Skip to main content
NCBI home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1995 Apr 1;129(1):147–156. doi: 10.1083/jcb.129.1.147

Effects of CapG overexpression on agonist-induced motility and second messenger generation

PMCID: PMC2120377  PMID: 7698981

Abstract

Actin modulating proteins that bind polyphosphoinositides, such as phosphatidylinositol 4, 5-bisphosphate (PIP2), can potentially participate in receptor signaling by restructuring the membrane cytoskeleton and modulating second messenger generation through the phosphoinositide cycle. We examined these possibilities by overexpressing CapG, an actin filament end capping, Ca(2+)- and polyphosphoinositide-binding protein of the gelsolin family. High level transient overexpression decreased actin filament staining in the center of the cells but not in the cell periphery. Moderate overexpression in clonally selected cell lines did not have a detectible effect on actin filament content or organization. Nevertheless, it promoted a dose-dependent increase in rates of wound healing and chemotaxis. The motile phenotype was similar to that observed with gelsolin overexpression, which in addition to capping, also severs and nucleates actin filaments. CapG overexpressing clones are more responsive to platelet-derived growth factor than control- transfected clones. They form more circular dorsal membrane ruffles, have higher phosphoinositide turnover, inositol 1,4,5-trisphosphate generation and Ca2+ signaling. These responses are consistent with enhanced PLC gamma activity. Direct measurements of PIP2 mass showed that the CapG effect on PLC gamma was not due primarily to an increase in the PIP2 substrate concentration. The observed changes in cell motility and membrane signaling are consistent with the hypothesis that PIP(2)-binding actin regulatory proteins modulate phosphoinositide turnover and second messenger generation in vivo. We infer that CapG and related proteins are poised to coordinate membrane signaling with actin filament dynamics following cell stimulation.

Full Text

The Full Text of this article is available as a PDF (2.7 MB).

Selected References

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

  1. Banno Y., Nakashima T., Kumada T., Ebisawa K., Nonomura Y., Nozawa Y. Effects of gelsolin on human platelet cytosolic phosphoinositide-phospholipase C isozymes. J Biol Chem. 1992 Apr 5;267(10):6488–6494. [PubMed] [Google Scholar]
  2. Carlier M. F., Pantaloni D. Actin assembly in response to extracellular signals: role of capping proteins, thymosin beta 4 and profilin. Semin Cell Biol. 1994 Jun;5(3):183–191. doi: 10.1006/scel.1994.1023. [DOI] [PubMed] [Google Scholar]
  3. Cayatte A. J., Kumbla L., Subbiah M. T. Marked acceleration of exogenous fatty acid incorporation into cellular triglycerides by fetuin. J Biol Chem. 1990 Apr 5;265(10):5883–5888. [PubMed] [Google Scholar]
  4. Chilvers E. R., Batty I. H., Challiss R. A., Barnes P. J., Nahorski S. R. Determination of mass changes in phosphatidylinositol 4,5-bisphosphate and evidence for agonist-stimulated metabolism of inositol 1,4,5-trisphosphate in airway smooth muscle. Biochem J. 1991 Apr 15;275(Pt 2):373–379. doi: 10.1042/bj2750373. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Cunningham C. C., Stossel T. P., Kwiatkowski D. J. Enhanced motility in NIH 3T3 fibroblasts that overexpress gelsolin. Science. 1991 Mar 8;251(4998):1233–1236. doi: 10.1126/science.1848726. [DOI] [PubMed] [Google Scholar]
  6. Dabiri G. A., Young C. L., Rosenbloom J., Southwick F. S. Molecular cloning of human macrophage capping protein cDNA. A unique member of the gelsolin/villin family expressed primarily in macrophages. J Biol Chem. 1992 Aug 15;267(23):16545–16552. [PubMed] [Google Scholar]
  7. Downes C. P., Hawkins P. T., Irvine R. F. Inositol 1,3,4,5-tetrakisphosphate and not phosphatidylinositol 3,4-bisphosphate is the probable precursor of inositol 1,3,4-trisphosphate in agonist-stimulated parotid gland. Biochem J. 1986 Sep 1;238(2):501–506. doi: 10.1042/bj2380501. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Finkel T., Theriot J. A., Dise K. R., Tomaselli G. F., Goldschmidt-Clermont P. J. Dynamic actin structures stabilized by profilin. Proc Natl Acad Sci U S A. 1994 Feb 15;91(4):1510–1514. doi: 10.1073/pnas.91.4.1510. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Füchtbauer A., Jockusch B. M., Maruta H., Kilimann M. W., Isenberg G. Disruption of microfilament organization after injection of F-actin capping proteins into living tissue culture cells. 1983 Jul 28-Aug 3Nature. 304(5924):361–364. doi: 10.1038/304361a0. [DOI] [PubMed] [Google Scholar]
  10. Goldschmidt-Clermont P. J., Kim J. W., Machesky L. M., Rhee S. G., Pollard T. D. Regulation of phospholipase C-gamma 1 by profilin and tyrosine phosphorylation. Science. 1991 Mar 8;251(4998):1231–1233. doi: 10.1126/science.1848725. [DOI] [PubMed] [Google Scholar]
  11. Goldschmidt-Clermont P. J., Machesky L. M., Baldassare J. J., Pollard T. D. The actin-binding protein profilin binds to PIP2 and inhibits its hydrolysis by phospholipase C. Science. 1990 Mar 30;247(4950):1575–1578. doi: 10.1126/science.2157283. [DOI] [PubMed] [Google Scholar]
  12. Gunning P., Leavitt J., Muscat G., Ng S. Y., Kedes L. A human beta-actin expression vector system directs high-level accumulation of antisense transcripts. Proc Natl Acad Sci U S A. 1987 Jul;84(14):4831–4835. doi: 10.1073/pnas.84.14.4831. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hedberg K. M., Bengtsson T., Safiejko-Mroczka B., Bell P. B., Lindroth M. PDGF and neomycin induce similar changes in the actin cytoskeleton in human fibroblasts. Cell Motil Cytoskeleton. 1993;24(2):139–149. doi: 10.1002/cm.970240207. [DOI] [PubMed] [Google Scholar]
  14. Huang C. L., Takenawa T., Ives H. E. Platelet-derived growth factor-mediated Ca2+ entry is blocked by antibodies to phosphatidylinositol 4,5-bisphosphate but does not involve heparin-sensitive inositol 1,4,5-trisphosphate receptors. J Biol Chem. 1991 Mar 5;266(7):4045–4048. [PubMed] [Google Scholar]
  15. Janmey P. A., Lamb J., Allen P. G., Matsudaira P. T. Phosphoinositide-binding peptides derived from the sequences of gelsolin and villin. J Biol Chem. 1992 Jun 15;267(17):11818–11823. [PubMed] [Google Scholar]
  16. Margolis B., Zilberstein A., Franks C., Felder S., Kremer S., Ullrich A., Rhee S. G., Skorecki K., Schlessinger J. Effect of phospholipase C-gamma overexpression on PDGF-induced second messengers and mitogenesis. Science. 1990 May 4;248(4955):607–610. doi: 10.1126/science.2333512. [DOI] [PubMed] [Google Scholar]
  17. Minta A., Kao J. P., Tsien R. Y. Fluorescent indicators for cytosolic calcium based on rhodamine and fluorescein chromophores. J Biol Chem. 1989 May 15;264(14):8171–8178. [PubMed] [Google Scholar]
  18. Norman J. C., Price L. S., Ridley A. J., Hall A., Koffer A. Actin filament organization in activated mast cells is regulated by heterotrimeric and small GTP-binding proteins. J Cell Biol. 1994 Aug;126(4):1005–1015. doi: 10.1083/jcb.126.4.1005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Onoda K., Yin H. L. gCap39 is phosphorylated. Stimulation by okadaic acid and preferential association with nuclei. J Biol Chem. 1993 Feb 25;268(6):4106–4112. [PubMed] [Google Scholar]
  20. Onoda K., Yu F. X., Yin H. L. gCap39 is a nuclear and cytoplasmic protein. Cell Motil Cytoskeleton. 1993;26(3):227–238. doi: 10.1002/cm.970260306. [DOI] [PubMed] [Google Scholar]
  21. Prendergast G. C., Ziff E. B. Mbh 1: a novel gelsolin/severin-related protein which binds actin in vitro and exhibits nuclear localization in vivo. EMBO J. 1991 Apr;10(4):757–766. doi: 10.1002/j.1460-2075.1991.tb08007.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Rhee S. G. Inositol phospholipids-specific phospholipase C: interaction of the gamma 1 isoform with tyrosine kinase. Trends Biochem Sci. 1991 Aug;16(8):297–301. doi: 10.1016/0968-0004(91)90122-c. [DOI] [PubMed] [Google Scholar]
  23. Shariff A., Luna E. J. Diacylglycerol-stimulated formation of actin nucleation sites at plasma membranes. Science. 1992 Apr 10;256(5054):245–247. doi: 10.1126/science.1373523. [DOI] [PubMed] [Google Scholar]
  24. Sharps E. S., McCarl R. L. A high-performance liquid chromatographic method to measure 32P incorporation into phosphorylated metabolites in cultured cells. Anal Biochem. 1982 Aug;124(2):421–424. doi: 10.1016/0003-2697(82)90059-8. [DOI] [PubMed] [Google Scholar]
  25. Simões A. P., Schnabel P., Pipkorn R., Camps M., Gierschik P. A peptide corresponding to a potential polyphosphoinositide binding site of phospholipase C-beta 2 enhances its catalytic activity. FEBS Lett. 1993 Oct 4;331(3):248–251. doi: 10.1016/0014-5793(93)80346-v. [DOI] [PubMed] [Google Scholar]
  26. Sohn R. H., Goldschmidt-Clermont P. J. Profilin: at the crossroads of signal transduction and the actin cytoskeleton. Bioessays. 1994 Jul;16(7):465–472. doi: 10.1002/bies.950160705. [DOI] [PubMed] [Google Scholar]
  27. Southwick F. S., DiNubile M. J. Rabbit alveolar macrophages contain a Ca2+-sensitive, 41,000-dalton protein which reversibly blocks the "barbed" ends of actin filaments but does not sever them. J Biol Chem. 1986 Oct 25;261(30):14191–14195. [PubMed] [Google Scholar]
  28. Stossel T. P. On the crawling of animal cells. Science. 1993 May 21;260(5111):1086–1094. doi: 10.1126/science.8493552. [DOI] [PubMed] [Google Scholar]
  29. Sun H. Q., Kwiatkowska K., Yin H. L. Actin monomer binding proteins. Curr Opin Cell Biol. 1995 Feb;7(1):102–110. doi: 10.1016/0955-0674(95)80051-4. [DOI] [PubMed] [Google Scholar]
  30. Theriot J. A., Mitchison T. J. The three faces of profilin. Cell. 1993 Dec 3;75(5):835–838. doi: 10.1016/0092-8674(93)90527-w. [DOI] [PubMed] [Google Scholar]
  31. Weeds A., Maciver S. F-actin capping proteins. Curr Opin Cell Biol. 1993 Feb;5(1):63–69. doi: 10.1016/s0955-0674(05)80009-2. [DOI] [PubMed] [Google Scholar]
  32. Yin H. L. Gelsolin: calcium- and polyphosphoinositide-regulated actin-modulating protein. Bioessays. 1987 Oct;7(4):176–179. doi: 10.1002/bies.950070409. [DOI] [PubMed] [Google Scholar]
  33. Young C. L., Feierstein A., Southwick F. S. Calcium regulation of actin filament capping and monomer binding by macrophage capping protein. J Biol Chem. 1994 May 13;269(19):13997–14002. [PubMed] [Google Scholar]
  34. Young C. L., Southwick F. S., Weber A. Kinetics of the interaction of a 41-kilodalton macrophage capping protein with actin: promotion of nucleation during prolongation of the lag period. Biochemistry. 1990 Mar 6;29(9):2232–2240. doi: 10.1021/bi00461a005. [DOI] [PubMed] [Google Scholar]
  35. Yu F. X., Johnston P. A., Südhof T. C., Yin H. L. gCap39, a calcium ion- and polyphosphoinositide-regulated actin capping protein. Science. 1990 Dec 7;250(4986):1413–1415. doi: 10.1126/science.2255912. [DOI] [PubMed] [Google Scholar]
  36. Yu F. X., Lin S. C., Morrison-Bogorad M., Yin H. L. Effects of thymosin beta 4 and thymosin beta 10 on actin structures in living cells. Cell Motil Cytoskeleton. 1994;27(1):13–25. doi: 10.1002/cm.970270103. [DOI] [PubMed] [Google Scholar]
  37. Yu F. X., Sun H. Q., Janmey P. A., Yin H. L. Identification of a polyphosphoinositide-binding sequence in an actin monomer-binding domain of gelsolin. J Biol Chem. 1992 Jul 25;267(21):14616–14621. [PubMed] [Google Scholar]
  38. Yu F. X., Zhou D. M., Yin H. L. Chimeric and truncated gCap39 elucidate the requirements for actin filament severing and end capping by the gelsolin family of proteins. J Biol Chem. 1991 Oct 15;266(29):19269–19275. [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES