Activated ERK2 Is a Monomer in Vitro with or without Divalent Cations and When Complexed to the Cytoplasmic Scaffold PEA-15

Abstract

The extracellular signal-regulated protein kinase, ERK2, fully activated by phosphorylation and without a His6-tag, shows little tendency to dimerize with or without either calcium or magnesium ions when analyzed by light scattering or analytical ultracentrifugation. Light scattering shows that ~ 90% of ERK2 is monomeric. Sedimentation equilibrium data (obtained at 4.8–11.2 μM ERK2) with or without magnesium (10 mM) are well described by an ideal one-component model with a fitted molar mass of 40,180 ± 240 Da (- Mg2+ ions) and 41,290 ± 330 Da (+ Mg2+ ions). These values, close to the sequence-derived mass of 41,711 Da, indicate that no significant dimerization of ERK2 occurs in solution. Analysis of sedimentation velocity data for a 15 μM solution of ERK2 with an enhanced van Holde-Weischet method determined the sedimentation coefficient (s) to be ~ 3.22 S for activated ERK2 with or without 10 mM MgCl2. The frictional coefficient ratio (f/f0) of 1.28 calculated from the sedimentation velocity and equilibrium data is close to that expected for a globular protein of ~ 42 kDa. The translational diffusion coefficient of ~ 8.3 × 10-7 cm2s-1 calculated from the experimentally determined molar mass and sedimentation coefficient agrees with the value determined by dynamic light scattering in the absence and presence of calcium or magnesium ions and a value determined by NMR spectrometry. ERK2 has been proposed to homodimerize and bind only to cytoplasmic but not nuclear proteins. Our light scattering data show, however, that ERK2 forms a strong 1:1 complex of ~ 57 kDa with the cytoplasmic scaffold protein PEA-15. Thus ERK2 binds PEA-15 as a monomer. Our data provide strong evidence that ERK2 is monomeric under physiological conditions. Analysis of the same ERK2 construct with the non-physiological His6-Tag shows substantial dimerization under the same ionic conditions.

Monomeric and Dimeric Models of ERK2 in Conjunction with Studies on Cellular Localization, Nuclear Translocation, and In Vitro Analysis

“…But, in several reports, the His-tag did affect biophysical/biochemical properties and has the propensity to promote protein dimerization/oligomerization (Amor-Mahjoub et al, 2006;Wu and Filutowicz, 1999). It is interesting to note that removal of the His-tag from Ets-1, one of well-defined ERK2 substrate in the nucleus, decreases its binding affinity to ERK2 by 1.5-fold (Kaoud et al, 2011). The influence of His-tag on ERK2 dimerization was also found in other studies in relation with divalent cations.…”

“…Although the authors also suggested the existence of a dimeric association of ERK2, its quantity was vanishingly small and negligible. A more thorough biophysical investigation on the monomer-dimer equilibrium of activated ERK2 was recently carried out by employing various experimental tools including light scattering, analytical ultracentrifuge and nuclear magnetic resonance spectroscopy (Kaoud et al, 2011). All the experimental results in this study indicated that tagless active ERK2 was a monomer and there was no propensity of homodimeric association.…”

“…But, many contradicting observations questioning the ERK2 dimer model compelled the inevitable reappraisal of the validity of this model. Especially, the thorough biophysical investigation of active ERK2 (Kaoud et al, 2011) significantly undermined the validity of the dimer hypothesis of active ERK2. According to the latter result, the effect of his-tag and the method of purification on ERK2 dimerization should be re-evaluated in the interpretation of experimental result.…”

“…Interestingly, the authors found that dimer formation of ERK2 was initiated by the association of monomeric ERK2 with a prebound ERK2-scaffold complex. But, dimeric interaction of ERK2 with scaffold proteins are not universal and, for example, PEA-15 was found to bind to monomeric ERK2 (Kaoud et al, 2011). So far, there has not been any observation that scaffold proteins and ERK2 dimerization are required in the activation of nuclear substrates.…”

“…In ERK2 binding, it requires MAPK insert in ERK2 (Whitehurst et al, 2004) and as a MAPK insert is necessary in MEK1 binding, PEA-15 binding was found to block activation of ERK2. A recent light scattering analysis (Kaoud et al, 2011) of active/ tagless ERK2/PEA-15 complex following fractionation with a size-exclusion column showed that ERK2 forms a strong 1:1 complex with PEA-15 of approximately 57 kDa, suggesting that ERK2 binds PEA-15 as a monomer.…”