An analysis of the phosphorylation and activation of extracellular-signal-regulated protein kinase 5 (ERK5) by mitogen-activated protein kinase kinase 5 (MKK5) in vitro.
MKK5 expressed as a glutathione S-transferase fusion protein in human embryonic kidney 293 cells activated full-length extracellular-signal-regulated protein kinase (ERK)5 (ERK5wt) as well as the isolated catalytic domain (ERK5cat) in vitro. Activation was accompanied by the phosphorylation of Thr(219) and Tyr(221), the former residue being phosphorylated preferentially. ERK5cat phosphorylated at Thr(219), but not Tyr(221), possessed 10% of the activity of the doubly phosphorylated protein towards myelin basic protein, whereas ERK5cat phosphorylated at Tyr(221) alone was much less active. Activated ERK5 phosphorylated itself at a number of residues, including Thr(28), Ser(421), Ser(433), Ser(496), Ser(731) and Thr(733). ERK5 phosphorylated at Thr(219), but not Tyr(221), phosphorylated itself at a similar rate to ERK5 phosphorylated at both Thr(219) and Tyr(221). Activated ERK5 also phosphorylated mitogen-activated protein kinase kinase 5 (MKK5) extensively at Ser(129), Ser(137), Ser(142) and Ser(149), which are located within the region in MKK5 that is thought to interact with ERK5.
Mody
Nimesh
N
MRC Protein Phosphorylation Unit, MSI/WTB Complex, University of Dundee, Dow Street, Scotland, UK.
Campbell
David G
DG
Morrice
Nick
N
Peggie
Mark
M
Cohen
Philip
P
D020134:Catalytic Domain / Q000502:physiology*; D002478:Cells, Cultured; D004789:Enzyme Activation / Q000187:drug effects; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D048054:Mitogen-Activated Protein Kinase 7; D020929:Mitogen-Activated Protein Kinase Kinases / Q000494:pharmacology*; D020928:Mitogen-Activated Protein Kinases / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013912:Threonine; D014357:Trypsin / Q000378:metabolism; D014443:Tyrosine
D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D013912:Threonine; D014443:Tyrosine; D005982:Glutathione Transferase; D048054:Mitogen-Activated Protein Kinase 7; D020928:Mitogen-Activated Protein Kinases; D020929:Mitogen-Activated Protein Kinase Kinases; D014357:Trypsin