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First published online 29 November 2005
doi: 10.1242/jcs.02683

Journal of Cell Science 118, 5767-5776 (2005)
Published by The Company of Biologists 2005
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Active ERK1 is dimerized in vivo: bisphosphodimers generate peak kinase activity and monophosphodimers maintain basal ERK1 activity

Rada Philipova and Michael Whitaker*

Institute of Cell and Molecular Biosciences, Faculty of Medical Sciences, University of Newcastle upon Tyne, Newcastle upon Tyne, NE2 4HH, UK



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  		Fig. 1. Human ERK1 activated in vitro dimerizes in the presence of cellular cofactors. The highest activity is associated with bisphosphodimers. (A) Western blotting of recombinant hERK1 with anti-dualphosphorylated ERK antibody. (Lane 1) Unactivated protein. (Lane 2) Unactivated protein incubated in cell extract for 15 minutes. (Lane 3) hERK1 after a 1 hour activation by recombinant MEK, not incubated in cell extract. (Lane 4) hERK1 treated as for lane 3, but with an additional incubation in cell extract for 15 minutes. (Lane 5) hERK1 treated as for lane 4, followed by dephosphorylation using hCL100. (Lane 6) hERK1 treated as for lane 4, followed by additional activation by MEK overnight at 4°C. (Lane 7) hERK1 sample treated identically to the sample in lane 6, but dissolved in sample buffer containing 5% ß-mercaptoethanol. (Lane 8) Control: low ERK1 activity extract (30 minutes post-fertilization, 15 µg total cellular protein loaded); active ERK1 is undetectable. (-AB control) hERK1 sample treated as for lane 4, but primary antibody was omitted. The positions of molecular mass markers (kDa) are shown. (B) MAP kinase assays with myelin basic protein (MBP) using samples identical to those in panel A. Relative protein kinase activity is also shown from densitometry. (C) Western blotting using anti-ERK antibody of samples identical to those of lanes 2, 4, 5, 6 and -AB in panel A. The positions of molecular mass markers are shown. (D) Western blotting using anti-GST antibody of samples identical to lanes 4-6 and -AB in panel A. The positions of molecular mass markers are shown. (E) GST-{Delta}PEHD-hERK1, a dimerization-deficient mutant, does not show enhanced activity when incubated in whole-cell extract. 6 pM of each ERK1 recombinant protein was used per sample. Western blotting with anti-ERK antibody. (Lane 1) Unactivated {Delta}PEHD-hERK1. (Lane 2) {Delta}PEHD-hERK1 after a 1 hour activation by active MEK not incubated in cell extract. (Lane 3) {Delta}PEHD-hERK1 treated as for lane 2, but with an additional incubation in cell extract for 15 minutes. (Lane 4) {Delta}PEHD-hERK1 treated as for Lane 3, followed by additional activation by MEK overnight at 4°C. (Lane 5) An equal amount of recombinant control hERK1 treated as for lane 3. (F) MAP kinase assays with MBP using samples identical to those in E. Relative protein kinase activity is also shown from densitometry. Note that the monomeric mutant protein is effectively activated by MEK (eightfold over baseline); however, incubation in cell extract does not result in additional increase in its activity. The same amount of activated control wild-type protein forms a complex in cell extract with much higher kinase activity.

 


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  		Fig. 2. ERK activity in vivo is associated with its homodimers. Monomers at 44 kDa are largely inactive. (A) Schematic diagram of ERK1 activity during the first mitotic cell cycle of sea urchin embryos. An inset with the original data (Philipova and Whitaker, 1998Go) is also shown. (B) Sea urchin embryos. Active ERK1 was immunoprecipitated during the first mitotic cell cycle at time points that corresponded to maximum (6 min, NEB) and minimum activity (Unfertilized, 30 min) using an anti-dualphosphorylated ERK antibody and detected by western blotting with a second, different anti-dualphosphorylated ERK antibody or with an anti-MEK antibody (NEB sample). Arrows, active ERK1 dimers. Cell extract: whole-cell extracts are rich in ERK1 monomers as a western blot of a 30 minute post-fertilization cell extract detected with anti-ERK antibody demonstrates. It was necessary to load 150 µg total cellular protein in order to obtain a detectable band at 91 kDa for comparison with the anti-dualphosphorylated ERK antibody immunoprecipitate. The positions of molecular mass markers are shown. (C) HeLa cells. Active ERK was immunoprecipitated from cell extracts using an anti-dualphosphorylated ERK antibody, and proteins detected on western blots using a second anti-dualphosphorylated ERK antibody, an anti-ERK antibody or an anti-MEK antibody. The immunoprecipitate was also run in the absence of ß-mercaptoethanol (ß-ME) before blotting (protein complex). The positions of molecular mass markers are shown (kDa).

 


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  		Fig. 3. In vitro phosphorylation by MEK of immunoprecipitated low-activity cellular ERK1 dimer (* – 0) results in the appearance of the shifted fully activated dimerized ERK1 (* – *). Total ERK1 was immunoprecipitated from 30 minute extracts from sea urchin embryos using an anti-ERK antibody and then treated in vitro with either active MEK (IP + MEK) or an inactive (kinase dead) mutant of MEK (IP + k.d.MEK); a control IP in the absence of the anti-ERK antibody is also shown (Control IP-AB). A and B show a comparison between western blots of the same samples with (A) anti-ERK antibody or (B) anti-dualphosphorylated ERK antibody. In both cases, the immunoprecipitates are compared with an untreated aliquot of the 30 minute extract used for immunoprecipitation. Note that immunoprecipitates using anti-dualphosphorylated ERK antibody are highly enriched in the 91 kDa dimeric form. (C) Relative activities of samples treated as in B, washed in MAP kinase buffer and used in protein kinase assays. The sample activated by MEK shows sixfold higher activity than those either untreated or

 


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  		Fig. 4. (A) The two ERK1 homodimers (* – 0 and * – *) copurify with the ERK1 monomer. Western blots of a fraction with high MAP kinase activity (fraction 23) eluted from a phenyl-Sepharose column during ERK1 purification from mitotic sea urchin embryo extracts probed with (a) an anti-ERK antibody and (b) an anti-dualphosphorylated ERK antibody. 4 µl of the fraction was loaded for each blot. Blot b was detected using the high-sensitivity ECL Advance Kit: a faint band of active monomer is visible (arrow). (B,C) Phosphorylation by MEK and dephosphorylation using CL100 dual specificity phosphatase cause the predicted gel shifts of the ERK1 dimers in the phenylsepharose column fraction. 0.5 µl of fraction 23 was used for each treatment; samples were blotted and probed with (B) anti-ERK antibody and (C) anti-dualphosphorylated ERK antibody. Samples treated with CL100 in the presence of vanadate to block phosphatase activity (CL100+Na3VO4) or with mutant-inactive (kinase dead) MEK (k.d. MEK) serve as controls. Treatment with CL100 led to the loss of the more slowly migrating band, while treatment with MEK enhanced the band. Note that after MEK treatment, dimeric forms accumulate in preference to the monomer.

 


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  		Fig. 5. Newly phosphorylated ERK1 accumulates as fully active (* – *) homodimers. Incorporation of {gamma}-[32P]ATP into purified ERK1 (fraction 23 from the phenyl-Sepharose column) during in vitro phosphorylation by MEK. 0.5 µl of the fraction was used per reaction as a substrate for in vitro MEK assays. Kinase reactions were carried out for 2, 4 and 6 minutes at 30°C, and then terminated by addition of sample buffer. Molecular mass markers are also shown. Note that radioactivity is incorporated predominantly into the band above 116 kDa. The radioactivity at the top of the gel represents insoluble material that has not entered the gel.

 

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© The Company of Biologists Ltd 2005