK293 cells expressing PTK6. Silencing of endogenous PTK6 expression in breast carcinoma cells decreased EGFR levels. These results demonstrate that PTK6 enhances EGFR signaling by inhibition of EGFR down-regulation through phosphorylation of ARAP1 in breast cancer cells.
PTK6（也称为 Brk）是一种非受体酪氨酸激酶，含有 SH3、SH2 和催化结构域，在超过 60%的乳腺癌中表达，但在正常乳腺组织中不表达。为了分析 PTK6 相互作用的蛋白质，我们在 HEK293 细胞中表达了 Flag 标记的 PTK6，并使用 Flag 抗体缀合的琼脂糖进行了共免疫沉淀测定。沉淀部分中的 164 kDa 蛋白通过 MALDI-TOF 质量分析被鉴定为 ARAP1（也称为半人马座 delta-2）。ARAP1 以 EGF/EGF 受体（EGFR）依赖性方式与 PTK6 相关联。此外，PTK6 的 SH2 结构域，特别是与酪氨酸残基的磷酸基团接触的 Arg（105）残基，对于这种关联是必不可少的。此外，PTK6 磷酸化了 ARAP1 N 端结构域中的 Tyr（231）残基。ARAP1 的表达，但不是 Y231F 突变体的表达，抑制了表达 PTK6 的 HEK293 细胞中 EGFR 的下调。乳腺癌细胞中内源性 PTK6 表达的沉默降低了 EGFR 水平。这些结果表明，PTK6 通过 ARAP1 的磷酸化抑制 EGFR 的下调，从而增强乳腺癌细胞中的 EGFR 信号。
