Role of Non-phosphorylated Activation Loop Residues in Determining ERK2 Dephosphorylation, Activity, and Subcellular Localization

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 EGFinduced 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.

Distinct Molecular Regulation of Glycogen Synthase Kinase-3α Isozyme Controlled by Its N-terminal Region

“…Phosphorylation of protein kinases is known to regulate their cellular distribution and their import/export from the nucleus (45)(46)(47)(48). Phosphorylation of GSK-3␣ at serine 21, located within its N-terminal region, inhibits its kinase activity via its ability to function as a pseudosubstrate (49,50).…”

SLK-mediated phosphorylation of paxillin is required for focal adhesion turnover and cell migration

“…Phosphorylation is a reversible and therefore, economical process for the cell: the dynamic interconversion between differentially phosphorylated and dephosphorylated forms of the same protein has the potential to increase the number of possible signaling outcomes without increasing the number of proteins required [58,59]. For example, phosphorylation of the MAPKs Erk1/2 by MEK results in signaling conducive to cell proliferation, survival and adhesion, whereas their dephosphorylation by MAPK phosphatases (MKPs) like MKP-1, has been shown to induce apoptosis [57].…”

The ERK Cascade: Distinct Functions within Various Subcellular Organelles

“…Importantly, other residues in close proximity to the activation loop of ERK1/2 are phosphorylated as well 67 and thereby may affect this important region of ERK1/2. 68 Notably, these Thr-Glu-Tyr-independent phosphorylations are usually not important merely for the activation mechanisms of ERK1/2 but rather play a role in the regulation of ERK1/2 localization or downregulation and thereby in the determination of their signaling specificity, as described below.…”