Selective activation of SHP2 activity by cisplatin revealed by a novel chemical probe-based assay.
Src homology-2 (SH2) domain-containing phosphatase 2 (SHP2) is known to participate in several different signaling pathways to mediate cell growth, survival, migration, and differentiation. However, due to the lack of proper analytical tools, it is unclear whether the phosphatase activity of SHP2 is activated in most studies. We have previously developed an activity-based probe LCL2 that formed covalent linkage with catalytically active protein tyrosine phosphatases (PTPs). Here, by combining LCL2 with a SHP2 specific antibody, we established an assay system that enables the direct monitoring of SHP2 activity upon cisplatin treatment of cancer cells. The protocol is advantageous over conventional colorimetric or in-gel PTP assays as it is specific and does not require the use of radioisotope reagents. Using this assay, we found SHP2 activity was selectively activated by cisplatin. Moreover, the activation of SHP2 appeared to be specific for cisplatin as other DNA damage agents failed to activate the activity. Although the role of SHP2 activation by cisplatin treatments is still unclear to us, our results provide the first direct evidence for the activation of SHP2 during cisplatin treatments. More importantly, the concept of using activity-based probe in conjunction with target-specific antibodies could be extended to other enzyme classes.
Kuo
Chun-Chen
CC
Institute of Biochemistry and Molecular Biology, National Yang-Ming University, Taipei 112, Taiwan.
Chu
Chi-Yuan
CY
Lin
Jing-Jer
JJ
Lo
Lee-Chiang
LC
D000906:Antibodies / Q000276:immunology; D000970:Antineoplastic Agents / Q000494:pharmacology*; D001681:Biological Assay; D001710:Biotin / Q000031:analogs & derivatives* / Q000737:chemistry; D045744:Cell Line, Tumor; D002945:Cisplatin / Q000494:pharmacology*; D004789:Enzyme Activation; D006801:Humans; D015335:Molecular Probes / Q000737:chemistry*; D009369:Neoplasms / Q000201:enzymology*; D010755:Organophosphates / Q000737:chemistry*; D010766:Phosphorylation; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11 / Q000096:biosynthesis* / Q000737:chemistry / Q000276:immunology; D017027:Protein Tyrosine Phosphatases / Q000378:metabolism
D000906:Antibodies; D000970:Antineoplastic Agents; D015335:Molecular Probes; D010755:Organophosphates; D001710:Biotin; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11; D017027:Protein Tyrosine Phosphatases; D002945:Cisplatin