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. 1995 Mar 1;181(3):1071–1079. doi: 10.1084/jem.181.3.1071

Nerve growth factor triggers microfilament assembly and paxillin phosphorylation in human B lymphocytes

PMCID: PMC2191930  PMID: 7869029

Abstract

Increasing evidence suggests that the nervous system is involved in allergic inflammation. One of the potential regulatory molecules of the neuroimmune system is nerve growth factor (NGF). Recent studies from our group demonstrated the presence of a functional NGF receptor (NGFR) on human B lymphocytes. Moreover, we showed that gp140trk tyrosine kinase, which serves as an NGFR, was involved in transduction of early signaling events in human B lymphocytes. The mechanisms by which NGF initiates the signaling cascade and the link between the neuroimmune systems are unknown. We have focused on the role of the cytoskeleton as a possible mediator for transduction of signals induced by NGF. Polymerized actin (F-actin) content was determined by fluorescent staining and immunoblotting with antiactin antibody. Addition of NGF caused a time- and concentration-dependent increase in F-actin content, and maximum effects were noted after 1 min. These increases in F-actin content and NGF-induced thymidine incorporation could be blocked by incubating the cells with cytochalasin D and botulinum C2 toxin before the addition of NGF. Incubation of human B lymphocytes with 10 nM K252a, an inhibitor of Trk kinase, decreased NGF-induced microfilament assembly by 75%. In immunoprecipitation experiments, addition of NGF to B cells induced a rapid increase in the tyrosine phosphorylation of paxillin, one of a group of focal adhesion proteins involved in linking actin filaments to the plasma membrane. Coimmunoprecipitation studies demonstrated the association between gp140trk kinase and paxillin. Together, these observations suggest that actin assembly is involved in NGF signaling in human B cells, and that paxillin may be essential in this pathway after phosphorylation by gp140trk kinase.

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Selected References

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  1. Aktories K., Bärmann M., Ohishi I., Tsuyama S., Jakobs K. H., Habermann E. Botulinum C2 toxin ADP-ribosylates actin. Nature. 1986 Jul 24;322(6077):390–392. doi: 10.1038/322390a0. [DOI] [PubMed] [Google Scholar]
  2. Bar-Sagi D., Rotin D., Batzer A., Mandiyan V., Schlessinger J. SH3 domains direct cellular localization of signaling molecules. Cell. 1993 Jul 16;74(1):83–91. doi: 10.1016/0092-8674(93)90296-3. [DOI] [PubMed] [Google Scholar]
  3. Barber B. H., Delovitch T. L. The identification of actin as a major lymphocyte component. J Immunol. 1979 Jan;122(1):320–325. [PubMed] [Google Scholar]
  4. Birge R. B., Fajardo J. E., Reichman C., Shoelson S. E., Songyang Z., Cantley L. C., Hanafusa H. Identification and characterization of a high-affinity interaction between v-Crk and tyrosine-phosphorylated paxillin in CT10-transformed fibroblasts. Mol Cell Biol. 1993 Aug;13(8):4648–4656. doi: 10.1128/mcb.13.8.4648. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Brodie C., Gelfand E. W. Functional nerve growth factor receptors on human B lymphocytes. Interaction with IL-2. J Immunol. 1992 Jun 1;148(11):3492–3497. [PubMed] [Google Scholar]
  6. Burridge K., Fath K., Kelly T., Nuckolls G., Turner C. Focal adhesions: transmembrane junctions between the extracellular matrix and the cytoskeleton. Annu Rev Cell Biol. 1988;4:487–525. doi: 10.1146/annurev.cb.04.110188.002415. [DOI] [PubMed] [Google Scholar]
  7. Burridge K., Turner C. E., Romer L. H. Tyrosine phosphorylation of paxillin and pp125FAK accompanies cell adhesion to extracellular matrix: a role in cytoskeletal assembly. J Cell Biol. 1992 Nov;119(4):893–903. doi: 10.1083/jcb.119.4.893. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Corps A. N., Metcalfe J. C., Pozzan T. Kinetic evidence for a common mechanism of capping on lymphocytes. Biochem J. 1982 Apr 15;204(1):229–237. doi: 10.1042/bj2040229. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Davies A. M., Lee K. F., Jaenisch R. p75-deficient trigeminal sensory neurons have an altered response to NGF but not to other neurotrophins. Neuron. 1993 Oct;11(4):565–574. doi: 10.1016/0896-6273(93)90069-4. [DOI] [PubMed] [Google Scholar]
  10. Felten D. L., Felten S. Y., Bellinger D. L., Carlson S. L., Ackerman K. D., Madden K. S., Olschowki J. A., Livnat S. Noradrenergic sympathetic neural interactions with the immune system: structure and function. Immunol Rev. 1987 Dec;100:225–260. doi: 10.1111/j.1600-065x.1987.tb00534.x. [DOI] [PubMed] [Google Scholar]
  11. Howard T. H., Oresajo C. O. The kinetics of chemotactic peptide-induced change in F-actin content, F-actin distribution, and the shape of neutrophils. J Cell Biol. 1985 Sep;101(3):1078–1085. doi: 10.1083/jcb.101.3.1078. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Jing S., Tapley P., Barbacid M. Nerve growth factor mediates signal transduction through trk homodimer receptors. Neuron. 1992 Dec;9(6):1067–1079. doi: 10.1016/0896-6273(92)90066-m. [DOI] [PubMed] [Google Scholar]
  13. Kaplan D. R., Hempstead B. L., Martin-Zanca D., Chao M. V., Parada L. F. The trk proto-oncogene product: a signal transducing receptor for nerve growth factor. Science. 1991 Apr 26;252(5005):554–558. doi: 10.1126/science.1850549. [DOI] [PubMed] [Google Scholar]
  14. Kaplan D. R., Martin-Zanca D., Parada L. F. Tyrosine phosphorylation and tyrosine kinase activity of the trk proto-oncogene product induced by NGF. Nature. 1991 Mar 14;350(6314):158–160. doi: 10.1038/350158a0. [DOI] [PubMed] [Google Scholar]
  15. Klein R., Jing S. Q., Nanduri V., O'Rourke E., Barbacid M. The trk proto-oncogene encodes a receptor for nerve growth factor. Cell. 1991 Apr 5;65(1):189–197. doi: 10.1016/0092-8674(91)90419-y. [DOI] [PubMed] [Google Scholar]
  16. Loeb D. M., Greene L. A. Transfection with trk restores "slow" NGF binding, efficient NGF uptake, and multiple NGF responses to NGF-nonresponsive PC12 cell mutants. J Neurosci. 1993 Jul;13(7):2919–2929. doi: 10.1523/JNEUROSCI.13-07-02919.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Loeb D. M., Maragos J., Martin-Zanca D., Chao M. V., Parada L. F., Greene L. A. The trk proto-oncogene rescues NGF responsiveness in mutant NGF-nonresponsive PC12 cell lines. Cell. 1991 Sep 6;66(5):961–966. doi: 10.1016/0092-8674(91)90441-z. [DOI] [PubMed] [Google Scholar]
  18. Melamed I., Downey G. P., Aktories K., Roifman C. M. Microfilament assembly is required for antigen-receptor-mediated activation of human B lymphocytes. J Immunol. 1991 Aug 15;147(4):1139–1146. [PubMed] [Google Scholar]
  19. Melamed I., Downey G. P., Roifman C. M. Tyrosine phosphorylation is essential for microfilament assembly in B lymphocytes. Biochem Biophys Res Commun. 1991 May 15;176(3):1424–1429. doi: 10.1016/0006-291x(91)90445-d. [DOI] [PubMed] [Google Scholar]
  20. Melamed I., Wang G., Roifman C. M. Antigen receptor-mediated protein tyrosine kinase activity is regulated by a pertussis toxin-sensitive G protein. J Immunol. 1992 Jul 1;149(1):169–174. [PubMed] [Google Scholar]
  21. Ohmichi M., Matuoka K., Takenawa T., Saltiel A. R. Growth factors differentially stimulate the phosphorylation of Shc proteins and their association with Grb2 in PC-12 pheochromocytoma cells. J Biol Chem. 1994 Jan 14;269(2):1143–1148. [PubMed] [Google Scholar]
  22. Otten U., Ehrhard P., Peck R. Nerve growth factor induces growth and differentiation of human B lymphocytes. Proc Natl Acad Sci U S A. 1989 Dec;86(24):10059–10063. doi: 10.1073/pnas.86.24.10059. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Paves H., Neuman T., Metsis M., Saarma M. Nerve growth factor-induced rapid reorganization of microfilaments in PC12 cells: possible roles of different second messenger systems. Exp Cell Res. 1990 Feb;186(2):218–226. doi: 10.1016/0014-4827(90)90299-p. [DOI] [PubMed] [Google Scholar]
  24. Rao K. M. Capping and mitogenesis: a model implicating microfilaments in lymphocyte activation. J Theor Biol. 1982 Sep 7;98(1):61–71. doi: 10.1016/0022-5193(82)90058-3. [DOI] [PubMed] [Google Scholar]
  25. Soltoff S. P., Rabin S. L., Cantley L. C., Kaplan D. R. Nerve growth factor promotes the activation of phosphatidylinositol 3-kinase and its association with the trk tyrosine kinase. J Biol Chem. 1992 Aug 25;267(24):17472–17477. [PubMed] [Google Scholar]
  26. Spoerri P. E., Roisen F. J. Cytoskeletal elements regulate the distribution of nerve growth factor receptors in PC12 cells. J Neurosci Res. 1992 Mar;31(3):494–501. doi: 10.1002/jnr.490310312. [DOI] [PubMed] [Google Scholar]
  27. Tapley P., Lamballe F., Barbacid M. K252a is a selective inhibitor of the tyrosine protein kinase activity of the trk family of oncogenes and neurotrophin receptors. Oncogene. 1992 Feb;7(2):371–381. [PubMed] [Google Scholar]
  28. Turner C. E., Glenney J. R., Jr, Burridge K. Paxillin: a new vinculin-binding protein present in focal adhesions. J Cell Biol. 1990 Sep;111(3):1059–1068. doi: 10.1083/jcb.111.3.1059. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Turner C. E., Miller J. T. Primary sequence of paxillin contains putative SH2 and SH3 domain binding motifs and multiple LIM domains: identification of a vinculin and pp125Fak-binding region. J Cell Sci. 1994 Jun;107(Pt 6):1583–1591. doi: 10.1242/jcs.107.6.1583. [DOI] [PubMed] [Google Scholar]
  30. Turner C. E. Paxillin is a major phosphotyrosine-containing protein during embryonic development. J Cell Biol. 1991 Oct;115(1):201–207. doi: 10.1083/jcb.115.1.201. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Turner C. E. Paxillin: a cytoskeletal target for tyrosine kinases. Bioessays. 1994 Jan;16(1):47–52. doi: 10.1002/bies.950160107. [DOI] [PubMed] [Google Scholar]
  32. Ullrich A., Schlessinger J. Signal transduction by receptors with tyrosine kinase activity. Cell. 1990 Apr 20;61(2):203–212. doi: 10.1016/0092-8674(90)90801-k. [DOI] [PubMed] [Google Scholar]
  33. Vale R. D., Ignatius M. J., Shooter E. M. Association of nerve growth factor receptors with the triton X-100 cytoskeleton of PC12 cells. J Neurosci. 1985 Oct;5(10):2762–2770. doi: 10.1523/JNEUROSCI.05-10-02762.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Weng Z., Taylor J. A., Turner C. E., Brugge J. S., Seidel-Dugan C. Detection of Src homology 3-binding proteins, including paxillin, in normal and v-Src-transformed Balb/c 3T3 cells. J Biol Chem. 1993 Jul 15;268(20):14956–14963. [PubMed] [Google Scholar]
  35. Zachary I., Sinnett-Smith J., Rozengurt E. Bombesin, vasopressin, and endothelin stimulation of tyrosine phosphorylation in Swiss 3T3 cells. Identification of a novel tyrosine kinase as a major substrate. J Biol Chem. 1992 Sep 25;267(27):19031–19034. [PubMed] [Google Scholar]
  36. Zachary I., Sinnett-Smith J., Turner C. E., Rozengurt E. Bombesin, vasopressin, and endothelin rapidly stimulate tyrosine phosphorylation of the focal adhesion-associated protein paxillin in Swiss 3T3 cells. J Biol Chem. 1993 Oct 15;268(29):22060–22065. [PubMed] [Google Scholar]

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