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

Abstract

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.

A Novel Role for Copper in Ras/Mitogen-Activated Protein Kinase Signaling

“…Human Erk2 and human Mek1 were obtained from Addgene (catalog numbers 23498 and 21208) and cloned into pGEX4T3 and pGEX6P1 from GE Life Sciences, respectively. Recombinant GST-human Erk2 (hErk2) and GSThuman Mek1 (hMek1) were purified from BL21(DE3) (New England BioLabs) bacteria as described previously (21,37), while unphosphorylated MBP was purchased from Millipore. Specifically, 500 ml of LB was inoculated with BL21(DE3) bacteria transformed with pGEX4T3-hErk2 or pGEX6P1-hMek1 and allowed to grow to an optical density at 600 nm (OD 600 ) of 0.6 was reached.…”

FcγRIIA and FcγRIIIB Mediate Nuclear Factor Activation through Separate Signaling Pathways in Human Neutrophils

“…Very recently, such ERK variants were reported to be identified by a specifically designed genetic screen (44). Six variants (each carrying a point mutation) of the yeast MAPK Mpk1/Erk were isolated and shown to present autophosphorylation activity (44). It would be interesting to explore whether such variants are also present in different compartments of leukocytes.…”

“…However, another interesting possibility is the existence of an ERK variant in the nucleus that can be activated independently of upstream phosphorylation, that is, independently of MEK. Very recently, such ERK variants were reported to be identified by a specifically designed genetic screen (44). Six variants (each carrying a point mutation) of the yeast MAPK Mpk1/Erk were isolated and shown to present autophosphorylation activity (44).…”

Computational Insights for the Discovery of Non-ATP Competitive Inhibitors of MAP Kinases

“…It was further suggested, based on crystal structures of intrinsically active mutants, that the conformational change in loop 16 plays a critical role in inducing autophosphorylation through an intermolecular mechanism [49]. Similarly, ERK mutants capable of auto-phosphorylation have been reported [50]. Emrick et al discovered that the mutation of a gatekeeper residue in ERK2 led to auto-phosphorylation.…”