p38alpha is active in vitro and in vivo when monophosphorylated at threonine 180.
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.
Askari
Nadav
N
Department of Biological Chemistry, The Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem, Israel 91904.
Beenstock
Jonah
J
Livnah
Oded
O
Engelberg
David
D
D004789:Enzyme Activation; D006801:Humans; D009154:Mutation; D010766:Phosphorylation; D013912:Threonine / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014443:Tyrosine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D048051:p38 Mitogen-Activated Protein Kinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D013912:Threonine; D014443:Tyrosine; D048051:p38 Mitogen-Activated Protein Kinases