MAP-quest: Could we produce constitutively active variants of MAP kinases?

Askari, Nadav; Diskin, Ron; Avitzour, Michal; Yaakov, Gilad; Livnah, Oded; Engelberg, David

doi:10.1016/j.mce.2006.03.015

Cited by 19 publications

(16 citation statements)

References 98 publications

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“…Also, although vast information is available with respect to the structural requirements for MAPK activation (45,46), it is not known how to impose these structural changes via mutagenesis (48). We therefore took a genetic approach and were able to generate mutants of the human MAPK p38␣, which were spontaneously active as recombinant proteins expressed in and purified from Escherichia coli cells (49).…”

mentioning

confidence: 99%

Hyperactive Variants of p38α Induce, whereas Hyperactive Variants of p38γ Suppress, Activating Protein 1-mediated Transcription

Askari

Diskin

Avitzour

et al. 2007

Journal of Biological Chemistry

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The p38 family of kinases is a subgroup of the mitogen-activated protein kinase family. It is composed of four isoforms and is involved in critical biological processes as well as in inflammatory diseases. The exact unique role of each p38 isoform in these processes is not understood well. To approach this question we have been developing intrinsically active variants of p38s. Recently we described a series of mutants of the human p38␣, which were spontaneously active as recombinant proteins purified from Escherichia coli cells. We show here that some of these mutants are spontaneously active in several mammalian cells in culture. The spontaneous activity of some mutants is higher than the activity of the fully activated wild type counterpart. We further produced mutants of the other p38 isoforms and found that p38␤ D176A , p38␥ D179A , p38␦ D176A , and p38␦ F324S are spontaneously active in vivo. The active mutants are also spontaneously phosphorylated. To test whether the mutants actually fulfill downstream duties of p38 proteins, we tested their effect on activating protein 1(AP-1)-mediated transcription. Active mutants of p38␣ induced AP-1-driven reporter genes, as well as the c-jun and c-fos promoters. An active variant of p38␥ suppressed AP-1-mediated transcription. When active variants of p38␣ and p38␥ were co-expressed, AP-1 activity was not induced, showing that p38␥ is dominant over p38␣ with respect to AP-1 activation. Thus, intrinsically active variants that are spontaneously active in vivo have been obtained for all p38 isoforms. These variants have disclosed different effects of each isoform on AP-1 activity.

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confidence: 99%

Hyperactive Variants of p38α Induce, whereas Hyperactive Variants of p38γ Suppress, Activating Protein 1-mediated Transcription

Askari

Diskin

Avitzour

et al. 2007

Journal of Biological Chemistry

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“…For a very long time and despite many efforts to obtain intrinsically active variants of Erks (30,37), such proteins could not be achieved. Now that such molecules are available for yeast, flies, and mammals, novel approaches are open for investigating, in a specific manner, the biological and biochemical functions of each isoform and splicing variants of these kinases.…”

Section: Discussionmentioning

confidence: 99%

“…Also, attempts to mimic phosphorylation by replacing the relevant Thr to Glu were not successful (25,29). A large number of studies applying other strategies were also just partially successful (30). Several years ago, we took a genetic approach for isolation of intrinsically active mutants of the yeast MAPK Hog1.…”

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confidence: 99%

Isolation of Intrinsically Active (MEK-independent) Variants of the ERK Family of Mitogen-activated Protein (MAP) Kinases

Levin-Salomon

Kogan

Ahn

et al. 2008

Journal of Biological Chemistry

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MAPKs are key components of cell signaling pathways with a unique activation mechanism: i.e. dual phosphorylation of neighboring threonine and tyrosine residues. The ERK enzymes form a subfamily of MAPKs involved in proliferation, differentiation, development, learning, and memory. The exact role of each Erk molecule in these processes is not clear. An efficient strategy for addressing this question is to activate individually each molecule, for example, by expressing intrinsically active variants of them. However, such molecules were not produced so far. Here, we report on the isolation, via a specifically designed genetic screen, of six variants (each carries a point mutation) of the yeast MAPK Mpk1/Erk that are active, independent of upstream phosphorylation. One of the activating mutations, R68S, occurred in a residue conserved in the mammalian Erk1 (Arg-84) and Erk2 (Arg-65) and in the Drosophila ERK Rolled (Arg-80). Replacing this conserved Arg with Ser rendered these MAPKs intrinsically active to very high levels when tested in vitro as recombinant proteins. Combination of the Arg to Ser mutation with the sevenmaker mutation (producing Erk2 R65S؉D319N and Rolled R80S؉D334N ) resulted in even higher activity (45 and 70%, respectively, in reference to fully active dually phosphorylated Erk2 or Rolled). Erk2 R65S and Erk2R65S؉D319N were found to be spontaneously active also when expressed in human HEK293 cells. We further revealed the mechanism of action of the mutants and show that it involves acquisition of autophosphorylation activity. Thus, a first generation of Erk molecules that are spontaneously active in vitro and in vivo has been obtained.

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“…It should be noted that numeration of the mutations in ERK1 (in text and in Table 1A) refer to the sequence of the human protein and numeration of mutations in ERK2 (in text and in Table 1B) refer to the sequence of rat protein. Notably, mutations that had been discovered (until 2006) in ERK orthologs in lower organisms were summarized in [98] and will not be discussed here.…”

Section: Almost All Known Mutations In Erks Have Been Identified Expementioning

confidence: 99%

Mutations That Confer Drug-Resistance, Oncogenicity and Intrinsic Activity on the ERK MAP Kinases—Current State of the Art

Smorodinsky-Atias

Soudah

Engelberg

2020

Cells

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Unique characteristics distinguish extracellular signal-regulated kinases (Erks) from other eukaryotic protein kinases (ePKs). Unlike most ePKs, Erks do not autoactivate and they manifest no basal activity; they become catalysts only when dually phosphorylated on neighboring Thr and Tyr residues and they possess unique structural motifs. Erks function as the sole targets of the receptor tyrosine kinases (RTKs)-Ras-Raf-MEK signaling cascade, which controls numerous physiological processes and is mutated in most cancers. Erks are therefore the executers of the pathway’s biology and pathology. As oncogenic mutations have not been identified in Erks themselves, combined with the tight regulation of their activity, Erks have been considered immune against mutations that would render them intrinsically active. Nevertheless, several such mutations have been generated on the basis of structure-function analysis, understanding of ePK evolution and, mostly, via genetic screens in lower eukaryotes. One of the mutations conferred oncogenic properties on Erk1. The number of interesting mutations in Erks has dramatically increased following the development of Erk-specific pharmacological inhibitors and identification of mutations that cause resistance to these compounds. Several mutations have been recently identified in cancer patients. Here we summarize the mutations identified in Erks so far, describe their properties and discuss their possible mechanism of action.

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MAP-quest: Could we produce constitutively active variants of MAP kinases?

Cited by 19 publications

References 98 publications

Hyperactive Variants of p38α Induce, whereas Hyperactive Variants of p38γ Suppress, Activating Protein 1-mediated Transcription

Hyperactive Variants of p38α Induce, whereas Hyperactive Variants of p38γ Suppress, Activating Protein 1-mediated Transcription

Isolation of Intrinsically Active (MEK-independent) Variants of the ERK Family of Mitogen-activated Protein (MAP) Kinases

Mutations That Confer Drug-Resistance, Oncogenicity and Intrinsic Activity on the ERK MAP Kinases—Current State of the Art

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