Sidhanth C, Bindhya S, Krishnapriya S, Manasa P, Shabna A, Alifia J, Patole C, Kumar V, Garg M, Ganesan T S
Laboratory for Cancer Biology, Departments of Medical Oncology and Clinical Research, Cancer Institute (WIA), Chennai, India.
Mass Spectrometry Facility Proteomics, National Centre for Biological Sciences (NCBS), Bangalore, India.
Biochim Biophys Acta Proteins Proteom. 2022 Apr 1;1870(4):140768. doi: 10.1016/j.bbapap.2022.140768. Epub 2022 Feb 11.
The gene for receptor tyrosine kinase ErbB2 is amplified in breast and ovarian tumours. The linear pathway by which signals are transduced through ErbB2 are well known. However, second generation questions that address spatial aspects of signaling remain. To address this, we have undertaken a mass spectrometry approach to identify phosphoproteins specific for ErbB2 using the inhibitors Lapatinib and CP724714 in ovarian cancer cells. The ErbB2 specific proteins identified in SKOV-3 cells were Myristoylated alanine-rich C-kinase substrate, Protein capicua homolog, Protein peptidyl isomerase G, Protein PRRC2C, Chromobox homolog1 and PRP4 homolog. We have evaluated three phosphoproteins PKM2, Aldose reductase and MARCKS in SKOV-3 cells. We observed that PKM2 was phosphorylated by EGF but was not inhibited by Lapatinib and CP724714. The activity of aldose reductase in reducing NADPH as a substrate was significantly higher in EGF stimulated cells which was inhibited by Lapatinib and CP724714 but not by Geftinib (EGFR inhibitor). MARCKS was phosphorylated on stimulation of SKOV-3 cells with EGF that was inhibited by Lapatinib and CP724714 which was dependent on the kinase activity of ErbB2. These results have identified phosphoproteins that are specific to ErbB2 which have not been previously reported and sets the basis for future experiments.
受体酪氨酸激酶ErbB2的基因在乳腺癌和卵巢肿瘤中会发生扩增。通过ErbB2转导信号的线性通路已为人熟知。然而，关于信号传导空间方面的第二代问题依然存在。为了解决这一问题，我们采用了一种质谱方法，在卵巢癌细胞中使用拉帕替尼和CP724714抑制剂来鉴定ErbB2特异性磷酸化蛋白。在SKOV-3细胞中鉴定出的ErbB2特异性蛋白有肉豆蔻酰化富含丙氨酸的C激酶底物、果蝇脑肿瘤抑制蛋白同源物、肽基脯氨酰顺反异构酶G、PRRC2C蛋白、染色体盒同源物1和PRP4同源物。我们在SKOV-3细胞中评估了三种磷酸化蛋白PKM2、醛糖还原酶和MARCKS。我们观察到PKM2被表皮生长因子（EGF）磷酸化，但不受拉帕替尼和CP724714抑制。在以NADPH为底物时，EGF刺激的细胞中醛糖还原酶的活性显著更高，该活性受到拉帕替尼和CP724714抑制，但不受吉非替尼（表皮生长因子受体抑制剂）抑制。在用EGF刺激SKOV-3细胞时，MARCKS发生磷酸化且受到拉帕替尼和CP724714抑制，这依赖于ErbB2的激酶活性。这些结果鉴定出了此前未报道过的ErbB2特异性磷酸化蛋白，并为未来的实验奠定了基础。
