Active ERK1 is dimerized in vivo: bisphosphodimers generate peak kinase activity and monophosphodimers maintain basal ERK1 activity.
ERK1 and ERK2 are widely involved in cell signalling. Using a recombinant approach, it has been shown that exogenous ERK2 is capable of dimerization and that preventing dimerization reduces its nuclear accumulation on stimulation. Dimerization occurs on phosphorylation; the dimer partner of phosphorylated ERK2 may be either phosphorylated or unphosphorylated. It has been assumed that monophosphodimers are hemiactive. Here we show that ERK1 is capable of dimerization both in vivo and in vitro. Dimerization of human recombinant ERK1 in vitro requires both ERK1 phosphorylation and cellular cofactor(s); it leads to the formation of a high molecular weight complex that can be dissociated by treatment with beta-mercaptoethanol. We demonstrate for the first time in both sea urchin embryos and human cells that native ERK forms dimers and that high ERK kinase activity is largely associated with bisphosphodimers, not with monophosphodimers or phosphorylated monomers. The activity of the bisphosphodimer is about 20-fold higher than that of the phosphorylated monomer in vitro and the bisphosphodimer shows 5- to 7-fold higher in vivo activity than the basal activity attributable to the monophosphodimer. Thus phosphorylation of both partners in the dimer is a hallmark of ERK activation. Judgments made about ERK kinase activity associated with phosphorylated monomers are at best a proxy for ERK activity.
Philipova
Rada
R
Institute of Cell and Molecular Biosciences, Faculty of Medical Sciences, University of Newcastle upon Tyne, Newcastle upon Tyne, NE2 4HH, UK.
Whitaker
Michael
M
D000818:Animals; D002474:Cell-Free System / Q000378:metabolism; D019281:Dimerization; D004789:Enzyme Activation / Q000502:physiology; D006367:HeLa Cells; D006801:Humans; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism*; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational / Q000502:physiology*; D011994:Recombinant Proteins / Q000378:metabolism; D012617:Sea Urchins
D011994:Recombinant Proteins; D048052:Mitogen-Activated Protein Kinase 3