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  • 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.

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.

The Biochemical journal (2000-11-04)
Y Fleming, C G Armstrong, N Morrice, A Paterson, M Goedert, P Cohen
ABSTRACT

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.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
p38γ/SAPK3 Protein, active, 10 µg, Active, recombinant GST fusion protein corresponding to full length human p38γ/SAPK3, for use in Kinase Assays.
Sigma-Aldrich
JNK1α1/SAPK1c Protein, active, 10 µg, Active, recombinant full-length human JNK1α1/SAPK1c with an N-terminal His-tag, for use in Kinase Assays.
Sigma-Aldrich
Casein Kinase 2 Protein, active, 10 µg, Active, recombinant full-length human Casein Kinase 2 protein, containing an α-subunit N-terminal 6His tag & a β-subunit N-terminal GST tag. For use in Kinase Assays.
Sigma-Aldrich
MKK4/SKK1 Protein, active, 10 µg, Active, N-terminal GST tagged, murine MKK4 residues 34-end, for use in Kinase Assays.
Sigma-Aldrich
JNK2α2/SAPK1a Protein, active, 10 µg, Active, N-terminal His-tagged, full length human JNK2α2/SAPK1a, for use in Kinase Assays.