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Characterization of MEK1 phosphorylation by the v-Mos protein
C D Pham1, R B Arlinghaus, C F Zheng
1Department of Molecular Pathology, University of Texas M.D. Anderson Cancer Center, Houston 77030, USA.
Abstract:
Activation of MAP kinase/Erk Kinase (MEK) via direct phosphorylation by Mos may be crucial for cellular transformation by the activated c-mos or v-mos gene. Recent studies on a number of different protein kinases showed that phosphorylation within a subdomain of the catalytic domain may represent a common mode of activation. In this regard, activation of MEK1 by Raf involves phosphorylation of serine residues 218 and 222. Here we show that recombinant kinase-inactive MEK1 is phosphorylated by v-Mos with equal efficiency at both Ser 218 and Ser 222 in vitro. Tryptic phosphopeptide analysis of glutathione-S-transferase (GST)-MEK1 K97R and its alanine-for-serine mutants indicated that Ser 222 is the preferred phosphorylation site. Wild-type GST-MEK1 was phosphorylated at the same sites but contained a significantly lower amount of doubly phosphorylated species then its K97R kinase-inactive mutant. The ratio of GST-MEK1 species phosphorylated at two serines to those phosphorylated at one serine was similar in auto-phosphorylated and v-Mos-phosphorylated GST-MEK1. Consistent with the in vitro data, phosphopeptide mapping of MEK1 immunoprecipitated from mos transformed cells showed an increased amount of singly phosphorylated phosphopeptide compared to nontransformed cels. MEK1 was found to be more highly activated in NIH3T3 cells transformed by an activated c-mos or v-mos gene than in cells growing normally in medium containing serum. Our data indicate that Mos activated MEK1 in vitro as well as in vivo by phosphorylating Ser 222.
Insights
Mos protein phosphorylates MEK1 at Serine 222, a key step for cellular transformation. This phosphorylation activates MEK1 (MAP kinase/Erk Kinase) in both lab experiments and living cells.
Area of Science:
- Molecular Biology
- Cell Signaling
- Oncogenesis
Background:
- Cellular transformation by c-mos or v-mos genes involves activation of MAP kinase/Erk Kinase (MEK).
- Protein kinase activation often occurs through phosphorylation within a catalytic domain subdomain.
- Raf-mediated MEK1 activation involves phosphorylation at Serine 218 and Serine 222.
Purpose of the Study:
- To investigate the specific phosphorylation sites and mechanism by which v-Mos activates MEK1.
- To determine if Serine 222 is a preferred phosphorylation site for v-Mos.
- To compare MEK1 activation in Mos-transformed cells versus normal cells.
Main Methods:
- In vitro phosphorylation assays using recombinant kinase-inactive MEK1 (GST-MEK1 K97R) and v-Mos.
- Tryptic phosphopeptide analysis of wild-type and mutant MEK1.
- Phosphopeptide mapping of MEK1 immunoprecipitated from Mos-transformed and non-transformed NIH3T3 cells.
Main Results:
- Recombinant kinase-inactive MEK1 was efficiently phosphorylated by v-Mos at both Ser 218 and Ser 222 in vitro.
- Serine 222 was identified as the preferred phosphorylation site for v-Mos.
- Mos-transformed cells exhibited higher MEK1 activation compared to normal cells, with an increased proportion of singly phosphorylated MEK1.
Conclusions:
- v-Mos directly phosphorylates MEK1, with Serine 222 being the primary site of activation.
- Mos-mediated MEK1 phosphorylation is a critical event in cellular transformation.
- MEK1 activation by Mos occurs both in vitro and in vivo.