The Specificity of Extracellular Signal-regulated Kinase 2 Dephosphorylation by Protein Phosphatases

Abstract

The extracellular signal-regulated protein kinase 2 (ERK2) is the founding member of a family of mitogenactivated 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 enzymecoupled 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 tyrosinespecific phosphatases (PTP1B, CD45, and HePTP), dual specificity MAPK phosphatases (VHR, MKP3, and MKP5), and Ser/Thr protein phosphatases (PP1, PP2A, PP2B, PP2C␣, and 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.

Electroconvulsive Seizures Increase the Expression of MAP Kinase Phosphatases in Limbic Regions of Rat Brain

“…In addition, phosphorylation of tyrosine residues on ERK inhibits the dephosphorylation of threonine residues by phosphatases such as PP2A. This indicates that MKP3, or other tyrosine phosphatases such as STEP, must dephosphorylate ERK before PP2A can influence ERK (Zhou et al, 2002). This further highlights the potential significance of the induction of MKP3 protein as well as mRNA expression in both the prefrontal cortex and hippocampus.…”

“…This possibility is supported by the results of the Western blot studies that demonstrate that all three MKPs can be detected in the sham-treated animals. The presence of MKP3 is particularly notable because this subtype has been reported to be one of the key regulators of ERK dephosphorylation (Zhou et al, 2002). This is due in part to the presence of an ERK kinase interaction domain in MKP3.…”

LB100, a small molecule inhibitor of PP2A with potent chemo- and radio-sensitizing potential

“…46,47 As such, overexpression of CIP2A has been demonstrated in numerous human malignancies, consistent with a role for PP2A as a tumor suppressor. 41,48,49 …”

“…39 Likewise, PP2A regulates the major signaling pathway, Ras/Raf/ MEK/ERK, by directly associating with ERK2 and MEK1 and indirectly with Ras and Raf. [40][41][42][43] Constitutive activation of the Ras/Raf/MEK/ERK pathway has been well characterized in malignant transformation of susceptible cells. 44 PP2A also targets c-Myc for proteasomal degradation by dephosphorylation and constitutive c-Myc expression is regarded as a significant event in oncogenic transformation.…”

Glucose lowers the threshold for human aortic vascular smooth muscle cell migration: inhibition by protein phosphatase-2A

“…At high PDGF-BB concentrations, both PP2A (a known negative regulator of ERK [34]) inhibition and PTP1B inhibition increased ERK activity and phosphorylation respectively in NG and HG. PTP1B demonstrated a small glucose-independent but PDGF-BB concentrationdependent response.…”