Abstract 3460: Regulatory roles of conserved phosphorylation sites in the activation loop of the MAP kinase
Authors: Lai Shenshen , Pelech Steven 
Published: May 2015

Abstract
The catalytic domains of most eukaryotic protein kinases are highly conserved in their primary structures. Phosphorylation of the activation loop, a variable region between kinase catalytic Subdomains VII and VIII, is a common mechanism for stimulation of protein phosphotransferase activity. Our investigation of the distribution and evolutionary conservation of phosphorylation sites in 492 typical human protein kinase catalytic domains revealed that about 75% of 303 known activating phosphosites are located within the activation loop. Extracellular signal-regulated kinase 1 (ERK1), which belongs to the highly 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 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. T207 and Y210 correspond to conserved phosphosites located just prior to the Subdomain VIII APE motif in many other protein-serine/threonine kinases including CDKs, CAMKs, PKB/Akts and PKCs, which may implicate a common regulatory role for of these phosphosites in a wide range of kinases. To investigate the possible roles of these obscure phosphosites in many protein kinases, we mutated T198, T207 and Y210 of ERK1 individually and in combinations. In vitro kinase assays with myelin basic protein (MBP) using the purified ERK1 phosphosite mutants indicated the functional importance of T207 and Y210, but not T198 in regulating ERK1 catalytic activity. By single substitution of the T207 to alanine, the activity of ERK1 towards MBP decreased dramatically without affecting the phosphorylation of the ERK1 at the T202 and Y204 sites by MEK1. However, the T207E mutant could not mimic phosphorylation of this site and also produced a less active form of ERK1. The Y210 site could be important for proper folding of ERK1 in this regulatory region, since the mutation of this residue caused decreases in protein solubility, and the Y210F mutant was not recognized by MEK1 for phosphorylation in vitro. Preliminary data indicated that ERK1 autophosphorylates at T207 phosphosite, after the phosphorylation of the T202 and Y204 sites by MEK1. We hypothesize that phosphorylation of the T207 site is required for full activation of the ERK1, and that subsequent slower phosphorylation of the Y210 sites may result in autoinactivation of the kinase. Hyper-phosphorylation within the kinase activation loop may serve as a general mechanism for protein kinases to achieve transient full stimulation by autophosphorylation following the initial activation by other upstream kinases, which is then reversed by inhibitory tyrosine autophosphorylation just prior to Subdomain VIII.
Citation Format: Shenshen Lai, Steven Pelech. Regulatory roles of conserved phosphorylation sites in the activation loop of the MAP kinase. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3460. doi:10.1158/1538-7445.AM2014-3460