T Cell Receptor-mediated Activation of p38α by Mono-phosphorylation of the Activation Loop Results in Altered Substrate Specificity

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 p38α and p38β 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 p38α 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. 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 p38α and p38β 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 p38α 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. MAPK 2The abbreviations used are:MAPKmitogen-activated protein kinaseMAPKKMAPK kinaseTCRT cell antigen receptorERKextracellular signal-regulated kinaseaaamino acidPBSphosphate-buffered salineIVKin vitro kinasePMAphorbol 12-myristate 13-acetateIFNinterferon. 2The abbreviations used are:MAPKmitogen-activated protein kinaseMAPKKMAPK kinaseTCRT cell antigen receptorERKextracellular signal-regulated kinaseaaamino acidPBSphosphate-buffered salineIVKin vitro kinasePMAphorbol 12-myristate 13-acetateIFNinterferon. are expressed in all eukaryotic cells and participate in responses to stimuli involved in cell activation, proliferation, differentiation, and death (1.Kyriakis J.M. Avruch J. Physiol. Rev. 2001; 81: 807-869Crossref PubMed Scopus (2833) Google Scholar, 2.Roux P.P. Blenis J. Microbiol. Mol. Biol. Rev. 2004; 68: 320-344Crossref PubMed Scopus (1878) Google Scholar). Of the three major MAPK families, the ERKs play a part in mitogenic signal propagation, whereas the JNKs (c-Jun N-terminal kinases) and p38 isoforms participate in responses to pro-inflammatory cytokines and stress. MAPK activity is closely coupled to changes in gene expression, consistent with the fact that their targets include numerous transcription factors and kinases that themselves regulate transcription factors. Aberrant p38 activity has been implicated in inflammation and cancer, for which p38 inhibitors are being explored as therapeutic agents (3.Engelberg D. Semin. Cancer Biol. 2004; 14: 271-282Crossref PubMed Scopus (88) Google Scholar, 4.Lee J.C. Kumar S. Griswold D.E. Underwood D.C. Votta B.J. Adams J.L. Immunopharmacology. 2000; 47: 185-201Crossref PubMed Scopus (436) Google Scholar). mitogen-activated protein kinase MAPK kinase T cell antigen receptor extracellular signal-regulated kinase amino acid phosphate-buffered saline in vitro kinase phorbol 12-myristate 13-acetate interferon. mitogen-activated protein kinase MAPK kinase T cell antigen receptor extracellular signal-regulated kinase amino acid phosphate-buffered saline in vitro kinase phorbol 12-myristate 13-acetate interferon. MAPK are activated by a three-tiered kinase signaling cascade. At the most membrane-proximal level are the MAPK kinase kinases (MAPKKK), serine/threonine kinases that phosphorylate and activate the second level, the MAPK kinases (MAPKK). MAPKK are dual specificity kinases that phosphorylate a MAPK Thr-X-Tyr motif (X being glycine for p38 family members) located on the flexible activation loop that borders the catalytic site and regulates access to substrate. The p38 MAPK family has four members as follows: α, β, γ, and δ. p38γ expression is limited primarily to skeletal muscle (5.Li Z. Jiang Y. Ulevitch R.J. Han J. Biochem. Biophys. Res. Commun. 1996; 228: 334-340Crossref PubMed Scopus (350) Google Scholar), whereas the others are more widely distributed. p38α is the major isoform in T cells, which also express lesser amounts of p38 β and δ (6.Hale K.K. Trollinger D. Rihanek M. Manthey C.L. J. Immunol. 1999; 162: 4246-4252PubMed Google Scholar). p38α and -β are the most closely related by amino acid sequence (74% homology), are subject to inhibition by the widely used inhibitor SB203580, and share a tripeptide motif, Pro-Tyr-Asp, involved in the T cell antigen receptor (TCR)-initiated alternative activation pathway (7.Salvador J.M. Mittelstadt P.R. Guszczynski T. Copeland T.D. Yamaguchi H. Appella E. Fornace A.J.J. Ashwell J.D. Nat. Immunol. 2005; 6: 390-395Crossref PubMed Scopus (226) Google Scholar). TCRs recognize peptide antigen presented by major histocompatibility molecules on the surface of antigen-presenting cells. Although it had generally been assumed that TCR-induced p38 activation utilized the canonical MAPK cascade (8.Rincon M. Flavell R.A. Davis R.A. Free Radic. Biol. Med. 2000; 28: 1328-1337Crossref PubMed Scopus (131) Google Scholar), we found that this is not the case (7.Salvador J.M. Mittelstadt P.R. Guszczynski T. Copeland T.D. Yamaguchi H. Appella E. Fornace A.J.J. Ashwell J.D. Nat. Immunol. 2005; 6: 390-395Crossref PubMed Scopus (226) Google Scholar). Rather, stimulation via the TCR results in Lck-dependent activation of ZAP70, which in turn phosphorylates p38α and -β on Tyr-323. Phosphorylation of Tyr-323 leads to p38α and p38β autophosphorylation on the activation loop and increased activity toward third party substrates. Recent evidence suggests that the scaffold protein Dlgh1 bridges Lck/ZAP70 and p38, and in its absence Tyr-323 is not phosphorylated, and p38 is not activated in response to TCR signaling (9.Round J.L. Humphries L.A. Tomassian T. Mittelstadt P. Zhang M. Miceli M.C. Nat. Immunol. 2007; 8: 154-161Crossref PubMed Scopus (94) Google Scholar). Interestingly, despite the fact that B cells express the ZAP70 relative Syk, the alternative pathway does not appear to exist in antigen receptor-stimulated B cells (7.Salvador J.M. Mittelstadt P.R. Guszczynski T. Copeland T.D. Yamaguchi H. Appella E. Fornace A.J.J. Ashwell J.D. Nat. Immunol. 2005; 6: 390-395Crossref PubMed Scopus (226) Google Scholar, 10.Jirmanova L. Sarma D.N. Jankovic D. Mittelstadt P.R. Ashwell J.D. Blood. 2008; PubMed Google Scholar). The importance of the alternative pathway has been demonstrated in mice lacking Gadd45α, in which the alternative pathway is constitutively active, resulting in spontaneous T cell p38 activity and autoimmunity (11.Salvador J.M. Mittelstadt P.R. Belova G.I. Fornace A.J.J. Ashwell J.D. Nat. Immunol. 2005; 6: 396-402Crossref PubMed Scopus (81) Google Scholar). More recently, we have found that p38α cannot be activated by TCR signaling in primary T cells from gene-targeted mice in which Tyr-323 has been replaced with Phe (10.Jirmanova L. Sarma D.N. Jankovic D. Mittelstadt P.R. Ashwell J.D. Blood. 2008; PubMed Google Scholar). An intriguing question is why T cells acquired a seemingly unique mechanism for p38 activation rather than utilizing the common signaling cascade. Here we find that the two activation pathways are qualitatively different, because unlike MAPKKs, p38α predominantly autophosphorylates a single residue in the activation loop, Thr-180, resulting in altered substrate selectivity. Primary T cells were purified from lymph nodes and spleens of 6–8-week-old C57/BL6 mice using negatively selecting T cell purification columns (CL101) from CedarLane. T cells and the Jurkat T lymphoma (ATCC) were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (Invitrogen), 2 mm glutamine, 50 μm β-mercaptoethanol, and 100 μm gentamicin. Antibodies against p38 (9217), phospho-p38 (9211), and phospho-Thr-71-ATF-2 (9221) were purchased from Cell Signaling. Anti-CD3 (clone 2C11) was from Pharmingen. Rabbit polyclonal antibodies against phospho-Tyr-182-p38 were generated as described (7.Salvador J.M. Mittelstadt P.R. Guszczynski T. Copeland T.D. Yamaguchi H. Appella E. Fornace A.J.J. Ashwell J.D. Nat. Immunol. 2005; 6: 390-395Crossref PubMed Scopus (226) Google Scholar). Jurkat TCR clonotype-specific antibodies were obtained from the supernatants of the hybridoma C305 (from ATCC) grown to high density (<106 cells/ml) in complete medium. PMA was purchased from Sigma and SB203508 from Calbiochem. p38α and other proteins were expressed in the bacterial strain BL21(DE3) using the vectors pGEX-4T-2 or pET15b. Point mutations for p38α (K53M, Y323F, T180A, and Y182F) and ATF2 (aa 1–109; T69A and T71A) were generated by site-directed mutagenesis using the QuikChange kit (Stratagene). Human MAPKAP kinase 2 (aa 46–400) (12.Lukas S.M. Kroe R.R. Wildeson J. Peet G.W. Frego L. Davidson W. Ingraham R.H. Pargellis C.A. Labadia M.E. Werneburg B.G. Biochemistry. 2004; 43: 9950-9960Crossref PubMed Scopus (46) Google Scholar) and STAT4 (aa 538–749) (13.Visconti R. Gadina M. Chiariello M. Chen E.H. Stancato L.F. Gutkind J.S. O'Shea J.J. Blood. 2000; 96: 1844-1852Crossref PubMed Google Scholar) were amplified from cDNA and cloned into pGEX-4T-2. Plasmids encoding GST-Bcl-2, Bcl-XL (14.Farley N. Pedraza-Alva G. Serrano-Gomez D. Nagaleekar V. Aronshtam A. Krahl T. Thornton T. Rincon M. Mol. Cell. Biol. 2006; 26: 2118-2129Crossref PubMed Scopus (69) Google Scholar), and GST-MEF2A (aa 151–411) (15.Marinissen M.J. Chiariello M. Pallante M. Gutkind J.S. Mol. Cell. Biol. 1999; 19: 4289-4301Crossref PubMed Scopus (188) Google Scholar) were generously provided by Mercedes Rincon and Silvio Gutkind, respectively. After cultures reached an A600 of 0.5–1.0, protein expression was induced with 0.3 mm isopropyl β-d-thiogalactopyranoside. His-p38α, His-MKK6 (S207E/T211E), and GST-ATF2 were incubated for a further 3 h at 25 °C and GST-MAPKAP kinase 2, GST-MEF2A, GST-STAT4, GST-Bcl-2, and GST-Bcl-XL for 12 h at 15 °C. Cells were resuspended in PBS (including 0.5 m NaCl if His-tagged), 0.1% Triton X-100, and 1 mm phenylmethylsulfonyl fluoride, sonicated, and centrifuged at 20,000 × g for 20 min at 4 °C. His-tagged proteins were purified with cobalt-charged chelating-Sepharose Fast Flow beads (Amersham Biosciences) and eluted with 0.3 m imidazole (in PBS with 0.5 m NaCl). GST-tagged proteins were purified with glutathione-Sepharose Fast Flow beads (Amersham Biosciences) and eluted with 50 mm Tris, pH 8, containing 20 mm glutathione. Proteins were concentrated and washed into PBS using Microcon YM-30 spin columns (Millipore). Substrate proteins (3–10 μg) were prewarmed at 30 °C in 30 μl of kinase buffer (20 mm Tris, pH 7.5, 10 mm MgCl2, 2 mm dithiothreitol, 5 mm β-glycerophosphate, 5 mm NaF, and 0.2 mm Na3VO4) with 1–5 μCi [32P]ATP and the indicated amounts of ATP. Reactions were initiated by addition of 100 ng of semisynthesized p38α or MKK6-activated p38, as indicated. At the indicated times aliquots were removed and mixed with sample buffer. Phosphorylated products were resolved by SDS-PAGE, transferred to nitrocellulose membranes, and visualized with a Storm PhosphorImager (GE Healthcare). To permit native chemical ligation, the natural histidine of the N-terminal residue of the synthetic peptide (aa 310–360) was mutated to cysteine. This mutation had no effect on the spontaneous activity of bacterially expressed full-length p38 (data not shown). Residues 1–309 of mouse p38α were cloned into the vector pTYB1 (New England Biolabs) using the NdeI and SapI restriction sites. To overcome insolubility, the resulting fusion protein of p38α (aa 1–309) with the VMA1 intein and the chitin-binding domain was grown in BL21(DE3) cells harboring the plasmid pREP4-GROEL/S, which encodes the chaperone complex GROEL/S. Cultures grown in LB with 100 μg/ml ampicillin and 25 μg/ml kanamycin to an A600 of 0.6 were induced with 0.1 mm isopropyl for 15 h at 12 and proteins were by in buffer 0.5 m 0.1% Triton proteins were on and p38α (aa 1–309) a was by at with 0.2 m After with buffer and to with YM-30 p38α (aa 1–309) was to a of the N-terminal of the (aa 310–360) with Tyr-323 or The were washed with YM-30 columns into PBS in in vitro kinase in complete medium supplemented with T cells were the TCR by to with 5 μg/ml in Cells were in buffer (20 mm Tris, pH 7.5, mm Triton X-100, 1 mm 10 mm NaF, 10 mm β-glycerophosphate, 5 mm and 1 mm supplemented with inhibitor and 1 mm were to protein in sample resolved by SDS-PAGE, and with the indicated were visualized with antibodies and or with antibodies and the activate p38α by the activation loop and G. M. Rev. 2001; PubMed Scopus Google Scholar). expressed p38α activated phosphorylated in vitro on Tyr-323 (7.Salvador J.M. Mittelstadt P.R. Guszczynski T. Copeland T.D. Yamaguchi H. Appella E. Fornace A.J.J. Ashwell J.D. Nat. Immunol. 2005; 6: 390-395Crossref PubMed Scopus (226) Google Scholar). p38α a of activation loop phosphorylation in (7.Salvador J.M. Mittelstadt P.R. Guszczynski T. Copeland T.D. Yamaguchi H. Appella E. Fornace A.J.J. Ashwell J.D. Nat. Immunol. 2005; 6: 390-395Crossref PubMed Scopus (226) Google Scholar) R. Mittelstadt and J. D. to because a of p38α activation loop phosphorylation or kinase activity (data not shown). To Tyr-323 phosphorylation in the absence of phosphorylation is to kinase we synthetic p38α phosphorylated on Tyr-323 using protein Rev. Biochem. PubMed Scopus Google Scholar). Recombinant p38α 1–309 were to full-length p38α with Tyr-323 phosphorylated or The effect of Tyr-323 phosphorylation on p38α activity was with an in vitro kinase using the substrate semisynthesized p38α Tyr-323 was the of a phosphorylated Tyr-323 in an kinase that phosphorylated ATF2 semisynthesized molecules were and of kinase because with which phosphorylates in the activation loop, phosphorylated ATF2 Tyr-323 phosphorylation in the absence of activation loop phosphorylation is to activate The site of p38α autophosphorylation by the alternative pathway was to the activation loop using an to recognize dual phosphorylated p38 (7.Salvador J.M. Mittelstadt P.R. Guszczynski T. Copeland T.D. Yamaguchi H. Appella E. Fornace A.J.J. Ashwell J.D. Nat. Immunol. 2005; 6: 390-395Crossref PubMed Scopus (226) Google Scholar). the that a against the phosphorylated activation loop also recognize mono-phosphorylated R. M. D. J. Mol. Biol. 2007; PubMed Scopus Google Scholar) to the question of were involved in p38α a polyclonal against a peptide containing the mouse p38α activation loop in which was phosphorylated of p38α with and phosphorylated or or phosphorylated was with the dual used to p38 phosphorylation in T cells (7.Salvador J.M. Mittelstadt P.R. Guszczynski T. Copeland T.D. Yamaguchi H. Appella E. Fornace A.J.J. Ashwell J.D. Nat. Immunol. 2005; 6: 390-395Crossref PubMed Scopus (226) Google Scholar) or with or had activity toward not The specificity of antibodies for the phosphorylated native protein was by p38α that mutations of or and that had been phosphorylated by primarily p38α mono-phosphorylated on the is to as TCR-induced p38α autophosphorylation phosphorylation of the loop residue Tyr-323 (7.Salvador J.M. Mittelstadt P.R. Guszczynski T. Copeland T.D. Yamaguchi H. Appella E. Fornace A.J.J. Ashwell J.D. Nat. Immunol. 2005; 6: 390-395Crossref PubMed Scopus (226) Google Scholar). that p38α expressed in grown at °C is phosphorylated on Tyr-323 and the activation loop because of because are not phosphorylated on the p38α (data not shown). of Tyr-323 to Phe also activation loop autophosphorylation and activation of p38α in (data not that autophosphorylation phosphorylation of Tyr-323. This that E. p38α as a for p38α activated by the alternative if p38α phosphorylate p38α and if on which GST-tagged p38α was as a substrate for p38α in an in vitro kinase After with was by not p38α phosphorylate in which is the mechanism of its autophosphorylation in T cells, on the activation loop residue not To the p38α the of the protein activated in cells, we the phosphorylation of p38 in T cells. phosphorylation in cells the TCR alternative or with PMA the MAPK a signal with p38α from T cells with PMA a signal with cells. unlike the MAPK cascade, alternative p38 activation primarily mono-phosphorylation of Thr-180. phosphorylated p38 is the of the activated that has been implicated in TCR signaling p38α mono-phosphorylation the that the in cells via the TCR with stimuli that the MAPK cascade. a we if dual and mono-phosphorylated p38α in their to phosphorylate p38α substrates. and mono-phosphorylated p38α were generated by and p38α with To of Tyr-323 Tyr-323 in proteins was replaced with phosphorylated proteins on to and dual phosphorylated p38α that were in an with the substrate J. 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