Department of Biological Chemistry, The Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem, Israel 91904.
Biochemistry. 2009 Mar 24;48(11):2497-504. doi: 10.1021/bi900024v.
A common feature of the regulation of many protein kinases is their phosphorylation on a conserved Thr residue in the activation loop. In the family of mitogen-activated protein kinases (MAPKs), another phosphorylation event, on a Tyr residue neighboring this Thr (in a TXY motif), is required for activity. Many studies suggested that this dual phosphorylation is an absolute requirement for MAPK activation, assigning an equal role for the Thr and Tyr of the phosphorylation motif. Here we tested this notion by producing p38alpha variants carrying a T180A or Y182F mutation or both and assessing their activity in vitro and in vivo. These mutations were inserted into the p38alpha(WT) molecule or into constitutively active variants of p38alpha. We found that p38alpha molecules carrying the T180A mutations lost their activity altogether. On the other hand, p38alpha(WT) and intrinsically active mutants carrying the Y182F mutation are activated by MKK6 in vitro and in vivo, although to low levels, mainly due to reduced affinity for the substrate. However, the intrinsically active variants carrying the Y182F mutation lost most of their autophosphorylation and intrinsic activities. Thus, Thr180 is essential for catalysis, whereas Tyr182 is required for autoactivation and substrate recognition. The p38alpha(Y182F) mutants are capable of activating reporter genes, suggesting that they are not only catalytically active to some degree but also capable of inducing the relevant downstream pathway. We suggest that p38s are active when only the Thr residue of the phosphorylation lip is phosphorylated, similar to many other kinases in nature.
许多蛋白激酶的调控的一个共同特点是它们在激活环中的保守 Thr 残基上发生磷酸化。在丝裂原激活蛋白激酶（MAPKs）家族中，另一个磷酸化事件，即在紧邻 Thr 的 Tyr 残基上（在 TXY 基序中），对于活性是必需的。许多研究表明，这种双重磷酸化是 MAPK 激活的绝对要求，赋予磷酸化模体中的 Thr 和 Tyr 同等作用。在这里，我们通过产生携带 T180A 或 Y182F 突变或两者的 p38alpha 变体，并在体外和体内评估它们的活性来检验这一概念。这些突变被插入到 p38alpha(WT)分子或组成性激活的 p38alpha 变体中。我们发现，携带 T180A 突变的 p38alpha 分子完全失去了活性。另一方面，携带 Y182F 突变的 p38alpha(WT)和固有活性突变体在体外和体内均可被 MKK6 激活，尽管水平较低，主要是由于对底物的亲和力降低。然而，携带 Y182F 突变的固有活性变体失去了大部分自身磷酸化和固有活性。因此，Thr180 对于催化是必不可少的，而 Tyr182 对于自动激活和底物识别是必需的。携带 Y182F 突变的 p38alpha 突变体能够激活报告基因，这表明它们不仅在某种程度上具有催化活性，而且还能够诱导相关的下游途径。我们建议，当磷酸化脂中的 Thr 残基被磷酸化时，p38 激酶是活跃的，类似于自然界中许多其他激酶。
