Abstract
Extracellular signal-regulated kinases (ERKs) activity is regulated by MAPK/ERK kinases (MEKs), which phosphorylate the regulatory Tyr and Thr residues in ERKs activation loop, and by various phosphatases that remove the incorporated phosphates. Although the role of the phosphorylated residues in the activation loop of ERKs is well studied, much less is known about the role of other residues within this loop. Here we substituted several residues within amino acids 173-177 of ERK2 and studied their role in ERK2 phosphorylation, substrate recognition, and subcellular localization. We found that substitution of residues 173-175 and particularly Pro(174) to alanines reduces the EGF-induced ERK2 phosphorylation, without modifying its in vitro phosphorylation by MEK1. Examining the ability of these mutants to be dephosphorylated revealed that 173-5A mutants are hypersensitive to phosphatases, indicating that these residues are important for setting the phosphorylation/dephosphorylation balance of ERKs. In addition, 173-5A mutants reduced ERK2 activity toward Elk-1, without affecting the activity of ERK2 toward MBP, while substitution of residues 176-8 decreased ERK2 activity toward both substrates. Substitution of Asp(177) to alanine increased nuclear localization of the construct in MEK1-overexpressing cells, suggesting that this residue together with His(176) is involved in the dissociation of ERK2 from MEKs. Combining CRS/CD motif and the activation loop mutations revealed that these two regions cooperate in determining the net phosphorylation of ERK2, but the role of the CRS/CD motif predominates that of the activation loop residues. Thus, we show here that residues 173-177 of ERK2 join other regulatory regions of ERKs in governing ERK activity.
Highlights
Humanities from the European Community’s Sixth Framework Program Project IST-2004-027265-SIMAP
In a previous study [32], we found that several activation loop residues besides Thr183 and Tyr185 participate in Extracellular signal-regulated kinases (ERKs) regulation
In a previous study we found that residue 176 plays a role in the release of ERK2 from its interaction with MAPK/ERK kinases (MEKs)
Summary
Humanities from the European Community’s Sixth Framework Program Project IST-2004-027265-SIMAP. Residues 173–175 of ERK2 Regulate Its Thr and Tyr Phosphorylation—To test the effect of the activation loop residues of ERK2 on its phosphorylation by MEKs, the constructs were co-transfected with HA-MEK1 into COS7 cells, which were serum-starved and stimulated with EGF.
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