Skip to main content
PMC home page
The Journal of Experimental Medicine logoLink to The Journal of Experimental Medicine
. 1996 Apr 1;183(4):1899–1904. doi: 10.1084/jem.183.4.1899

Association of mitogen-activated protein kinases with microtubules in mouse macrophages

PMCID: PMC2192474  PMID: 8666946

Abstract

Taxol, a microtubule-binding diterpene, mimics many effects of lipopolysaccharide (LPS) on mouse macrophages. The LPS-mimetic effects of taxol appear to be under the same genetic control as responses to LPS itself. Thus we have postulated a role for microtubule-associated proteins (MAP) in the response of macrophages to LPS. Stimulation of macrophages by LPS quickly induces the activation of mitogen-activated protein kinases (MAPK). MAPK are generally considered cytosolic enzymes. Herein we report that much of the LPS-activatable pool of MAPK in primary mouse peritoneal macrophages is microtubule associated. By immunofluorescence, MAPK were localized to colchicine- and nocodazole- disruptible filaments. From both mouse brain and RAW 264.7 macrophages, MAPK could be coisolated with polymerized tubulin. Fractionation of primary macrophages into cytosol-, microfilament-, microtubule-, and intermediated filament-rich extracts revealed that approximately 10% of MAPK but none of MAPK kinase (MEK1A and MEK2) was microtubule bound. Exposure of macrophages to LPS did not change the proportion of MAPK bound to microtubules, but preferentially activated the microtubule- associated pool. These findings confirm the prediction that LPS activates a kinase bound to microtubules. Together with LPS-mimetic actions of taxol and the shared genetic control of responses to LPS and taxol, these results support the hypothesis that a major LPS-signaling pathway in mouse macrophages may involve activation of one or more microtubule-associated kinases.

Full Text

The Full Text of this article is available as a PDF (1.3 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Ahn N. G., Krebs E. G. Evidence for an epidermal growth factor-stimulated protein kinase cascade in Swiss 3T3 cells. Activation of serine peptide kinase activity by myelin basic protein kinases in vitro. J Biol Chem. 1990 Jul 15;265(20):11495–11501. [PubMed] [Google Scholar]
  2. Ahn N. G., Seger R., Bratlien R. L., Krebs E. G. Growth factor-stimulated phosphorylation cascades: activation of growth factor-stimulated MAP kinase. Ciba Found Symp. 1992;164:113–131. doi: 10.1002/9780470514207.ch8. [DOI] [PubMed] [Google Scholar]
  3. Alberola-Ila J., Forbush K. A., Seger R., Krebs E. G., Perlmutter R. M. Selective requirement for MAP kinase activation in thymocyte differentiation. Nature. 1995 Feb 16;373(6515):620–623. doi: 10.1038/373620a0. [DOI] [PubMed] [Google Scholar]
  4. Alexander-Miller M. A., Burke K., Koszinowski U. H., Hansen T. H., Connolly J. M. Alloreactive cytotoxic T lymphocytes generated in the presence of viral-derived peptides show exquisite peptide and MHC specificity. J Immunol. 1993 Jul 1;151(1):1–10. [PubMed] [Google Scholar]
  5. Alvarez E., Northwood I. C., Gonzalez F. A., Latour D. A., Seth A., Abate C., Curran T., Davis R. J. Pro-Leu-Ser/Thr-Pro is a consensus primary sequence for substrate protein phosphorylation. Characterization of the phosphorylation of c-myc and c-jun proteins by an epidermal growth factor receptor threonine 669 protein kinase. J Biol Chem. 1991 Aug 15;266(23):15277–15285. [PubMed] [Google Scholar]
  6. Anderson N. G., Maller J. L., Tonks N. K., Sturgill T. W. Requirement for integration of signals from two distinct phosphorylation pathways for activation of MAP kinase. Nature. 1990 Feb 15;343(6259):651–653. doi: 10.1038/343651a0. [DOI] [PubMed] [Google Scholar]
  7. Balasubramanian S. V., Straubinger R. M. Taxol-lipid interactions: taxol-dependent effects on the physical properties of model membranes. Biochemistry. 1994 Aug 2;33(30):8941–8947. doi: 10.1021/bi00196a011. [DOI] [PubMed] [Google Scholar]
  8. Blenis J. Signal transduction via the MAP kinases: proceed at your own RSK. Proc Natl Acad Sci U S A. 1993 Jul 1;90(13):5889–5892. doi: 10.1073/pnas.90.13.5889. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Bogdan C., Ding A. Taxol, a microtubule-stabilizing antineoplastic agent, induces expression of tumor necrosis factor alpha and interleukin-1 in macrophages. J Leukoc Biol. 1992 Jul;52(1):119–121. doi: 10.1002/jlb.52.1.119. [DOI] [PubMed] [Google Scholar]
  10. Childs T. J., Watson M. H., Sanghera J. S., Campbell D. L., Pelech S. L., Mak A. S. Phosphorylation of smooth muscle caldesmon by mitogen-activated protein (MAP) kinase and expression of MAP kinase in differentiated smooth muscle cells. J Biol Chem. 1992 Nov 15;267(32):22853–22859. [PubMed] [Google Scholar]
  11. Davis R. J. MAPKs: new JNK expands the group. Trends Biochem Sci. 1994 Nov;19(11):470–473. doi: 10.1016/0968-0004(94)90132-5. [DOI] [PubMed] [Google Scholar]
  12. Ding A. H., Nathan C. F., Stuehr D. J. Release of reactive nitrogen intermediates and reactive oxygen intermediates from mouse peritoneal macrophages. Comparison of activating cytokines and evidence for independent production. J Immunol. 1988 Oct 1;141(7):2407–2412. [PubMed] [Google Scholar]
  13. Ding A. H., Porteu F., Sanchez E., Nathan C. F. Shared actions of endotoxin and taxol on TNF receptors and TNF release. Science. 1990 Apr 20;248(4953):370–372. doi: 10.1126/science.1970196. [DOI] [PubMed] [Google Scholar]
  14. Ding A., Sanchez E., Tancinco M., Nathan C. Interactions of bacterial lipopolysaccharide with microtubule proteins. J Immunol. 1992 May 1;148(9):2853–2858. [PubMed] [Google Scholar]
  15. Drechsel D. N., Hyman A. A., Cobb M. H., Kirschner M. W. Modulation of the dynamic instability of tubulin assembly by the microtubule-associated protein tau. Mol Biol Cell. 1992 Oct;3(10):1141–1154. doi: 10.1091/mbc.3.10.1141. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Fiore R. S., Bayer V. E., Pelech S. L., Posada J., Cooper J. A., Baraban J. M. p42 mitogen-activated protein kinase in brain: prominent localization in neuronal cell bodies and dendrites. Neuroscience. 1993 Jul;55(2):463–472. doi: 10.1016/0306-4522(93)90516-i. [DOI] [PubMed] [Google Scholar]
  17. Geppert T. D., Whitehurst C. E., Thompson P., Beutler B. Lipopolysaccharide signals activation of tumor necrosis factor biosynthesis through the ras/raf-1/MEK/MAPK pathway. Mol Med. 1994 Nov;1(1):93–103. [PMC free article] [PubMed] [Google Scholar]
  18. Gonzalez F. A., Seth A., Raden D. L., Bowman D. S., Fay F. S., Davis R. J. Serum-induced translocation of mitogen-activated protein kinase to the cell surface ruffling membrane and the nucleus. J Cell Biol. 1993 Sep;122(5):1089–1101. doi: 10.1083/jcb.122.5.1089. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Gotoh Y., Nishida E., Matsuda S., Shiina N., Kosako H., Shiokawa K., Akiyama T., Ohta K., Sakai H. In vitro effects on microtubule dynamics of purified Xenopus M phase-activated MAP kinase. Nature. 1991 Jan 17;349(6306):251–254. doi: 10.1038/349251a0. [DOI] [PubMed] [Google Scholar]
  20. Grove J. R., Price D. J., Goodman H. M., Avruch J. Recombinant fragment of protein kinase inhibitor blocks cyclic AMP-dependent gene transcription. Science. 1987 Oct 23;238(4826):530–533. doi: 10.1126/science.2821622. [DOI] [PubMed] [Google Scholar]
  21. Haccard O., Sarcevic B., Lewellyn A., Hartley R., Roy L., Izumi T., Erikson E., Maller J. L. Induction of metaphase arrest in cleaving Xenopus embryos by MAP kinase. Science. 1993 Nov 19;262(5137):1262–1265. doi: 10.1126/science.8235656. [DOI] [PubMed] [Google Scholar]
  22. Hoshi M., Ohta K., Gotoh Y., Mori A., Murofushi H., Sakai H., Nishida E. Mitogen-activated-protein-kinase-catalyzed phosphorylation of microtubule-associated proteins, microtubule-associated protein 2 and microtubule-associated protein 4, induces an alteration in their function. Eur J Biochem. 1992 Jan 15;203(1-2):43–52. doi: 10.1111/j.1432-1033.1992.tb19825.x. [DOI] [PubMed] [Google Scholar]
  23. Huby R. D., Carlile G. W., Ley S. C. Interactions between the protein-tyrosine kinase ZAP-70, the proto-oncoprotein Vav, and tubulin in Jurkat T cells. J Biol Chem. 1995 Dec 22;270(51):30241–30244. doi: 10.1074/jbc.270.51.30241. [DOI] [PubMed] [Google Scholar]
  24. Hwang S., Ding A. Activation of NF-kappa B in murine macrophages by taxol. Cancer Biochem Biophys. 1995 Jan;14(4):265–272. [PubMed] [Google Scholar]
  25. Jameson L., Caplow M. Modification of microtubule steady-state dynamics by phosphorylation of the microtubule-associated proteins. Proc Natl Acad Sci U S A. 1981 Jun;78(6):3413–3417. doi: 10.1073/pnas.78.6.3413. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Kang Y. H., Dwivedi R. S., Lee C. H. Ultrastructural and immunocytochemical study of the uptake and distribution of bacterial lipopolysaccharide in human monocytes. J Leukoc Biol. 1990 Oct;48(4):316–332. doi: 10.1002/jlb.48.4.316. [DOI] [PubMed] [Google Scholar]
  27. Lenormand P., Sardet C., Pagès G., L'Allemain G., Brunet A., Pouysségur J. Growth factors induce nuclear translocation of MAP kinases (p42mapk and p44mapk) but not of their activator MAP kinase kinase (p45mapkk) in fibroblasts. J Cell Biol. 1993 Sep;122(5):1079–1088. doi: 10.1083/jcb.122.5.1079. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Ley S. C., Marsh M., Bebbington C. R., Proudfoot K., Jordan P. Distinct intracellular localization of Lck and Fyn protein tyrosine kinases in human T lymphocytes. J Cell Biol. 1994 May;125(3):639–649. doi: 10.1083/jcb.125.3.639. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Manthey C. L., Brandes M. E., Perera P. Y., Vogel S. N. Taxol increases steady-state levels of lipopolysaccharide-inducible genes and protein-tyrosine phosphorylation in murine macrophages. J Immunol. 1992 Oct 1;149(7):2459–2465. [PubMed] [Google Scholar]
  30. Manthey C. L., Qureshi N., Stütz P. L., Vogel S. N. Lipopolysaccharide antagonists block taxol-induced signaling in murine macrophages. J Exp Med. 1993 Aug 1;178(2):695–702. doi: 10.1084/jem.178.2.695. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Nishida E., Gotoh Y. The MAP kinase cascade is essential for diverse signal transduction pathways. Trends Biochem Sci. 1993 Apr;18(4):128–131. doi: 10.1016/0968-0004(93)90019-j. [DOI] [PubMed] [Google Scholar]
  32. Northwood I. C., Gonzalez F. A., Wartmann M., Raden D. L., Davis R. J. Isolation and characterization of two growth factor-stimulated protein kinases that phosphorylate the epidermal growth factor receptor at threonine 669. J Biol Chem. 1991 Aug 15;266(23):15266–15276. [PubMed] [Google Scholar]
  33. Olesen O. F. Expression of low molecular weight isoforms of microtubule-associated protein 2. Phosphorylation and induction of microtubule assembly in vitro. J Biol Chem. 1994 Dec 30;269(52):32904–32908. [PubMed] [Google Scholar]
  34. Olmsted J. B. Microtubule-associated proteins. Annu Rev Cell Biol. 1986;2:421–457. doi: 10.1146/annurev.cb.02.110186.002225. [DOI] [PubMed] [Google Scholar]
  35. Ookata K., Hisanaga S., Bulinski J. C., Murofushi H., Aizawa H., Itoh T. J., Hotani H., Okumura E., Tachibana K., Kishimoto T. Cyclin B interaction with microtubule-associated protein 4 (MAP4) targets p34cdc2 kinase to microtubules and is a potential regulator of M-phase microtubule dynamics. J Cell Biol. 1995 Mar;128(5):849–862. doi: 10.1083/jcb.128.5.849. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Perez J., Tinelli D., Cagnoli C., Pecin P., Brunello N., Racagni G. Evidence for the existence of cAMP-dependent protein kinase phosphorylation system associated with specific phosphoproteins in stable microtubules from rat cerebral cortex. Brain Res. 1993 Jan 29;602(1):77–83. doi: 10.1016/0006-8993(93)90244-h. [DOI] [PubMed] [Google Scholar]
  37. Pulverer B. J., Kyriakis J. M., Avruch J., Nikolakaki E., Woodgett J. R. Phosphorylation of c-jun mediated by MAP kinases. Nature. 1991 Oct 17;353(6345):670–674. doi: 10.1038/353670a0. [DOI] [PubMed] [Google Scholar]
  38. Qui M. S., Green S. H. PC12 cell neuronal differentiation is associated with prolonged p21ras activity and consequent prolonged ERK activity. Neuron. 1992 Oct;9(4):705–717. doi: 10.1016/0896-6273(92)90033-a. [DOI] [PubMed] [Google Scholar]
  39. Rao S., Krauss N. E., Heerding J. M., Swindell C. S., Ringel I., Orr G. A., Horwitz S. B. 3'-(p-azidobenzamido)taxol photolabels the N-terminal 31 amino acids of beta-tubulin. J Biol Chem. 1994 Feb 4;269(5):3132–3134. [PubMed] [Google Scholar]
  40. Rao S., Orr G. A., Chaudhary A. G., Kingston D. G., Horwitz S. B. Characterization of the taxol binding site on the microtubule. 2-(m-Azidobenzoyl)taxol photolabels a peptide (amino acids 217-231) of beta-tubulin. J Biol Chem. 1995 Sep 1;270(35):20235–20238. doi: 10.1074/jbc.270.35.20235. [DOI] [PubMed] [Google Scholar]
  41. Reimann T., Büscher D., Hipskind R. A., Krautwald S., Lohmann-Matthes M. L., Baccarini M. Lipopolysaccharide induces activation of the Raf-1/MAP kinase pathway. A putative role for Raf-1 in the induction of the IL-1 beta and the TNF-alpha genes. J Immunol. 1994 Dec 15;153(12):5740–5749. [PubMed] [Google Scholar]
  42. Reszka A. A., Seger R., Diltz C. D., Krebs E. G., Fischer E. H. Association of mitogen-activated protein kinase with the microtubule cytoskeleton. Proc Natl Acad Sci U S A. 1995 Sep 12;92(19):8881–8885. doi: 10.1073/pnas.92.19.8881. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Robbins D. J., Zhen E., Cheng M., Xu S., Vanderbilt C. A., Ebert D., Garcia C., Dang A., Cobb M. H. Regulation and properties of extracellular signal-regulated protein kinases 1, 2, and 3. J Am Soc Nephrol. 1993 Nov;4(5):1104–1110. doi: 10.1681/ASN.V451104. [DOI] [PubMed] [Google Scholar]
  44. Seger R., Krebs E. G. The MAPK signaling cascade. FASEB J. 1995 Jun;9(9):726–735. [PubMed] [Google Scholar]
  45. Shibuya E. K., Ruderman J. V. Mos induces the in vitro activation of mitogen-activated protein kinases in lysates of frog oocytes and mammalian somatic cells. Mol Biol Cell. 1993 Aug;4(8):781–790. doi: 10.1091/mbc.4.8.781. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Sontag E., Nunbhakdi-Craig V., Bloom G. S., Mumby M. C. A novel pool of protein phosphatase 2A is associated with microtubules and is regulated during the cell cycle. J Cell Biol. 1995 Mar;128(6):1131–1144. doi: 10.1083/jcb.128.6.1131. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Sturgill T. W., Ray L. B., Erikson E., Maller J. L. Insulin-stimulated MAP-2 kinase phosphorylates and activates ribosomal protein S6 kinase II. Nature. 1988 Aug 25;334(6184):715–718. doi: 10.1038/334715a0. [DOI] [PubMed] [Google Scholar]
  48. Takishima K., Griswold-Prenner I., Ingebritsen T., Rosner M. R. Epidermal growth factor (EGF) receptor T669 peptide kinase from 3T3-L1 cells is an EGF-stimulated "MAP" kinase. Proc Natl Acad Sci U S A. 1991 Mar 15;88(6):2520–2524. doi: 10.1073/pnas.88.6.2520. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Tsai M., Chen R. H., Tam S. Y., Blenis J., Galli S. J. Activation of MAP kinases, pp90rsk and pp70-S6 kinases in mouse mast cells by signaling through the c-kit receptor tyrosine kinase or Fc epsilon RI: rapamycin inhibits activation of pp70-S6 kinase and proliferation in mouse mast cells. Eur J Immunol. 1993 Dec;23(12):3286–3291. doi: 10.1002/eji.1830231234. [DOI] [PubMed] [Google Scholar]
  50. Tsao H., Aletta J. M., Greene L. A. Nerve growth factor and fibroblast growth factor selectively activate a protein kinase that phosphorylates high molecular weight microtubule-associated proteins. Detection, partial purification, and characterization in PC12 cells. J Biol Chem. 1990 Sep 15;265(26):15471–15480. [PubMed] [Google Scholar]
  51. Vallee R. B. Purification of brain microtubules and microtubule-associated protein 1 using taxol. Methods Enzymol. 1986;134:104–115. doi: 10.1016/0076-6879(86)34079-5. [DOI] [PubMed] [Google Scholar]
  52. Verlhac M. H., de Pennart H., Maro B., Cobb M. H., Clarke H. J. MAP kinase becomes stably activated at metaphase and is associated with microtubule-organizing centers during meiotic maturation of mouse oocytes. Dev Biol. 1993 Aug;158(2):330–340. doi: 10.1006/dbio.1993.1192. [DOI] [PubMed] [Google Scholar]
  53. Weinstein S. L., Gold M. R., DeFranco A. L. Bacterial lipopolysaccharide stimulates protein tyrosine phosphorylation in macrophages. Proc Natl Acad Sci U S A. 1991 May 15;88(10):4148–4152. doi: 10.1073/pnas.88.10.4148. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Weinstein S. L., Sanghera J. S., Lemke K., DeFranco A. L., Pelech S. L. Bacterial lipopolysaccharide induces tyrosine phosphorylation and activation of mitogen-activated protein kinases in macrophages. J Biol Chem. 1992 Jul 25;267(21):14955–14962. [PubMed] [Google Scholar]
  55. Zhou R. P., Oskarsson M., Paules R. S., Schulz N., Cleveland D., Vande Woude G. F. Ability of the c-mos product to associate with and phosphorylate tubulin. Science. 1991 Feb 8;251(4994):671–675. doi: 10.1126/science.1825142. [DOI] [PubMed] [Google Scholar]
  56. van Bergen en Henegouwen P. M., den Hartigh J. C., Romeyn P., Verkleij A. J., Boonstra J. The epidermal growth factor receptor is associated with actin filaments. Exp Cell Res. 1992 Mar;199(1):90–97. doi: 10.1016/0014-4827(92)90465-k. [DOI] [PubMed] [Google Scholar]

Articles from The Journal of Experimental Medicine are provided here courtesy of The Rockefeller University Press

RESOURCES