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Regulatory roles of conserved phosphorylation sites in the activation T-loop of the MAP kinase ERK1.

ABSTRACT: The catalytic domains of most eukaryotic protein kinases are highly conserved in their primary structures. Their phosphorylation within the well-known activation T-loop, a variable region between protein kinase catalytic subdomains VII and VIII, is a common mechanism for stimulation of their phosphotransferase activities. Extracellular signal-regulated kinase 1 (ERK1), a member of the extensively studied mitogen-activated protein kinase (MAPK) family, serves as a paradigm for regulation of protein kinases in signaling modules. In addition to the well-documented T202 and Y204 stimulatory phosphorylation sites in the activation T-loop of ERK1 and its closest relative, ERK2, three additional flanking phosphosites have been confirmed (T198, T207, and Y210 from ERK1) by high-throughput mass spectrometry. In vitro kinase assays revealed the functional importance of T207 and Y210, but not T198, in negatively regulating ERK1 catalytic activity. The Y210 site could be important for proper conformational arrangement of the active site, and a Y210F mutant could not be recognized by MEK1 for phosphorylation of T202 and Y204 in vitro. Autophosphorylation of T207 reduces the catalytic activity and stability of activated ERK1. We propose that after the activation of ERK1 by MEK1, subsequent slower phosphorylation of the flanking sites results in inhibition of the kinase. Because the T207 and Y210 phosphosites of ERK1 are highly conserved within the eukaryotic protein kinase family, hyperphosphorylation within the kinase activation T-loop may serve as a general mechanism for protein kinase down-regulation after initial activation by their upstream kinases.

SUBMITTER: Lai S

PROVIDER: S-EPMC4791125 | biostudies-literature | 2016 Mar

REPOSITORIES: biostudies-literature

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Regulatory roles of conserved phosphorylation sites in the activation T-loop of the MAP kinase ERK1.

Lai Shenshen S Pelech Steven S

Molecular biology of the cell 20160128 6

The catalytic domains of most eukaryotic protein kinases are highly conserved in their primary structures. Their phosphorylation within the well-known activation T-loop, a variable region between protein kinase catalytic subdomains VII and VIII, is a common mechanism for stimulation of their phosphotransferase activities. Extracellular signal-regulated kinase 1 (ERK1), a member of the extensively studied mitogen-activated protein kinase (MAPK) family, serves as a paradigm for regulation of prote ...[more]

PMID: 26823016

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Project description:In the present study, we determined the cellular regulators of ERK1/2 and Akt signaling pathways in response to human CRF1 receptor (CRF1R) activation in transfected COS-7 cells. We found that Pertussis Toxin (PTX) treatment or sequestering G?? reduced CRF1R-mediated activation of ERK1/2, suggesting the involvement of a Gi-linked cascade. Neither Gs/PKA nor Gq/PKC were associated with ERK1/2 activation. Besides, CRF induced EGF receptor (EGFR) phosphorylation at Tyr1068, and selective inhibition of EGFR kinase activity by AG1478 strongly inhibited the CRF1R-mediated phosphorylation of ERK1/2, indicating the participation of EGFR transactivation. Furthermore, CRF-induced ERK1/2 phosphorylation was not altered by pretreatment with batimastat, GM6001, or an HB-EGF antibody indicating that metalloproteinase processing of HB-EGF ligands is not required for the CRF-mediated EGFR transactivation. We also observed that CRF induced Src and PYK2 phosphorylation in a G??-dependent manner. Additionally, using the specific Src kinase inhibitor PP2 and the dominant-negative-SrcYF-KM, it was revealed that CRF-stimulated ERK1/2 phosphorylation depends on Src activation. PP2 also blocked the effect of CRF on Src and EGFR (Tyr845) phosphorylation, further demonstrating the centrality of Src. We identified the formation of a protein complex consisting of CRF1R, Src, and EGFR facilitates EGFR transactivation and CRF1R-mediated signaling. CRF stimulated Akt phosphorylation, which was dependent on Gi/?? subunits, and Src activation, however, was only slightly dependent on EGFR transactivation. Moreover, PI3K inhibitors were able to inhibit not only the CRF-induced phosphorylation of Akt, as expected, but also ERK1/2 activation by CRF suggesting a PI3K dependency in the CRF1R ERK signaling. Finally, CRF-stimulated ERK1/2 activation was similar in the wild-type CRF1R and the phosphorylation-deficient CRF1R-?386 mutant, which has impaired agonist-dependent ?-arrestin-2 recruitment; however, this situation may have resulted from the low ?-arrestin expression in the COS-7 cells. When ?-arrestin-2 was overexpressed in COS-7 cells, CRF-stimulated ERK1/2 phosphorylation was markedly upregulated. These findings indicate that on the base of a constitutive CRF1R/EGFR interaction, the Gi/?? subunits upstream activation of Src, PYK2, PI3K, and transactivation of the EGFR are required for CRF1R signaling via the ERK1/2-MAP kinase pathway. In contrast, Akt activation via CRF1R is mediated by the Src/PI3K pathway with little contribution of EGFR transactivation.

Dataset Information

Regulatory roles of conserved phosphorylation sites in the activation T-loop of the MAP kinase ERK1.

Publications

Regulatory roles of conserved phosphorylation sites in the activation T-loop of the MAP kinase ERK1.

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