Activated ERK2 is a monomer in vitro with or without divalent cations and when complexed to the cytoplasmic scaffold PEA-15

Tamer S. Kaoud, Ashwini K. Devkota, Richard Harris, Mitra S. Rana, Olga Abramczyk, Mangalika Warthaka, Sunbae Lee, Mark E. Girvin, Austen F. Riggs, Kevin N. Dalby

Research output: Contribution to journalArticle

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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 that̃90% 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 40180 ± 240 Da (without Mg2+ ions) or 41290 ( 330 Da (with Mg2+ ions). These values, close to the sequence-derived mass of 41711 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 10mMMgCl2. The frictional coefficient ratio (f/f0) of 1.28 calculated from the sedimentation velocity and equilibriumdata is close to that expected for an ~42 kDa globular protein. The translational diffusion coefficient of ̃8.3 × 10-7 cm2 s-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 byNMRspectrometry. ERK2 has been proposed to homodimerize and bind only to cytoplasmic but not nuclear proteins [Casar, B., et al. (2008) Mol. Cell 31, 708-721]. 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 nonphysiological His6 tag shows substantial dimerization under the same ionic conditions.

Original languageEnglish (US)
Pages (from-to)4568-4578
Number of pages11
JournalBiochemistry
Volume50
Issue number21
DOIs
StatePublished - May 31 2011

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His-His-His-His-His-His
Divalent Cations
Sedimentation
Scaffolds
Magnesium
Monomers
Dimerization
Ions
Light
Light scattering
Molar mass
Calcium
Ultracentrifugation
Extracellular Signal-Regulated MAP Kinases
Nuclear Proteins
Protein Kinases
Proteins
Phosphorylation
Dynamic light scattering
In Vitro Techniques

ASJC Scopus subject areas

  • Biochemistry

Cite this

Kaoud, T. S., Devkota, A. K., Harris, R., Rana, M. S., Abramczyk, O., Warthaka, M., ... Dalby, K. N. (2011). Activated ERK2 is a monomer in vitro with or without divalent cations and when complexed to the cytoplasmic scaffold PEA-15. Biochemistry, 50(21), 4568-4578. https://doi.org/10.1021/bi200202y

Activated ERK2 is a monomer in vitro with or without divalent cations and when complexed to the cytoplasmic scaffold PEA-15. / Kaoud, Tamer S.; Devkota, Ashwini K.; Harris, Richard; Rana, Mitra S.; Abramczyk, Olga; Warthaka, Mangalika; Lee, Sunbae; Girvin, Mark E.; Riggs, Austen F.; Dalby, Kevin N.

In: Biochemistry, Vol. 50, No. 21, 31.05.2011, p. 4568-4578.

Research output: Contribution to journalArticle

Kaoud, TS, Devkota, AK, Harris, R, Rana, MS, Abramczyk, O, Warthaka, M, Lee, S, Girvin, ME, Riggs, AF & Dalby, KN 2011, 'Activated ERK2 is a monomer in vitro with or without divalent cations and when complexed to the cytoplasmic scaffold PEA-15', Biochemistry, vol. 50, no. 21, pp. 4568-4578. https://doi.org/10.1021/bi200202y
Kaoud, Tamer S. ; Devkota, Ashwini K. ; Harris, Richard ; Rana, Mitra S. ; Abramczyk, Olga ; Warthaka, Mangalika ; Lee, Sunbae ; Girvin, Mark E. ; Riggs, Austen F. ; Dalby, Kevin N. / Activated ERK2 is a monomer in vitro with or without divalent cations and when complexed to the cytoplasmic scaffold PEA-15. In: Biochemistry. 2011 ; Vol. 50, No. 21. pp. 4568-4578.
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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 that̃90{\%} 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 40180 ± 240 Da (without Mg2+ ions) or 41290 ( 330 Da (with Mg2+ ions). These values, close to the sequence-derived mass of 41711 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 10mMMgCl2. The frictional coefficient ratio (f/f0) of 1.28 calculated from the sedimentation velocity and equilibriumdata is close to that expected for an ~42 kDa globular protein. The translational diffusion coefficient of ̃8.3 × 10-7 cm2 s-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 byNMRspectrometry. ERK2 has been proposed to homodimerize and bind only to cytoplasmic but not nuclear proteins [Casar, B., et al. (2008) Mol. Cell 31, 708-721]. 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 nonphysiological His6 tag shows substantial dimerization under the same ionic conditions.",
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AU - Kaoud, Tamer S.

AU - Devkota, Ashwini K.

AU - Harris, Richard

AU - Rana, Mitra S.

AU - Abramczyk, Olga

AU - Warthaka, Mangalika

AU - Lee, Sunbae

AU - Girvin, Mark E.

AU - Riggs, Austen F.

AU - Dalby, Kevin N.

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N2 - 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 that̃90% 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 40180 ± 240 Da (without Mg2+ ions) or 41290 ( 330 Da (with Mg2+ ions). These values, close to the sequence-derived mass of 41711 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 10mMMgCl2. The frictional coefficient ratio (f/f0) of 1.28 calculated from the sedimentation velocity and equilibriumdata is close to that expected for an ~42 kDa globular protein. The translational diffusion coefficient of ̃8.3 × 10-7 cm2 s-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 byNMRspectrometry. ERK2 has been proposed to homodimerize and bind only to cytoplasmic but not nuclear proteins [Casar, B., et al. (2008) Mol. Cell 31, 708-721]. 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 nonphysiological His6 tag shows substantial dimerization under the same ionic conditions.

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