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. 2004 May 15;380(Pt 1):19–30. doi: 10.1042/BJ20031628

Stress- and mitogen-induced phosphorylation of the synapse-associated protein SAP90/PSD-95 by activation of SAPK3/p38gamma and ERK1/ERK2.

Guadalupe Sabio 1, Suzana Reuver 1, Carmen Feijoo 1, Masato Hasegawa 1, Gareth M Thomas 1, Francisco Centeno 1, Sven Kuhlendahl 1, Sergio Leal-Ortiz 1, Michel Goedert 1, Craig Garner 1, Ana Cuenda 1
PMCID: PMC1224136  PMID: 14741046

Abstract

SAPK3 (stress-activated protein kinase-3, also known as p38gamma) is a member of the mitogen-activated protein kinase family; it phosphorylates substrates in response to cellular stress, and has been shown to bind through its C-terminal sequence to the PDZ domain of alpha1-syntrophin. In the present study, we show that SAP90 [(synapse-associated protein 90; also known as PSD-95 (postsynaptic density-95)] is a novel physiological substrate for both SAPK3/p38gamma and the ERK (extracellular-signal-regulated protein kinase). SAPK3/p38gamma binds preferentially to the third PDZ domain of SAP90 and phosphorylates residues Thr287 and Ser290 in vitro, and Ser290 in cells in response to cellular stresses. Phosphorylation of SAP90 is dependent on the binding of SAPK3/p38gamma to the PDZ domain of SAP90. It is not blocked by SB 203580, which inhibits SAPK2a/p38alpha and SAPK2b/p38beta but not SAPK3/p38gamma, or by the ERK pathway inhibitor PD 184352. However, phosphorylation is abolished when cells are treated with a cell-permeant Tat fusion peptide that disrupts the interaction of SAPK3/p38gamma with SAP90. ERK2 also phosphorylates SAP90 at Thr287 and Ser290 in vitro, but this does not require PDZ-dependent binding. SAP90 also becomes phosphorylated in response to mitogens, and this phosphorylation is prevented by pretreatment of the cells with PD 184352, but not with SB 203580. In neurons, SAP90 and SAPK3/p38gamma co-localize and they are co-immunoprecipitated from brain synaptic junctional preparations. These results demonstrate that SAP90 is a novel binding partner for SAPK3/p38gamma, a first physiological substrate described for SAPK3/p38gamma and a novel substrate for ERK1/ERK2, and that phosphorylation of SAP90 may play a role in regulating protein-protein interactions at the synapse in response to adverse stress- or mitogen-related stimuli.

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Selected References

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  1. Aarts Michelle, Liu Yitao, Liu Lidong, Besshoh Shintaro, Arundine Mark, Gurd James W., Wang Yu-Tian, Salter Michael W., Tymianski Michael. Treatment of ischemic brain damage by perturbing NMDA receptor- PSD-95 protein interactions. Science. 2002 Oct 25;298(5594):846–850. doi: 10.1126/science.1072873. [DOI] [PubMed] [Google Scholar]
  2. Abe J., Kusuhara M., Ulevitch R. J., Berk B. C., Lee J. D. Big mitogen-activated protein kinase 1 (BMK1) is a redox-sensitive kinase. J Biol Chem. 1996 Jul 12;271(28):16586–16590. doi: 10.1074/jbc.271.28.16586. [DOI] [PubMed] [Google Scholar]
  3. Adamski F. M., Zhu M. Y., Bahiraei F., Shieh B. H. Interaction of eye protein kinase C and INAD in Drosophila. Localization of binding domains and electrophysiological characterization of a loss of association in transgenic flies. J Biol Chem. 1998 Jul 10;273(28):17713–17719. doi: 10.1074/jbc.273.28.17713. [DOI] [PubMed] [Google Scholar]
  4. Burnett P. E., Blackshaw S., Lai M. M., Qureshi I. A., Burnett A. F., Sabatini D. M., Snyder S. H. Neurabin is a synaptic protein linking p70 S6 kinase and the neuronal cytoskeleton. Proc Natl Acad Sci U S A. 1998 Jul 7;95(14):8351–8356. doi: 10.1073/pnas.95.14.8351. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Buée-Scherrer Valérie, Goedert Michel. Phosphorylation of microtubule-associated protein tau by stress-activated protein kinases in intact cells. FEBS Lett. 2002 Mar 27;515(1-3):151–154. doi: 10.1016/s0014-5793(02)02460-2. [DOI] [PubMed] [Google Scholar]
  6. Cao T. T., Deacon H. W., Reczek D., Bretscher A., von Zastrow M. A kinase-regulated PDZ-domain interaction controls endocytic sorting of the beta2-adrenergic receptor. Nature. 1999 Sep 16;401(6750):286–290. doi: 10.1038/45816. [DOI] [PubMed] [Google Scholar]
  7. Chung H. J., Xia J., Scannevin R. H., Zhang X., Huganir R. L. Phosphorylation of the AMPA receptor subunit GluR2 differentially regulates its interaction with PDZ domain-containing proteins. J Neurosci. 2000 Oct 1;20(19):7258–7267. doi: 10.1523/JNEUROSCI.20-19-07258.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cohen N. A., Brenman J. E., Snyder S. H., Bredt D. S. Binding of the inward rectifier K+ channel Kir 2.3 to PSD-95 is regulated by protein kinase A phosphorylation. Neuron. 1996 Oct;17(4):759–767. doi: 10.1016/s0896-6273(00)80207-x. [DOI] [PubMed] [Google Scholar]
  9. Cohen P. The search for physiological substrates of MAP and SAP kinases in mammalian cells. Trends Cell Biol. 1997 Sep;7(9):353–361. doi: 10.1016/S0962-8924(97)01105-7. [DOI] [PubMed] [Google Scholar]
  10. Colledge M., Dean R. A., Scott G. K., Langeberg L. K., Huganir R. L., Scott J. D. Targeting of PKA to glutamate receptors through a MAGUK-AKAP complex. Neuron. 2000 Jul;27(1):107–119. doi: 10.1016/s0896-6273(00)00013-1. [DOI] [PubMed] [Google Scholar]
  11. Cuenda A., Cohen P., Buée-Scherrer V., Goedert M. Activation of stress-activated protein kinase-3 (SAPK3) by cytokines and cellular stresses is mediated via SAPKK3 (MKK6); comparison of the specificities of SAPK3 and SAPK2 (RK/p38). EMBO J. 1997 Jan 15;16(2):295–305. doi: 10.1093/emboj/16.2.295. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Cuenda A., Rouse J., Doza Y. N., Meier R., Cohen P., Gallagher T. F., Young P. R., Lee J. C. SB 203580 is a specific inhibitor of a MAP kinase homologue which is stimulated by cellular stresses and interleukin-1. FEBS Lett. 1995 May 8;364(2):229–233. doi: 10.1016/0014-5793(95)00357-f. [DOI] [PubMed] [Google Scholar]
  13. Davies S. P., Reddy H., Caivano M., Cohen P. Specificity and mechanism of action of some commonly used protein kinase inhibitors. Biochem J. 2000 Oct 1;351(Pt 1):95–105. doi: 10.1042/0264-6021:3510095. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Eyers P. A., Craxton M., Morrice N., Cohen P., Goedert M. Conversion of SB 203580-insensitive MAP kinase family members to drug-sensitive forms by a single amino-acid substitution. Chem Biol. 1998 Jun;5(6):321–328. doi: 10.1016/s1074-5521(98)90170-3. [DOI] [PubMed] [Google Scholar]
  15. Garner C. C., Nash J., Huganir R. L. PDZ domains in synapse assembly and signalling. Trends Cell Biol. 2000 Jul;10(7):274–280. doi: 10.1016/s0962-8924(00)01783-9. [DOI] [PubMed] [Google Scholar]
  16. Goedert M., Cuenda A., Craxton M., Jakes R., Cohen P. Activation of the novel stress-activated protein kinase SAPK4 by cytokines and cellular stresses is mediated by SKK3 (MKK6); comparison of its substrate specificity with that of other SAP kinases. EMBO J. 1997 Jun 16;16(12):3563–3571. doi: 10.1093/emboj/16.12.3563. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Goedert M., Hasegawa M., Jakes R., Lawler S., Cuenda A., Cohen P. Phosphorylation of microtubule-associated protein tau by stress-activated protein kinases. FEBS Lett. 1997 Jun 2;409(1):57–62. doi: 10.1016/s0014-5793(97)00483-3. [DOI] [PubMed] [Google Scholar]
  18. Hall R. A., Premont R. T., Chow C. W., Blitzer J. T., Pitcher J. A., Claing A., Stoffel R. H., Barak L. S., Shenolikar S., Weinman E. J. The beta2-adrenergic receptor interacts with the Na+/H+-exchanger regulatory factor to control Na+/H+ exchange. Nature. 1998 Apr 9;392(6676):626–630. doi: 10.1038/33458. [DOI] [PubMed] [Google Scholar]
  19. Hasegawa M., Cuenda A., Spillantini M. G., Thomas G. M., Buée-Scherrer V., Cohen P., Goedert M. Stress-activated protein kinase-3 interacts with the PDZ domain of alpha1-syntrophin. A mechanism for specific substrate recognition. J Biol Chem. 1999 Apr 30;274(18):12626–12631. doi: 10.1074/jbc.274.18.12626. [DOI] [PubMed] [Google Scholar]
  20. Hirbec Hélène, Francis Joanna C., Lauri Sari E., Braithwaite Steven P., Coussen Françoise, Mulle Christophe, Dev Kumlesh K., Coutinho Victoria, Meyer Guido, Isaac John T. R. Rapid and differential regulation of AMPA and kainate receptors at hippocampal mossy fibre synapses by PICK1 and GRIP. Neuron. 2003 Feb 20;37(4):625–638. doi: 10.1016/s0896-6273(02)01191-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Hsueh Y. P., Kim E., Sheng M. Disulfide-linked head-to-head multimerization in the mechanism of ion channel clustering by PSD-95. Neuron. 1997 May;18(5):803–814. doi: 10.1016/s0896-6273(00)80319-0. [DOI] [PubMed] [Google Scholar]
  22. Huber A., Sander P., Gobert A., Bähner M., Hermann R., Paulsen R. The transient receptor potential protein (Trp), a putative store-operated Ca2+ channel essential for phosphoinositide-mediated photoreception, forms a signaling complex with NorpA, InaC and InaD. EMBO J. 1996 Dec 16;15(24):7036–7045. [PMC free article] [PubMed] [Google Scholar]
  23. Husi H., Grant S. G. Isolation of 2000-kDa complexes of N-methyl-D-aspartate receptor and postsynaptic density 95 from mouse brain. J Neurochem. 2001 Apr;77(1):281–291. doi: 10.1046/j.1471-4159.2001.t01-1-00248.x. [DOI] [PubMed] [Google Scholar]
  24. Kim E., Naisbitt S., Hsueh Y. P., Rao A., Rothschild A., Craig A. M., Sheng M. GKAP, a novel synaptic protein that interacts with the guanylate kinase-like domain of the PSD-95/SAP90 family of channel clustering molecules. J Cell Biol. 1997 Feb 10;136(3):669–678. doi: 10.1083/jcb.136.3.669. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Kim E., Niethammer M., Rothschild A., Jan Y. N., Sheng M. Clustering of Shaker-type K+ channels by interaction with a family of membrane-associated guanylate kinases. Nature. 1995 Nov 2;378(6552):85–88. doi: 10.1038/378085a0. [DOI] [PubMed] [Google Scholar]
  26. Kim J. H., Liao D., Lau L. F., Huganir R. L. SynGAP: a synaptic RasGAP that associates with the PSD-95/SAP90 protein family. Neuron. 1998 Apr;20(4):683–691. doi: 10.1016/s0896-6273(00)81008-9. [DOI] [PubMed] [Google Scholar]
  27. Kistner U., Wenzel B. M., Veh R. W., Cases-Langhoff C., Garner A. M., Appeltauer U., Voss B., Gundelfinger E. D., Garner C. C. SAP90, a rat presynaptic protein related to the product of the Drosophila tumor suppressor gene dlg-A. J Biol Chem. 1993 Mar 5;268(7):4580–4583. [PubMed] [Google Scholar]
  28. Knebel A., Morrice N., Cohen P. A novel method to identify protein kinase substrates: eEF2 kinase is phosphorylated and inhibited by SAPK4/p38delta. EMBO J. 2001 Aug 15;20(16):4360–4369. doi: 10.1093/emboj/20.16.4360. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Knebel Axel, Haydon Claire E., Morrice Nick, Cohen Philip. Stress-induced regulation of eukaryotic elongation factor 2 kinase by SB 203580-sensitive and -insensitive pathways. Biochem J. 2002 Oct 15;367(Pt 2):525–532. doi: 10.1042/BJ20020916. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Kornau H. C., Schenker L. T., Kennedy M. B., Seeburg P. H. Domain interaction between NMDA receptor subunits and the postsynaptic density protein PSD-95. Science. 1995 Sep 22;269(5231):1737–1740. doi: 10.1126/science.7569905. [DOI] [PubMed] [Google Scholar]
  31. Kyriakis J. M., Avruch J. Mammalian mitogen-activated protein kinase signal transduction pathways activated by stress and inflammation. Physiol Rev. 2001 Apr;81(2):807–869. doi: 10.1152/physrev.2001.81.2.807. [DOI] [PubMed] [Google Scholar]
  32. Lechner C., Zahalka M. A., Giot J. F., Møller N. P., Ullrich A. ERK6, a mitogen-activated protein kinase involved in C2C12 myoblast differentiation. Proc Natl Acad Sci U S A. 1996 Apr 30;93(9):4355–4359. doi: 10.1073/pnas.93.9.4355. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Marshall C. J. Specificity of receptor tyrosine kinase signaling: transient versus sustained extracellular signal-regulated kinase activation. Cell. 1995 Jan 27;80(2):179–185. doi: 10.1016/0092-8674(95)90401-8. [DOI] [PubMed] [Google Scholar]
  34. Matsuda S., Mikawa S., Hirai H. Phosphorylation of serine-880 in GluR2 by protein kinase C prevents its C terminus from binding with glutamate receptor-interacting protein. J Neurochem. 1999 Oct;73(4):1765–1768. doi: 10.1046/j.1471-4159.1999.731765.x. [DOI] [PubMed] [Google Scholar]
  35. Mertens S., Craxton M., Goedert M. SAP kinase-3, a new member of the family of mammalian stress-activated protein kinases. FEBS Lett. 1996 Apr 1;383(3):273–276. doi: 10.1016/0014-5793(96)00255-4. [DOI] [PubMed] [Google Scholar]
  36. Mody N., Leitch J., Armstrong C., Dixon J., Cohen P. Effects of MAP kinase cascade inhibitors on the MKK5/ERK5 pathway. FEBS Lett. 2001 Jul 27;502(1-2):21–24. doi: 10.1016/s0014-5793(01)02651-5. [DOI] [PubMed] [Google Scholar]
  37. Mora A., Sabio G., González-Polo R. A., Cuenda A., Alessi D. R., Alonso J. C., Fuentes J. M., Soler G., Centeno F. Lithium inhibits caspase 3 activation and dephosphorylation of PKB and GSK3 induced by K+ deprivation in cerebellar granule cells. J Neurochem. 2001 Jul;78(1):199–206. doi: 10.1046/j.1471-4159.2001.00410.x. [DOI] [PubMed] [Google Scholar]
  38. Nagy A. K., Shuster T. A., Delgado-Escueta A. V. Rat brain synaptosomal ATP:AMP-phosphotransferase activity. J Neurochem. 1989 Oct;53(4):1166–1172. doi: 10.1111/j.1471-4159.1989.tb07410.x. [DOI] [PubMed] [Google Scholar]
  39. Pak D. T., Yang S., Rudolph-Correia S., Kim E., Sheng M. Regulation of dendritic spine morphology by SPAR, a PSD-95-associated RapGAP. Neuron. 2001 Aug 2;31(2):289–303. doi: 10.1016/s0896-6273(01)00355-5. [DOI] [PubMed] [Google Scholar]
  40. Parker C. G., Hunt J., Diener K., McGinley M., Soriano B., Keesler G. A., Bray J., Yao Z., Wang X. S., Kohno T. Identification of stathmin as a novel substrate for p38 delta. Biochem Biophys Res Commun. 1998 Aug 28;249(3):791–796. doi: 10.1006/bbrc.1998.9250. [DOI] [PubMed] [Google Scholar]
  41. Schwarze S. R., Ho A., Vocero-Akbani A., Dowdy S. F. In vivo protein transduction: delivery of a biologically active protein into the mouse. Science. 1999 Sep 3;285(5433):1569–1572. doi: 10.1126/science.285.5433.1569. [DOI] [PubMed] [Google Scholar]
  42. Sheng M., Pak D. T. Ligand-gated ion channel interactions with cytoskeletal and signaling proteins. Annu Rev Physiol. 2000;62:755–778. doi: 10.1146/annurev.physiol.62.1.755. [DOI] [PubMed] [Google Scholar]
  43. Sheng M., Sala C. PDZ domains and the organization of supramolecular complexes. Annu Rev Neurosci. 2001;24:1–29. doi: 10.1146/annurev.neuro.24.1.1. [DOI] [PubMed] [Google Scholar]
  44. Songyang Z., Fanning A. S., Fu C., Xu J., Marfatia S. M., Chishti A. H., Crompton A., Chan A. C., Anderson J. M., Cantley L. C. Recognition of unique carboxyl-terminal motifs by distinct PDZ domains. Science. 1997 Jan 3;275(5296):73–77. doi: 10.1126/science.275.5296.73. [DOI] [PubMed] [Google Scholar]
  45. Staudinger J., Lu J., Olson E. N. Specific interaction of the PDZ domain protein PICK1 with the COOH terminus of protein kinase C-alpha. J Biol Chem. 1997 Dec 19;272(51):32019–32024. doi: 10.1074/jbc.272.51.32019. [DOI] [PubMed] [Google Scholar]
  46. Tsunoda S., Sierralta J., Sun Y., Bodner R., Suzuki E., Becker A., Socolich M., Zuker C. S. A multivalent PDZ-domain protein assembles signalling complexes in a G-protein-coupled cascade. Nature. 1997 Jul 17;388(6639):243–249. doi: 10.1038/40805. [DOI] [PubMed] [Google Scholar]
  47. Xu X. Z., Choudhury A., Li X., Montell C. Coordination of an array of signaling proteins through homo- and heteromeric interactions between PDZ domains and target proteins. J Cell Biol. 1998 Jul 27;142(2):545–555. doi: 10.1083/jcb.142.2.545. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Zhai R., Olias G., Chung W. J., Lester R. A., tom Dieck S., Langnaese K., Kreutz M. R., Kindler S., Gundelfinger E. D., Garner C. C. Temporal appearance of the presynaptic cytomatrix protein bassoon during synaptogenesis. Mol Cell Neurosci. 2000 May;15(5):417–428. doi: 10.1006/mcne.2000.0839. [DOI] [PubMed] [Google Scholar]

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