Synergistic activation of stress-activated protein kinase 1/c-Jun N-terminal kinase (SAPK1/JNK) isoforms by mitogen-activated protein kinase kinase 4 (MKK4) and MKK7.
Stress-activated protein kinase 1 (SAPK1), also called c-Jun N-terminal kinase (JNK), becomes activated in vivo in response to pro-inflammatory cytokines or cellular stresses. Its full activation requires the phosphorylation of a threonine and a tyrosine residue in a Thr-Pro-Tyr motif, which can be catalysed by the protein kinases mitogen-activated protein kinase kinase (MKK)4 and MKK7. Here we report that MKK4 shows a striking preference for the tyrosine residue (Tyr-185), and MKK7 a striking preference for the threonine residue (Thr-183) in three SAPK1/JNK1 isoforms tested (JNK1 alpha 1, JNK2 alpha 2 and JNK3 alpha 1). For this reason, MKK4 and MKK7 together produce a synergistic increase in the activity of each SAPK1/JNK isoform in vitro. The MKK7 beta variant, which is several hundred-fold more efficient in activating all three SAPK1/JNK isoforms than is MKK7 alpha', is equally specific for Thr-183. MKK7 also phosphorylates JNK2 alpha 2 at Thr-404 and Ser-407 in vitro, Ser-407 being phosphorylated much more rapidly than Thr-183 in vitro. Thr-404/Ser-407 are phosphorylated in unstimulated human KB cells and HEK-293 cells, and phosphorylation is increased in response to an osmotic stress (0.5 M sorbitol). However, in contrast with Thr-183 and Tyr-185, the phosphorylation of Thr-404 and Ser-407 is not increased in response to other agonists that activate MKK7 and SAPK1/JNK, suggesting that phosphorylation of these residues is catalysed by another protein kinase, such as CK2, which also phosphorylates Thr-404 and Ser-407 in vitro. MKK3, MKK4 and MKK6 all show a strong preference for phosphorylation of the tyrosine residue of the Thr-Gly-Tyr motifs in their known substrates SAPK2a/p38, SAPK3/p38 gamma and SAPK4/p38 delta. MKK7 also phosphorylates SAPK2a/p38 at a low rate (but not SAPK3/p38 gamma or SAPK4/p38 delta), and phosphorylation occurs exclusively at the tyrosine residue, demonstrating that MKK7 is intrinsically a 'dual-specific' protein kinase.
Fleming
Y
Y
MRC Protein Phosphorylation Unit, Department of Biochemistry, MSI/WTB Complex, University of Dundee, Dow Street, Dundee DD1 5EH, Scotland, UK. Y.Fleming@dundee.ac.uk
Armstrong
C G
CG
Morrice
N
N
Paterson
A
A
Goedert
M
M
Cohen
P
P
D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular; D004789:Enzyme Activation; D006801:Humans; D007313:Insecta; D048031:JNK Mitogen-Activated Protein Kinases; D048670:MAP Kinase Kinase 4; D048688:MAP Kinase Kinase 7; D048055:Mitogen-Activated Protein Kinase 8; D020929:Mitogen-Activated Protein Kinase Kinases / Q000737:chemistry* / Q000378:metabolism*; D020928:Mitogen-Activated Protein Kinases / Q000737:chemistry* / Q000378:metabolism*; D009995:Osmosis; D010449:Peptide Mapping; D010766:Phosphorylation; D020033:Protein Isoforms; D012694:Serine / Q000737:chemistry; D013379:Substrate Specificity; D013912:Threonine / Q000737:chemistry; D014357:Trypsin / Q000494:pharmacology; D014443:Tyrosine / Q000737:chemistry
D020033:Protein Isoforms; D013912:Threonine; D014443:Tyrosine; D012694:Serine; D048031:JNK Mitogen-Activated Protein Kinases; D048055:Mitogen-Activated Protein Kinase 8; D020928:Mitogen-Activated Protein Kinases; D048670:MAP Kinase Kinase 4; D048688:MAP Kinase Kinase 7; C482559:MAP2K4 protein, human; C482569:MAP2K7 protein, human; D020929:Mitogen-Activated Protein Kinase Kinases; D014357:Trypsin