Dataset Information

Distinct patterns of activation-dependent changes in conformational mobility between ERK1 and ERK2.

ABSTRACT: Hydrogen/deuterium exchange measurements by mass spectrometry (HX-MS) can be used to report localized conformational mobility within folded proteins, where exchange predominantly occurs through low energy fluctuations in structure, allowing transient solvent exposure. Changes in conformational mobility may impact protein function, even in cases where structural changes are unobservable. Previous studies of the MAP kinase, ERK2, revealed increases in HX upon activation occured at the hinge between conserved N- and C-terminal domains, which could be ascribed to enhanced backbone flexibility. This implied that kinase activation modulates interdomain closure, and was supported by evidence for two modes of nucleotide binding that were consistent with closed vs open conformations in active vs inactive forms of ERK2, respectively. Thus, phosphorylation of ERK2 releases constraints to interdomain closure, by modulating hinge flexibility. In this study, we examined ERK1, which shares 90% sequence identity with ERK2. HX-MS measurements of ERK1 showed similarities with ERK2 in overall deuteration, consistent with their similar tertiary structures. However, the patterns of HX that were altered upon activation of ERK1 differed from those in ERK2. In particular, alterations in HX at the hinge region upon activation of ERK2 did not occur in ERK1, suggesting that the two enzymes differ with respect to their regulation of hinge mobility and interdomain closure. In agreement, HX-MS measurements of nucleotide binding suggested revealed domain closure in both inactive and active forms of ERK1. We conclude that although ERK1 and ERK2 are closely related with respect to primary sequence and tertiary structure, they utilize distinct mechanisms for controlling enzyme function through interdomain interactions.

SUBMITTER: Ring AY

PROVIDER: S-EPMC3139246 | biostudies-literature | 2011 Apr

REPOSITORIES: biostudies-literature

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Distinct patterns of activation-dependent changes in conformational mobility between ERK1 and ERK2.

Ring Adam Y AY Sours Kevin M KM Lee Thomas T Ahn Natalie G NG

International journal of mass spectrometry 20110401 1-3

Hydrogen/deuterium exchange measurements by mass spectrometry (HX-MS) can be used to report localized conformational mobility within folded proteins, where exchange predominantly occurs through low energy fluctuations in structure, allowing transient solvent exposure. Changes in conformational mobility may impact protein function, even in cases where structural changes are unobservable. Previous studies of the MAP kinase, ERK2, revealed increases in HX upon activation occured at the hinge betwee ...[more]

PMID: 21785572

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Project description:Hydrogen-deuterium exchange measurements represent a powerful approach to investigating changes in conformation and conformational mobility in proteins. Here, we examine p38alpha MAP kinase (MAPK) by hydrogen-exchange (HX) mass spectrometry to determine whether changes in conformational mobility may be induced by kinase phosphorylation and activation. Factors influencing sequence coverage in the HX mass spectrometry experiment, which show that varying sampling depths, instruments, and peptide search strategies yield the highest coverage of exchangeable amides, are examined. Patterns of regional deuteration in p38alpha are consistent with tertiary structure and similar to deuteration patterns previously determined for extracellular-signal-regulated kinase (ERK) 2, indicating that MAPKs are conserved with respect to the extent of local amide HX. Activation of p38alpha alters HX in five regions, which are interpreted by comparing X-ray structures of unphosphorylated p38alpha and X-ray structures of phosphorylated p38gamma. Conformational differences account for altered HX within the activation lip, the P+1 site, and the active site. In contrast, HX alterations are ascribed to activation-induced effects on conformational mobility, within substrate-docking sites (alphaF-alphaG, beta7-beta8), the C-terminal core (alphaE), and the N-terminal core region (beta4-beta5, alphaL16, alphaC). Activation also decreases HX in a 3-10 helix at the C-terminal extension of p38alpha. Although this helix in ERK2 forms a dimerization interface that becomes protected from HX upon activation, analytical ultracentrifugation shows that this does not occur in p38alpha because both unphosphorylated and diphosphorylated forms are monomeric. Finally, HX patterns in monophosphorylated p38alpha are similar to those in unphosphorylated kinase, indicating that the major activation lip remodeling events occur only after diphosphorylation. Importantly, patterns of activation-induced HX show differences between p38alpha and ERK2 despite their similarities in overall deuteration, suggesting that although MAPKs are closely related with respect to primary sequence and tertiary structure, they have distinct mechanisms for dynamic control of enzyme function.

Project description:BackgroundThe Ras/Raf/MEK/ERK signaling pathway is involved in essential cell processes and it is abnormally activated in ~30 % of cancers and cognitive disorders. Two ERK isoforms have been described, ERK1 and ERK2; ERK2 being regarded by many as essential due to the embryonic lethality of ERK2 knock-out mice, whereas mice lacking ERK1 are viable and fertile. The controversial question of why we have two ERKs and whether they have differential functions or display functional redundancy has not yet been resolved.ResultsTo investigate this question we used a novel approach based on comparing the evolution of ERK isoforms' sequences and protein expression across vertebrates. We gathered and cloned erk1 and erk2 coding sequences and we examined protein expression of isoforms in brain extracts in all major clades of vertebrate evolution. For the first time, we measured each isoforms' relative protein level in phylogenetically distant animals using anti-phospho antibodies targeting active ERKs. We demonstrate that squamates (lizards, snakes and geckos), despite having both genes, do not express ERK2 protein whereas other tetrapods either do not express ERK1 protein or have lost the erk1 gene. To demonstrate the unexpected squamates' lack of ERK2 expression, we targeted each ERK isoform in lizard primary fibroblasts by specific siRNA-mediated knockdown. We also found that undetectable expression of ERK2 in lizard is compensated by a greater strength of lizard's erk1 promoter. Finally, phylogenetic analysis revealed that ERK1 amino acids sequences evolve faster than ERK2's likely due to genomic factors, including a large difference in gene size, rather than from functional differences since amino acids essential for function are kept invariant.ConclusionsERK isoforms appeared by a single gene duplication at the onset of vertebrate evolution at least 400 Mya. Our results demonstrate that tetrapods can live by expressing either one or both ERK isoforms, supporting the notion that ERK1/2 act interchangeably. Substrate recognition sites and catalytic cleft are nearly invariant in all vertebrate ERKs further suggesting functional redundancy. We suggest that future ERK research should shift towards understanding the role and regulation of total ERK quantity, especially in light of newly described erk2 gene amplification identified in tumors.

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Distinct patterns of activation-dependent changes in conformational mobility between ERK1 and ERK2.

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Distinct patterns of activation-dependent changes in conformational mobility between ERK1 and ERK2.

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