T cell receptor-mediated activation of p38{alpha} by mono-phosphorylation of the activation loop results in altered substrate specificity

Abstract

p38 MAPKs are typically activated by upstream MAPK kinases that phosphorylate a Thr-X-Tyr motif in the activation loop. An exception is the T cell antigen receptor signaling pathway, which bypasses the MAPK cascade and activates p38alpha and p38beta by phosphorylation of Tyr-323 and subsequent autophosphorylation of the activation loop. Here we show that, unlike the classic MAPK cascade, the alternative pathway results primarily in mono-phosphorylation of the activation loop residue Thr-180. Recombinant mono-phosphorylated and dual phosphorylated p38alpha differed widely with regard to activity and substrate preference. Altered substrate specificity was reproduced in T cells in which p38 was activated by the alternative or classical MAPK pathways. These findings suggest that T cells have evolved a mechanism to utilize p38 in a specialized manner independent of and distinct from the classical p38 MAPK signaling cascade.

FIGURE 1.

Tyr-323 phosphorylation activates p38α. Left, semisynthesized unphosphorylated or Tyr-323-phosphorylated p38 was incubated with GST-ATF2 in an IVK reaction in the presence of 1 μm ATP. [³²P]ATP incorporation was detected by SDS-PAGE and autoradiography. Right, semisynthesized p38 proteins were phosphorylated (P) on the activation loop by MKK6 and then incubated with GST-ATF2 an in vitro kinase reaction.

FIGURE 2.

Specificity of anti-phospho-p38 antibodies. A, dot blots of immobilized p38 peptides were immunoblotted (IB) with the antibodies recognizing p38 phosphorylated (P) on Thr-180 (9211) or Tyr-182 (P-Tyr). B, immunoblots of phosphorylated activation loop mutants of p38α. Kinase-dead GST-p38 (K53M) and the indicated activation loop mutants, either left untreated or phosphorylated on available activation loop phosphoacceptor sites by MKK6, were immunoblotted as in A (upper two panels) and with antibodies recognizing total p38 (lowest panel).

FIGURE 3.

Trans-autophosphorylation of p38α takes place on Thr-180. Kinase-dead GST-p38 (Lys-53-Met) was left unphosphorylated or phosphorylated by active His-p38 in an IVK reaction. Reactions were immunoblotted (IB) with the indicated anti-phospho-p38α antibodies (upper panels) and with antibodies recognizing total p38 (lower panels).

FIGURE 4.

In vivo alternatively activated p38 becomes phosphorylated on Thr-180 and not on Tyr-182. Purified T cells were stimulated with PMA or plate-bound anti-CD3 for 15 min. Lysates were immunoblotted with antibodies recognizing p38 Thr(P)-180 or Tyr(P)-182. All samples were from the same experiment, and some intervening lanes were removed from the figure for clarity.

FIGURE 5.

Thr-180 mono-phosphorylated p38α displays altered substrate specificity. A, Thr-180 phosphorylation status of the Thr-180 mono-phosphorylated or dual phosphorylated p38α (Y323F) proteins phosphorylated by MKK6 was evaluated by immunoblotting (IB). B, indicated substrate proteins were incubated with either Thr-180 mono-phosphorylated or dual phosphorylated p38α in an in vitro kinase reaction with 3 mm ATP, and [³²P]ATP incorporation was detected as in Fig. 1. C, determination of the phosphorylation status of migratory variants of wild type (WT) ATF2. GST-ATF2 and the phosphoacceptor site mutants T69A, T71A, and T69A/T71A were incubated with dual phosphorylated p38α in an IVK reaction for 10 min.

FIGURE 6.

In vivo TCR-activated p38α displays altered substrate specificity. T cells were stimulated as in Fig. 4, and lysates were immunoblotted with antibodies recognizing the indicated phosphorylated proteins.