Fleming Y, Armstrong C G, Morrice N, Paterson A, Goedert M, Cohen P
MRC Protein Phosphorylation Unit, Department of Biochemistry, MSI/WTB Complex, University of Dundee, Dow Street, Dundee DD1 5EH, Scotland, UK.
Biochem J. 2000 Nov 15;352 Pt 1(Pt 1):145-54.
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.
应激激活蛋白激酶1（SAPK1），也称为c-Jun氨基末端激酶（JNK），在体内会因促炎细胞因子或细胞应激而被激活。其完全激活需要苏氨酸-脯氨酸-酪氨酸基序中的一个苏氨酸残基和一个酪氨酸残基发生磷酸化，这可由丝裂原活化蛋白激酶激酶（MKK）4和MKK7催化。在此我们报告，在测试的三种SAPK1/JNK1亚型（JNK1α1、JNK2α2和JNK3α1）中，MKK4对酪氨酸残基（Tyr-185）表现出显著偏好，而MKK7对苏氨酸残基（Thr-183）表现出显著偏好。因此，MKK4和MKK7共同在体外协同提高每种SAPK1/JNK亚型的活性。MKK7β变体在激活所有三种SAPK1/JNK亚型方面比MKK7α'高效数百倍，它对Thr-183同样具有特异性。MKK7在体外还能使JNK2α2的Thr-404和Ser-407发生磷酸化，Ser-407在体外的磷酸化速度比Thr-183快得多。Thr-404/Ser-407在未受刺激的人KB细胞和HEK-293细胞中发生磷酸化，并且在渗透压应激（0.5 M山梨醇）刺激下磷酸化增加。然而，与Thr-183和Tyr-185不同，Thr-404和Ser-407的磷酸化在激活MKK7和SAPK1/JNK的其他激动剂刺激下并未增加，这表明这些残基的磷酸化是由另一种蛋白激酶（如CK2）催化的，CK2在体外也能使Thr-404和Ser-407发生磷酸化。MKK3、MKK4和MKK6在其已知底物SAPK2a/p38、SAPK3/p38γ和SAPK4/p38δ中，都对苏氨酸-甘氨酸-酪氨酸基序的酪氨酸残基磷酸化表现出强烈偏好。MKK7也能以低速率使SAPK2a/p38发生磷酸化（但不能使SAPK3/p38γ或SAPK4/p38δ发生磷酸化），且磷酸化仅发生在酪氨酸残基上，这表明MKK7本质上是一种“双特异性”蛋白激酶。
