Monophosphothreonyl extracellular signal-regulated kinases 1 and 2 (ERK1/2) are formed endogenously in intact cardiac myocytes and are enzymically active

Institute for Cardiovascular and Metabolic Research, School of Biological Sciences, University of Reading, Whiteknights, Reading RG6 6UB, UK.
Cellular Signalling (Impact Factor: 4.32). 10/2010; 23(2):468-77. DOI: 10.1016/j.cellsig.2010.10.024
Source: PubMed


ERK1 and ERK2 (ERK1/2) are central to the regulation of cell division, growth and survival. They are activated by phosphorylation of the Thr- and the Tyr- residues in their Thr-Glu-Tyr activation loops. The dogma is that dually-phosphorylated ERK1/2 constitute the principal activities in intact cells. We previously showed that, in neonatal rat cardiac myocytes, endothelin-1 and phorbol 12-myristate 13-acetate (PMA) powerfully and rapidly (maximal at ~5 min) activate ERK1/2. Here, we show that dually-phosphorylated ERK1/2 rapidly (< 2 min) appear in the nucleus following stimulation with endothelin-1. We characterized the active ERK1/2 species in myocytes exposed to endothelin-1 or PMA using MonoQ FPLC. Unexpectedly, two peaks of ERK1 and two peaks of ERK2 activity were resolved using in vitro kinase assays. One of each of these represented the dually-phosphorylated species. The other two represented activities for ERK1 or ERK2 which were phosphorylated solely on the Thr- residue. Monophosphothreonyl ERK1/2 represented maximally ~30% of total ERK1/2 activity after stimulation with endothelin-1 or PMA, and their k(cat) values were estimated to be minimally ~30% of the dually-phosphorylated species. Appearance of monophosphothreonyl ERK1/2 was rapid but delayed in comparison with dually-phosphorylated ERK1/2. Of 10 agonists studied, endothelin-1 and PMA were most effective in terms of ERK1/2 activation and in stimulating the appearance of monophosphothreonyl and dually-phosphorylated ERK1/2. Thus, enzymically active monophosphothreonyl ERK1/2 are formed endogenously following activation of the ERK1/2 cascade and we suggest that monophosphothreonyl ERK1/2 arise by protein tyrosine phosphatase-mediated dephosphorylation of dually-phosphorylated ERK1/2.

Download full-text


Available from: Angela Clerk,
  • Source
    • "e l s e v i e r . c o m / l o c a t e / c e l l s i g dephosphorylation by specific MAPK tyrosine phosphatases (PTP) [16]. All these precedents suggest that phosphorylation of the two phosphoacceptors of MAPKs are not equally important, and that threonine phosphorylation would confer higher catalytical and biological activity than tyrosine phosphorylation. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Mitogen-activated protein kinases (MAPKs) define a specific group of eukaryotic protein kinases which regulate a number of cellular functions by transducing extracellular signals to intracellular responses. Unlike other protein kinases, catalytic activation of MAPKs by MAPKKs depends on dual phosphorylation at two tyrosine and threonine residues within the conserved TXY motif, and this has been proposed to occur in an ordered fashion, where the initial phosphorylation on tyrosine is followed by phosphorylation at the threonine residue. However, monophosphorylated MAPKs also exist in vivo, and although threonine phosphorylated isoforms retain some catalytic activity, their functional significance remains to be further elucidated. In the fission yeast Schizosaccharomyces pombe MAPKs Sty1 and Pmk1 control multiple aspects of fission yeast life cycle, including morphogenesis, cell cycle, and cellular response to a variety of stressful situations. In this work we show that a trapping mechanism increases MAPKK binding and tyrosine phosphorylation of both Sty1 and Pmk1 when subsequent phosphorylation at threonine is hampered, indicating that a sequential and likely processive mechanism might be responsible for MAPK activation in this simple organism. Whereas threonine-monophosphorylated Sty1 showed a limited biological activity particularly at the transcriptional level, threonine-monophosphorylated Pmk1 was able to execute most of the biological functions of the dually phosphorylated kinase. Thus, threonine monophosphorylated MAPKs might display distinct functional relevance among eukaryotes.
    Cellular Signalling 10/2015; 27(12). DOI:10.1016/j.cellsig.2015.09.017 · 4.32 Impact Factor
  • Source
    • "Typically, in MEK-mediated phosphorylation of Erk1/2, the phosphorylation of the activation loop tyrosine precedes the phosphorylation of the activation loop threonine (Haystead et al., 1992). However, threonine mono-phosphorylated Erk1/2 was also has been detected in cells (Sugden et al., 2011). To further locate the phosphorylation site, recombinant Erk1 mutants whose activation loop residues were mutated (T202A and Y204F). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Abnormal metabolism and sustained proliferation are hallmarks of cancer. Pyruvate kinase M2 (PKM2) is a metabolic enzyme that plays important roles in both processes. Recently, PKM2 was shown to have protein kinase activity phosphorylating histone H3 and promoting cancer cell proliferation. However, the mechanism and extent of this protein kinase in cancer cells remain unclear. Here, we report that binding of succinyl-5-aminoimidazole-4-carboxamide-1-ribose-5'-phosphate (SAICAR), a metabolite abundant in proliferating cells, induces PKM2's protein kinase activity in vitro and in cells. Protein microarray experiments revealed that more than 100 human proteins, mostly protein kinases, are phosphorylated by PKM2-SAICAR. In particular, PKM2-SAICAR phosphorylates and activates Erk1/2, which in turn sensitizes PKM2 for SAICAR binding through phosphorylation. Additionally, PKM2-SAICAR was necessary to induce sustained Erk1/2 activation and mitogen-induced cell proliferation. Thus, the ligand-induced protein kinase activity from PKM2 is a mechanism that directly couples cell proliferation with intracellular metabolic status.
    Molecular cell 03/2014; 53(5):700-709. DOI:10.1016/j.molcel.2014.02.015 · 14.02 Impact Factor
  • Source
    • "Our previous transcriptomics studies focused on ET-1 and the role of ERK1/2 signalling [13–15,20] because maximally effective concentrations activate the entire pool of ERK1/2 in cardiomyocytes (thus a maximal transcriptomics response should be obtained), and rapid (within 2 min) nuclear localization of phosphorylated ERK1/2 was detectable by immunostaining [12]. In contrast, α1-AR agonists such as PE do not activate the entire pool of ERK1/2 and we could not detect a significant nuclear signal for phospho-ERK1/2 using immunostaining (results not shown). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Extracellular signal-regulated kinases 1/2 (ERK1/2) and their substrates, p90 ribosomal S6 kinases (RSKs), phosphorylate different transcription factors, contributing differentially to transcriptomic profiles. In cardiomyocytes, ERK1/2 are required for >70% of the transcriptomic response to endothelin-1. Here, we investigated the role of RSKs in the transcriptomic responses to Gq protein-coupled receptor agonists, endothelin-1, phenylephrine (generic α1-adrenergic receptor agonist) and A61603 (α1A-adrenergic receptor selective). Phospho-ERK1/2 and phospho-RSKs appeared in cardiomyocyte nuclei within 2-3 min of stimulation (endothelin-1>a61603≈phenylephrine). All agonists increased nuclear RSK2, but only endothelin-1 increased nuclear RSK1 content. PD184352 (inhibits ERK1/2 activation) and BI-D1870 (inhibits RSKs) were used to dissect the contribution of RSKs to the endothelin-1-responsive transcriptome. Of 213 RNAs upregulated at 1 h, 51% required RSKs for upregulation whereas 29% required ERK1/2 but not RSKs. The transcriptomic response to phenylephrine overlapped with, but was not identical to, endothelin-1. As with endothelin-1, PD184352 inhibited upregulation of most phenylephrine-responsive transcripts, but the greater variation in effects of BI-D1870 suggests that differential RSK signalling influences global gene expression. A61603 induced similar changes in RNA expression in cardiomyocytes as phenylephrine, indicating that the signal was mediated largely through α1A-adrenergic receptors. A61603 also increased expression of immediate early genes in perfused adult rat hearts and, as in cardiomyocytes, upregulation of the majority of genes was inhibited by PD184352. PD184352 or BI-D1870 prevented the increased surface area induced by endothelin-1 in cardiomyocytes. Thus, RSKs play a significant role in regulating cardiomyocyte gene expression and hypertrophy in response to Gq protein-coupled receptor stimulation.
    Biochemical Journal 12/2012; 450(Pt 2). DOI:10.1042/BJ20121371 · 4.40 Impact Factor
Show more