Department of Microbiology, Biochemistry and Molecular Genetics, Cancer Center, Rutgers New Jersey Medical School, Newark, New Jersey.
Protein Quality Control and Diseases Laboratory, Cancer Center, School of Laboratory Medicine and Life Sciences, Wenzhou Medical University, Wenzhou, Zhejiang, China.
Mol Cancer Res. 2018 Jan;16(1):173-183. doi: 10.1158/1541-7786.MCR-17-0242. Epub 2017 Oct 3.
The activity of Src family kinases (Src being the prototypical member) is tightly regulated by differential phosphorylation on Tyr416 (positive) and Tyr527 (negative), a duet that reciprocally regulates kinase activity. The latter negative regulation of Src on Tyr527 is mediated by C-terminal Src kinase (CSK) that phosphorylates Tyr527 and maintains Src in a clamped negative regulated state by promoting an intramolecular association. Here it is demonstrated that the SH2- and SH3-domain containing adaptor protein CrkII, by virtue of its phosphorylation on Tyr239, regulates the Csk/Src signaling axis to control Src activation. Once phosphorylated, the motif (PIpYARVIQ) forms a consensus sequence for the SH2 domain of CSK to form a pTyr239-CSK complex. Functionally, when expressed in Crk MEFs or in Crk HS683 cells, Crk Y239F delayed PDGF-BB-inducible Src Tyr416 phosphorylation. Moreover, expression of Crk Y239F in HS683 cells delayed Src kinase activation and suppressed the cell-invasive and -transforming phenotypes. Finally, through loss-of-function and epistasis experiments using CRISPR-Cas9-engineered 4T1 murine breast cancer cells, Crk Tyr239 is implicated in breast cancer tumor growth and metastasis in orthotopic immunocompetent 4T1 mice model of breast adenocarcinoma. These findings delineate a novel role for Crk Tyr239 phosphorylation in the regulation of Src kinases, as well as a potential molecular explanation for a long-standing question as to how Crk regulates the activation of Src kinases. These findings provide new perspectives on the versatility of Crk in cancer by demonstrating how Crk mechanistically drives, through a tyrosine phosphorylation-dependent manner, tumor growth, and metastasis. .
Src 家族激酶（Src 是典型成员）的活性受到 Tyr416（正）和 Tyr527（负）的差异磷酸化的严格调节，这种双重调节相互调节激酶活性。Src 对 Tyr527 的负调节是由 C 端Src 激酶（CSK）介导的，CSK 磷酸化 Tyr527 并通过促进分子内缔合将 Src 维持在夹闭的负调节状态。在这里，证明含有 SH2 和 SH3 结构域的衔接蛋白 CrkII 通过其 Tyr239 的磷酸化来调节 Csk/Src 信号轴以控制 Src 激活。一旦磷酸化，该基序（PIpYARVIQ）就形成 CSK 的 SH2 结构域的一个共识序列，形成 pTyr239-CSK 复合物。在功能上，当在 Crk MEFs 或 Crk HS683 细胞中表达时，Crk Y239F 延迟了 PDGF-BB 诱导的 Src Tyr416 磷酸化。此外，Crk Y239F 在 HS683 细胞中的表达延迟了 Src 激酶的激活，并抑制了细胞侵袭和转化表型。最后，通过使用 CRISPR-Cas9 工程化的 4T1 小鼠乳腺癌细胞进行的功能丧失和上位性实验，Crk Tyr239 参与了乳腺癌肿瘤生长和在乳腺癌腺癌的同源免疫活性 4T1 小鼠模型中的转移。这些发现描绘了 Crk Tyr239 磷酸化在 Src 激酶调节中的新作用，以及对长期以来关于 Crk 如何调节 Src 激酶激活的问题的潜在分子解释。这些发现通过证明 Crk 如何通过一种依赖于酪氨酸磷酸化的方式在机制上驱动肿瘤生长和转移，为 Crk 在癌症中的多功能性提供了新的视角。
