The specificity of extracellular signal-regulated kinase 2 dephosphorylation by protein phosphatases.
The extracellular signal-regulated protein kinase 2 (ERK2) is the founding member of a family of mitogen-activated protein kinases (MAPKs) that are central components of signal transduction pathways for cell proliferation, stress responses, and differentiation. The MAPKs are unique among the Ser/Thr protein kinases in that they require both Thr and Tyr phosphorylation for full activation. The dual phosphorylation of Thr-183 and Tyr-185 in ERK2 is catalyzed by MAPK/ERK kinase 1 (MEK1). However, the identity and relative activity of protein phosphatases that inactivate ERK2 are less well established. In this study, we performed a kinetic analysis of ERK2 dephosphorylation by protein phosphatases using a continuous spectrophotometric enzyme-coupled assay that measures the inorganic phosphate produced in the reaction. Eleven different protein phosphatases, many previously suggested to be involved in ERK2 regulation, were compared, including tyrosine-specific phosphatases (PTP1B, CD45, and HePTP), dual specificity MAPK phosphatases (VHR, MKP3, and MKP5), and Ser/Thr protein phosphatases (PP1, PP2A, PP2B, PP2C alpha, and lambda PP). The results provide biochemical evidence that protein phosphatases display exquisite specificity in their substrate recognition and implicate HePTP, MKP3, and PP2A as ERK2 phosphatases. The fact that ERK2 inactivation could be carried out by multiple specific phosphatases shows that signals can be integrated into the pathway at the phosphatase level to determine the cellular response to external stimuli. Important insights into the roles of various protein phosphatases in ERK2 kinase signaling are obtained, and further analysis of the mechanism by which different protein phosphatases recognize and inactivate MAPKs will increase our understanding of how this kinase family is regulated.
Zhou
Bo
B
Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
Wang
Zhi-Xin
ZX
Zhao
Yu
Y
Brautigan
David L
DL
Zhang
Zhong-Yin
ZY
D005982:Glutathione Transferase / Q000235:genetics; D007700:Kinetics; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism*; D010749:Phosphoprotein Phosphatases / Q000145:classification / Q000378:metabolism*; D010766:Phosphorylation; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013379:Substrate Specificity
D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase; D019950:Mitogen-Activated Protein Kinase 1; D010749:Phosphoprotein Phosphatases