A one-step method to identify MAP kinase residues involved in inactivation by tyrosine- and dual-specificity protein phosphatases.
MAP kinases (MAPKs) are enzymes directly involved in the control of cellular homeostasis in response to external cues, from differentiation and developmental processes to cell transformation. The activation status of MAPKs, both in magnitude and in duration, reflects the balance of phosphorylation at their Thr and Tyr regulatory residues by specific MAPK kinases and their dephosphorylation by inactivating protein serine/threonine phosphatases (PPs) and protein tyrosine phosphatases (PTPs). The dephosphorylation of MAPKs by PTPs relies on molecular docking between the two enzymes at specific interaction sites. Here we outline a one-step method to identify ERK1/2 and p38alpha mutations that prevent binding and inactivation by PTPs (tyrosine- or dual-specificity phosphatases) based on the use of anti-pTyr antibodies and cell lysis buffers lacking or containing the broad PTP inhibitor sodium orthovanadate (Na3VO4).
Tárrega
Céline
C
Centro de Investigacion Principe Felipe, Molecular Biology of Cancer, Avda. Autopista del Saler 16-3, 46013 Valencia, Spain.
Pulido
Rafael
R
D000818:Animals; D002460:Cell Line; D054637:Dual-Specificity Phosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D051379:Mice; D019950:Mitogen-Activated Protein Kinase 1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D048308:Mitogen-Activated Protein Kinase 14 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D010766:Phosphorylation; D017027:Protein Tyrosine Phosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013379:Substrate Specificity; D014638:Vanadates / Q000494:pharmacology
D004791:Enzyme Inhibitors; D014638:Vanadates; D019950:Mitogen-Activated Protein Kinase 1; D048308:Mitogen-Activated Protein Kinase 14; D054637:Dual-Specificity Phosphatases; D017027:Protein Tyrosine Phosphatases