Skip to main content
PMC home page
The EMBO Journal logoLink to The EMBO Journal
. 1998 Jan 2;17(1):149–158. doi: 10.1093/emboj/17.1.149

The MAP kinase kinase kinase MLK2 co-localizes with activated JNK along microtubules and associates with kinesin superfamily motor KIF3.

K Nagata 1, A Puls 1, C Futter 1, P Aspenstrom 1, E Schaefer 1, T Nakata 1, N Hirokawa 1, A Hall 1
PMCID: PMC1170366  PMID: 9427749

Abstract

The MLK (mixed lineage) ser/thr kinases are most closely related to the MAP kinase kinase kinase family. In addition to a kinase domain, MLK1, MLK2 and MLK3 each contain an SH3 domain, a leucine zipper domain and a potential Rac/Cdc42 GTPase-binding (CRIB) motif. The C-terminal regions of the proteins are essentially unrelated. Using yeast two-hybrid analysis and in vitro dot-blots, we show that MLK2 and MLK3 interact with the activated (GTP-bound) forms of Rac and Cdc42, with a slight preference for Rac. Transfection of MLK2 into COS cells leads to strong and constitutive activation of the JNK (c-Jun N-terminal kinase) MAP kinase cascade, but also to activation of ERK (extracellular signal-regulated kinase) and p38. When expressed in fibroblasts, MLK2 co-localizes with active, dually phosphorylated JNK1/2 to punctate structures along microtubules. In an attempt to identify proteins that affect the activity and localization of MLK2, we have screened a yeast two-hybrid cDNA library. MLK2 and MLK3 interact with members of the KIF3 family of kinesin superfamily motor proteins and with KAP3A, the putative targeting component of KIF3 motor complexes, suggesting a potential link between stress activation and motor protein function.

Full Text

The Full Text of this article is available as a PDF (781.3 KB).

Selected References

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

  1. Aitken A. 14-3-3 and its possible role in co-ordinating multiple signalling pathways. Trends Cell Biol. 1996 Sep;6(9):341–347. doi: 10.1016/0962-8924(96)10029-5. [DOI] [PubMed] [Google Scholar]
  2. Aizawa H., Sekine Y., Takemura R., Zhang Z., Nangaku M., Hirokawa N. Kinesin family in murine central nervous system. J Cell Biol. 1992 Dec;119(5):1287–1296. doi: 10.1083/jcb.119.5.1287. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Aspenström P., Olson M. F. Yeast two-hybrid system to detect protein-protein interactions with Rho GTPases. Methods Enzymol. 1995;256:228–241. doi: 10.1016/0076-6879(95)56027-0. [DOI] [PubMed] [Google Scholar]
  4. Bagrodia S., Dérijard B., Davis R. J., Cerione R. A. Cdc42 and PAK-mediated signaling leads to Jun kinase and p38 mitogen-activated protein kinase activation. J Biol Chem. 1995 Nov 24;270(47):27995–27998. doi: 10.1074/jbc.270.47.27995. [DOI] [PubMed] [Google Scholar]
  5. Brady S. T. A kinesin medley: biochemical and functional heterogeneity. Trends Cell Biol. 1995 Apr;5(4):159–164. doi: 10.1016/s0962-8924(00)88980-1. [DOI] [PubMed] [Google Scholar]
  6. Brown J. L., Stowers L., Baer M., Trejo J., Coughlin S., Chant J. Human Ste20 homologue hPAK1 links GTPases to the JNK MAP kinase pathway. Curr Biol. 1996 May 1;6(5):598–605. doi: 10.1016/s0960-9822(02)00546-8. [DOI] [PubMed] [Google Scholar]
  7. Burbelo P. D., Drechsel D., Hall A. A conserved binding motif defines numerous candidate target proteins for both Cdc42 and Rac GTPases. J Biol Chem. 1995 Dec 8;270(49):29071–29074. doi: 10.1074/jbc.270.49.29071. [DOI] [PubMed] [Google Scholar]
  8. Burkhardt J. K. In search of membrane receptors for microtubule-based motors - is kinectin a kinesin receptor? Trends Cell Biol. 1996 Apr;6(4):127–131. doi: 10.1016/0962-8924(96)20002-9. [DOI] [PubMed] [Google Scholar]
  9. Chang H. C., Rubin G. M. 14-3-3 epsilon positively regulates Ras-mediated signaling in Drosophila. Genes Dev. 1997 May 1;11(9):1132–1139. doi: 10.1101/gad.11.9.1132. [DOI] [PubMed] [Google Scholar]
  10. Coso O. A., Chiariello M., Yu J. C., Teramoto H., Crespo P., Xu N., Miki T., Gutkind J. S. The small GTP-binding proteins Rac1 and Cdc42 regulate the activity of the JNK/SAPK signaling pathway. Cell. 1995 Jun 30;81(7):1137–1146. doi: 10.1016/s0092-8674(05)80018-2. [DOI] [PubMed] [Google Scholar]
  11. Diekmann D., Nobes C. D., Burbelo P. D., Abo A., Hall A. Rac GTPase interacts with GAPs and target proteins through multiple effector sites. EMBO J. 1995 Nov 1;14(21):5297–5305. doi: 10.1002/j.1460-2075.1995.tb00214.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Dizhoor A. M., Ray S., Kumar S., Niemi G., Spencer M., Brolley D., Walsh K. A., Philipov P. P., Hurley J. B., Stryer L. Recoverin: a calcium sensitive activator of retinal rod guanylate cyclase. Science. 1991 Feb 22;251(4996):915–918. doi: 10.1126/science.1672047. [DOI] [PubMed] [Google Scholar]
  13. Fanger G. R., Johnson N. L., Johnson G. L. MEK kinases are regulated by EGF and selectively interact with Rac/Cdc42. EMBO J. 1997 Aug 15;16(16):4961–4972. doi: 10.1093/emboj/16.16.4961. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Galcheva-Gargova Z., Dérijard B., Wu I. H., Davis R. J. An osmosensing signal transduction pathway in mammalian cells. Science. 1994 Aug 5;265(5173):806–808. doi: 10.1126/science.8047888. [DOI] [PubMed] [Google Scholar]
  15. Gerwins P., Blank J. L., Johnson G. L. Cloning of a novel mitogen-activated protein kinase kinase kinase, MEKK4, that selectively regulates the c-Jun amino terminal kinase pathway. J Biol Chem. 1997 Mar 28;272(13):8288–8295. doi: 10.1074/jbc.272.13.8288. [DOI] [PubMed] [Google Scholar]
  16. Hirai S., Izawa M., Osada S., Spyrou G., Ohno S. Activation of the JNK pathway by distantly related protein kinases, MEKK and MUK. Oncogene. 1996 Feb 1;12(3):641–650. [PubMed] [Google Scholar]
  17. Hirokawa N. Organelle transport along microtubules - the role of KIFs. Trends Cell Biol. 1996 Apr;6(4):135–141. doi: 10.1016/0962-8924(96)10003-9. [DOI] [PubMed] [Google Scholar]
  18. Hotta K., Tanaka K., Mino A., Kohno H., Takai Y. Interaction of the Rho family small G proteins with kinectin, an anchoring protein of kinesin motor. Biochem Biophys Res Commun. 1996 Aug 5;225(1):69–74. doi: 10.1006/bbrc.1996.1132. [DOI] [PubMed] [Google Scholar]
  19. Ichijo H., Nishida E., Irie K., ten Dijke P., Saitoh M., Moriguchi T., Takagi M., Matsumoto K., Miyazono K., Gotoh Y. Induction of apoptosis by ASK1, a mammalian MAPKKK that activates SAPK/JNK and p38 signaling pathways. Science. 1997 Jan 3;275(5296):90–94. doi: 10.1126/science.275.5296.90. [DOI] [PubMed] [Google Scholar]
  20. Joneson T., McDonough M., Bar-Sagi D., Van Aelst L. RAC regulation of actin polymerization and proliferation by a pathway distinct from Jun kinase. Science. 1996 Nov 22;274(5291):1374–1376. doi: 10.1126/science.274.5291.1374. [DOI] [PubMed] [Google Scholar]
  21. Katoh M., Hirai M., Sugimura T., Terada M. Cloning and characterization of MST, a novel (putative) serine/threonine kinase with SH3 domain. Oncogene. 1995 Apr 6;10(7):1447–1451. [PubMed] [Google Scholar]
  22. Khokhlatchev A., Xu S., English J., Wu P., Schaefer E., Cobb M. H. Reconstitution of mitogen-activated protein kinase phosphorylation cascades in bacteria. Efficient synthesis of active protein kinases. J Biol Chem. 1997 Apr 25;272(17):11057–11062. doi: 10.1074/jbc.272.17.11057. [DOI] [PubMed] [Google Scholar]
  23. Kobayashi M., Takamatsu K., Saitoh S., Miura M., Noguchi T. Molecular cloning of hippocalcin, a novel calcium-binding protein of the recoverin family exclusively expressed in hippocampus. Biochem Biophys Res Commun. 1992 Nov 30;189(1):511–517. doi: 10.1016/0006-291x(92)91587-g. [DOI] [PubMed] [Google Scholar]
  24. Kobayashi M., Takamatsu K., Saitoh S., Noguchi T. Myristoylation of hippocalcin is linked to its calcium-dependent membrane association properties. J Biol Chem. 1993 Sep 5;268(25):18898–18904. [PubMed] [Google Scholar]
  25. Kockel L., Vorbrüggen G., Jäckle H., Mlodzik M., Bohmann D. Requirement for Drosophila 14-3-3 zeta in Raf-dependent photoreceptor development. Genes Dev. 1997 May 1;11(9):1140–1147. doi: 10.1101/gad.11.9.1140. [DOI] [PubMed] [Google Scholar]
  26. Kozma R., Ahmed S., Best A., Lim L. The Ras-related protein Cdc42Hs and bradykinin promote formation of peripheral actin microspikes and filopodia in Swiss 3T3 fibroblasts. Mol Cell Biol. 1995 Apr;15(4):1942–1952. doi: 10.1128/mcb.15.4.1942. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Kyriakis J. M., Avruch J. Protein kinase cascades activated by stress and inflammatory cytokines. Bioessays. 1996 Jul;18(7):567–577. doi: 10.1002/bies.950180708. [DOI] [PubMed] [Google Scholar]
  28. Lamarche N., Tapon N., Stowers L., Burbelo P. D., Aspenström P., Bridges T., Chant J., Hall A. Rac and Cdc42 induce actin polymerization and G1 cell cycle progression independently of p65PAK and the JNK/SAPK MAP kinase cascade. Cell. 1996 Nov 1;87(3):519–529. doi: 10.1016/s0092-8674(00)81371-9. [DOI] [PubMed] [Google Scholar]
  29. Leevers S. J., Paterson H. F., Marshall C. J. Requirement for Ras in Raf activation is overcome by targeting Raf to the plasma membrane. Nature. 1994 Jun 2;369(6479):411–414. doi: 10.1038/369411a0. [DOI] [PubMed] [Google Scholar]
  30. Manser E., Leung T., Salihuddin H., Zhao Z. S., Lim L. A brain serine/threonine protein kinase activated by Cdc42 and Rac1. Nature. 1994 Jan 6;367(6458):40–46. doi: 10.1038/367040a0. [DOI] [PubMed] [Google Scholar]
  31. Marais R., Light Y., Paterson H. F., Marshall C. J. Ras recruits Raf-1 to the plasma membrane for activation by tyrosine phosphorylation. EMBO J. 1995 Jul 3;14(13):3136–3145. doi: 10.1002/j.1460-2075.1995.tb07316.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. 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]
  33. Minden A., Lin A., Claret F. X., Abo A., Karin M. Selective activation of the JNK signaling cascade and c-Jun transcriptional activity by the small GTPases Rac and Cdc42Hs. Cell. 1995 Jun 30;81(7):1147–1157. doi: 10.1016/s0092-8674(05)80019-4. [DOI] [PubMed] [Google Scholar]
  34. Moriguchi T., Kuroyanagi N., Yamaguchi K., Gotoh Y., Irie K., Kano T., Shirakabe K., Muro Y., Shibuya H., Matsumoto K. A novel kinase cascade mediated by mitogen-activated protein kinase kinase 6 and MKK3. J Biol Chem. 1996 Jun 7;271(23):13675–13679. doi: 10.1074/jbc.271.23.13675. [DOI] [PubMed] [Google Scholar]
  35. Nobes C. D., Hall A. Rho, rac, and cdc42 GTPases regulate the assembly of multimolecular focal complexes associated with actin stress fibers, lamellipodia, and filopodia. Cell. 1995 Apr 7;81(1):53–62. doi: 10.1016/0092-8674(95)90370-4. [DOI] [PubMed] [Google Scholar]
  36. Olson M. F., Ashworth A., Hall A. An essential role for Rho, Rac, and Cdc42 GTPases in cell cycle progression through G1. Science. 1995 Sep 1;269(5228):1270–1272. doi: 10.1126/science.7652575. [DOI] [PubMed] [Google Scholar]
  37. Peter M., Neiman A. M., Park H. O., van Lohuizen M., Herskowitz I. Functional analysis of the interaction between the small GTP binding protein Cdc42 and the Ste20 protein kinase in yeast. EMBO J. 1996 Dec 16;15(24):7046–7059. [PMC free article] [PubMed] [Google Scholar]
  38. Rana A., Gallo K., Godowski P., Hirai S., Ohno S., Zon L., Kyriakis J. M., Avruch J. The mixed lineage kinase SPRK phosphorylates and activates the stress-activated protein kinase activator, SEK-1. J Biol Chem. 1996 Aug 9;271(32):19025–19028. doi: 10.1074/jbc.271.32.19025. [DOI] [PubMed] [Google Scholar]
  39. Ridley A. J., Paterson H. F., Johnston C. L., Diekmann D., Hall A. The small GTP-binding protein rac regulates growth factor-induced membrane ruffling. Cell. 1992 Aug 7;70(3):401–410. doi: 10.1016/0092-8674(92)90164-8. [DOI] [PubMed] [Google Scholar]
  40. Robbins D. J., Nybakken K. E., Kobayashi R., Sisson J. C., Bishop J. M., Thérond P. P. Hedgehog elicits signal transduction by means of a large complex containing the kinesin-related protein costal2. Cell. 1997 Jul 25;90(2):225–234. doi: 10.1016/s0092-8674(00)80331-1. [DOI] [PubMed] [Google Scholar]
  41. Robinson M. J., Cobb M. H. Mitogen-activated protein kinase pathways. Curr Opin Cell Biol. 1997 Apr;9(2):180–186. doi: 10.1016/s0955-0674(97)80061-0. [DOI] [PubMed] [Google Scholar]
  42. Salmeron A., Ahmad T. B., Carlile G. W., Pappin D., Narsimhan R. P., Ley S. C. Activation of MEK-1 and SEK-1 by Tpl-2 proto-oncoprotein, a novel MAP kinase kinase kinase. EMBO J. 1996 Feb 15;15(4):817–826. [PMC free article] [PubMed] [Google Scholar]
  43. Stokoe D., Macdonald S. G., Cadwallader K., Symons M., Hancock J. F. Activation of Raf as a result of recruitment to the plasma membrane. Science. 1994 Jun 3;264(5164):1463–1467. doi: 10.1126/science.7811320. [DOI] [PubMed] [Google Scholar]
  44. Tabish M., Siddiqui Z. K., Nishikawa K., Siddiqui S. S. Exclusive expression of C. elegans osm-3 kinesin gene in chemosensory neurons open to the external environment. J Mol Biol. 1995 Mar 31;247(3):377–389. doi: 10.1006/jmbi.1994.0146. [DOI] [PubMed] [Google Scholar]
  45. Teramoto H., Coso O. A., Miyata H., Igishi T., Miki T., Gutkind J. S. Signaling from the small GTP-binding proteins Rac1 and Cdc42 to the c-Jun N-terminal kinase/stress-activated protein kinase pathway. A role for mixed lineage kinase 3/protein-tyrosine kinase 1, a novel member of the mixed lineage kinase family. J Biol Chem. 1996 Nov 1;271(44):27225–27228. doi: 10.1074/jbc.271.44.27225. [DOI] [PubMed] [Google Scholar]
  46. Tibbles L. A., Ing Y. L., Kiefer F., Chan J., Iscove N., Woodgett J. R., Lassam N. J. MLK-3 activates the SAPK/JNK and p38/RK pathways via SEK1 and MKK3/6. EMBO J. 1996 Dec 16;15(24):7026–7035. [PMC free article] [PubMed] [Google Scholar]
  47. Waskiewicz A. J., Cooper J. A. Mitogen and stress response pathways: MAP kinase cascades and phosphatase regulation in mammals and yeast. Curr Opin Cell Biol. 1995 Dec;7(6):798–805. doi: 10.1016/0955-0674(95)80063-8. [DOI] [PubMed] [Google Scholar]
  48. Westwick J. K., Lambert Q. T., Clark G. J., Symons M., Van Aelst L., Pestell R. G., Der C. J. Rac regulation of transformation, gene expression, and actin organization by multiple, PAK-independent pathways. Mol Cell Biol. 1997 Mar;17(3):1324–1335. doi: 10.1128/mcb.17.3.1324. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Yamazaki H., Nakata T., Okada Y., Hirokawa N. Cloning and characterization of KAP3: a novel kinesin superfamily-associated protein of KIF3A/3B. Proc Natl Acad Sci U S A. 1996 Aug 6;93(16):8443–8448. doi: 10.1073/pnas.93.16.8443. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Yamazaki H., Nakata T., Okada Y., Hirokawa N. KIF3A/B: a heterodimeric kinesin superfamily protein that works as a microtubule plus end-directed motor for membrane organelle transport. J Cell Biol. 1995 Sep;130(6):1387–1399. doi: 10.1083/jcb.130.6.1387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Zhang S., Han J., Sells M. A., Chernoff J., Knaus U. G., Ulevitch R. J., Bokoch G. M. Rho family GTPases regulate p38 mitogen-activated protein kinase through the downstream mediator Pak1. J Biol Chem. 1995 Oct 13;270(41):23934–23936. doi: 10.1074/jbc.270.41.23934. [DOI] [PubMed] [Google Scholar]

Articles from The EMBO Journal are provided here courtesy of Nature Publishing Group

RESOURCES