Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 1991 Sep 1;278(Pt 2):447–452. doi: 10.1042/bj2780447

Characterization of the tyrosine phosphorylation of calpactin I (annexin II) induced by platelet-derived growth factor.

R Brambilla 1, R Zippel 1, E Sturani 1, L Morello 1, A Peres 1, L Alberghina 1
PMCID: PMC1151364  PMID: 1654883

Abstract

Stimulation in vivo of Swiss 3T3 fibroblasts with platelet-derived growth factor (PDGF) in the presence of orthovanadate induces the tyrosine phosphorylation of a 39 kDa protein, identified as the phosphorylated slow-migrating form of calpactin I (annexin II) heavy chain, p36. In fact, in PDGF-stimulated cells, anti-(calpactin I) antibodies recognize a doublet of bands, p36 and p39, and the latter disappears upon treatment with phosphatase. In many regards phosphorylation of p39 differs from the rapid and transient phosphorylation of the PDGF receptor and of other substrates: (a) it has slower kinetics but is then stable for longer periods of time; (b) it occurs at 37 degrees C but not at 4 degrees C; and (c) whereas most of the tyrosine-phosphorylated proteins are associated with membrane-enriched preparations, membrane association of p39 only occurs in the presence of Ca2+. Moreover, calpactin I leaks out of permeabilized cells at 0.1 microM free Ca2+, whereas it remains associated with the cells at concentrations of Ca2+ greater than or equal to 1 microM. PDGF does not stimulate phosphoinositide turnover (and thus Ca2+ mobilization) at 4 degrees C; thus it can be suggested that the Ca(2+)-dependent translocation of the protein to membrane/cytoskeletal structures is a necessary condition for its phosphorylation. In addition, calpactin I may not be a direct substrate for the PDGF receptor kinase, but rather the substrate of another tyrosine kinase activated by the receptor.

Full text

PDF
447

Images in this article

448Fig. 1.
on p.448
449Fig. 2.
on p.449
449Fig. 3.
on p.449
450Fig. 4.
on p.450
450Fig. 5.
on p.450
450Fig. 6.
on p.450

Selected References

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

  1. Ali S. M., Geisow M. J., Burgoyne R. D. A role for calpactin in calcium-dependent exocytosis in adrenal chromaffin cells. Nature. 1989 Jul 27;340(6231):313–315. doi: 10.1038/340313a0. [DOI] [PubMed] [Google Scholar]
  2. Berridge M. J., Heslop J. P., Irvine R. F., Brown K. D. Inositol trisphosphate formation and calcium mobilization in Swiss 3T3 cells in response to platelet-derived growth factor. Biochem J. 1984 Aug 15;222(1):195–201. doi: 10.1042/bj2220195. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Berridge M. J., Irvine R. F. Inositol phosphates and cell signalling. Nature. 1989 Sep 21;341(6239):197–205. doi: 10.1038/341197a0. [DOI] [PubMed] [Google Scholar]
  4. Blay J., Valentine-Braun K. A., Northup J. K., Hollenberg M. D. Epidermal-growth-factor-stimulated phosphorylation of calpactin II in membrane vesicles shed from cultured A-431 cells. Biochem J. 1989 Apr 15;259(2):577–583. doi: 10.1042/bj2590577. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Blinks J. R., Wier W. G., Hess P., Prendergast F. G. Measurement of Ca2+ concentrations in living cells. Prog Biophys Mol Biol. 1982;40(1-2):1–114. doi: 10.1016/0079-6107(82)90011-6. [DOI] [PubMed] [Google Scholar]
  6. Cattaneo M. G., Vicentini L. M. Differential mechanisms of inositol phosphate generation at the receptors for bombesin and platelet-derived growth factor. Biochem J. 1989 Sep 1;262(2):665–668. doi: 10.1042/bj2620665. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Comoglio P. M., Di Renzo M. F., Tarone G., Giancotti F. G., Naldini L., Marchisio P. C. Detection of phosphotyrosine-containing proteins in the detergent-insoluble fraction of RSV-transformed fibroblasts by azobenzene phosphonate antibodies. EMBO J. 1984 Mar;3(3):483–489. doi: 10.1002/j.1460-2075.1984.tb01834.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cooper J. A., Hunter T. Changes in protein phosphorylation in Rous sarcoma virus-transformed chicken embryo cells. Mol Cell Biol. 1981 Feb;1(2):165–178. doi: 10.1128/mcb.1.2.165-178.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Cooper J. A., King C. S. Dephosphorylation or antibody binding to the carboxy terminus stimulates pp60c-src. Mol Cell Biol. 1986 Dec;6(12):4467–4477. doi: 10.1128/mcb.6.12.4467-4477.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Downing J. R., Margolis B. L., Zilberstein A., Ashmun R. A., Ullrich A., Sherr C. J., Schlessinger J. Phospholipase C-gamma, a substrate for PDGF receptor kinase, is not phosphorylated on tyrosine during the mitogenic response to CSF-1. EMBO J. 1989 Nov;8(11):3345–3350. doi: 10.1002/j.1460-2075.1989.tb08496.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Drust D. S., Creutz C. E. Aggregation of chromaffin granules by calpactin at micromolar levels of calcium. Nature. 1988 Jan 7;331(6151):88–91. doi: 10.1038/331088a0. [DOI] [PubMed] [Google Scholar]
  12. Erusalimsky J. D., Friedberg I., Rozengurt E. Bombesin, diacylglycerols, and phorbol esters rapidly stimulate the phosphorylation of an Mr = 80,000 protein kinase C substrate in permeabilized 3T3 cells. Effect of guanine nucleotides. J Biol Chem. 1988 Dec 15;263(35):19188–19194. [PubMed] [Google Scholar]
  13. Fava R. A., Cohen S. Isolation of a calcium-dependent 35-kilodalton substrate for the epidermal growth factor receptor/kinase from A-431 cells. J Biol Chem. 1984 Feb 25;259(4):2636–2645. [PubMed] [Google Scholar]
  14. Glenney J. R., Jr Calpactins: calcium-regulated membrane-skeletal proteins. Biochem Soc Trans. 1987 Oct;15(5):798–800. doi: 10.1042/bst0150798. [DOI] [PubMed] [Google Scholar]
  15. Glenney J. R., Jr Phosphorylation of p36 in vitro with pp60src. Regulation by Ca2+ and phospholipid. FEBS Lett. 1985 Nov 11;192(1):79–82. doi: 10.1016/0014-5793(85)80047-8. [DOI] [PubMed] [Google Scholar]
  16. Glenney J. R., Jr, Tack B. F. Amino-terminal sequence of p36 and associated p10: identification of the site of tyrosine phosphorylation and homology with S-100. Proc Natl Acad Sci U S A. 1985 Dec;82(23):7884–7888. doi: 10.1073/pnas.82.23.7884. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Glenney J. Phospholipid-dependent Ca2+ binding by the 36-kDa tyrosine kinase substrate (calpactin) and its 33-kDa core. J Biol Chem. 1986 Jun 5;261(16):7247–7252. [PubMed] [Google Scholar]
  18. Glenney J. Two related but distinct forms of the Mr 36,000 tyrosine kinase substrate (calpactin) that interact with phospholipid and actin in a Ca2+-dependent manner. Proc Natl Acad Sci U S A. 1986 Jun;83(12):4258–4262. doi: 10.1073/pnas.83.12.4258. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Gould K. L., Hunter T. Platelet-derived growth factor induces multisite phosphorylation of pp60c-src and increases its protein-tyrosine kinase activity. Mol Cell Biol. 1988 Aug;8(8):3345–3356. doi: 10.1128/mcb.8.8.3345-3356.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Gould K. L., Woodgett J. R., Isacke C. M., Hunter T. The protein-tyrosine kinase substrate p36 is also a substrate for protein kinase C in vitro and in vivo. Mol Cell Biol. 1986 Jul;6(7):2738–2744. doi: 10.1128/mcb.6.7.2738-2744.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Heldin C. H., Westermark B. Platelet-derived growth factor: mechanism of action and possible in vivo function. Cell Regul. 1990 Jul;1(8):555–566. doi: 10.1091/mbc.1.8.555. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Howell T. W., Gomperts B. D. Rat mast cells permeabilised with streptolysin O secrete histamine in response to Ca2+ at concentrations buffered in the micromolar range. Biochim Biophys Acta. 1987 Feb 18;927(2):177–183. doi: 10.1016/0167-4889(87)90132-7. [DOI] [PubMed] [Google Scholar]
  23. Isacke C. M., Trowbridge I. S., Hunter T. Modulation of p36 phosphorylation in human cells: studies using anti-p36 monoclonal antibodies. Mol Cell Biol. 1986 Jul;6(7):2745–2751. doi: 10.1128/mcb.6.7.2745-2751.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Klee C. B. Ca2+-dependent phospholipid- (and membrane-) binding proteins. Biochemistry. 1988 Sep 6;27(18):6645–6653. doi: 10.1021/bi00418a001. [DOI] [PubMed] [Google Scholar]
  25. Kumjian D. A., Wahl M. I., Rhee S. G., Daniel T. O. Platelet-derived growth factor (PDGF) binding promotes physical association of PDGF receptor with phospholipase C. Proc Natl Acad Sci U S A. 1989 Nov;86(21):8232–8236. doi: 10.1073/pnas.86.21.8232. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Kypta R. M., Goldberg Y., Ulug E. T., Courtneidge S. A. Association between the PDGF receptor and members of the src family of tyrosine kinases. Cell. 1990 Aug 10;62(3):481–492. doi: 10.1016/0092-8674(90)90013-5. [DOI] [PubMed] [Google Scholar]
  27. Lau K. H., Farley J. R., Baylink D. J. Phosphotyrosyl protein phosphatases. Biochem J. 1989 Jan 1;257(1):23–36. doi: 10.1042/bj2570023. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Meisenhelder J., Suh P. G., Rhee S. G., Hunter T. Phospholipase C-gamma is a substrate for the PDGF and EGF receptor protein-tyrosine kinases in vivo and in vitro. Cell. 1989 Jun 30;57(7):1109–1122. doi: 10.1016/0092-8674(89)90048-2. [DOI] [PubMed] [Google Scholar]
  29. Nechay B. R. Mechanisms of action of vanadium. Annu Rev Pharmacol Toxicol. 1984;24:501–524. doi: 10.1146/annurev.pa.24.040184.002441. [DOI] [PubMed] [Google Scholar]
  30. Paris S., Pouysségur J. Further evidence for a phospholipase C-coupled G protein in hamster fibroblasts. Induction of inositol phosphate formation by fluoroaluminate and vanadate and inhibition by pertussis toxin. J Biol Chem. 1987 Feb 15;262(5):1970–1976. [PubMed] [Google Scholar]
  31. Peres A., Racca C., Zippel R., Sturani E. Cytosolic calcium and membrane conductance in response to platelet-derived growth factor and bradykinin stimulation in single human fibroblasts. Eur J Cell Biol. 1990 Dec;53(2):290–295. [PubMed] [Google Scholar]
  32. Peres A., Sturani E., Zippel R. Properties of the voltage-dependent calcium channel of mouse Swiss 3T3 fibroblasts. J Physiol. 1988 Jul;401:639–655. doi: 10.1113/jphysiol.1988.sp017184. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Powell M. A., Glenney J. R. Regulation of calpactin I phospholipid binding by calpactin I light-chain binding and phosphorylation by p60v-src. Biochem J. 1987 Oct 15;247(2):321–328. doi: 10.1042/bj2470321. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Ralston R., Bishop J. M. The product of the protooncogene c-src is modified during the cellular response to platelet-derived growth factor. Proc Natl Acad Sci U S A. 1985 Dec;82(23):7845–7849. doi: 10.1073/pnas.82.23.7845. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Sturani E., Vicentini L. M., Zippel R., Toschi L., Pandiella-Alonso A., Comoglio P. M., Meldolesi J. PDGF-induced receptor phosphorylation and phosphoinositide hydrolysis are unaffected by protein kinase C activation in mouse Swiss 3T3 and human skin fibroblasts. Biochem Biophys Res Commun. 1986 May 29;137(1):343–350. doi: 10.1016/0006-291x(86)91216-7. [DOI] [PubMed] [Google Scholar]
  36. Sturani E., Zippel R., Morello L., Brambillo R., Alberghina L. Dissociation of the ligand and dephosphorylation of the platelet-derived growth factor receptor. FEBS Lett. 1988 Jun 20;233(2):371–374. doi: 10.1016/0014-5793(88)80463-0. [DOI] [PubMed] [Google Scholar]
  37. Sturani E., Zippel R., Toschi L., Morello L., Comoglio P. M., Alberghina L. Kinetics and regulation of the tyrosine phosphorylation of epidermal growth factor receptor in intact A431 cells. Mol Cell Biol. 1988 Mar;8(3):1345–1351. doi: 10.1128/mcb.8.3.1345-1351.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Swarup G., Cohen S., Garbers D. L. Inhibition of membrane phosphotyrosyl-protein phosphatase activity by vanadate. Biochem Biophys Res Commun. 1982 Aug;107(3):1104–1109. doi: 10.1016/0006-291x(82)90635-0. [DOI] [PubMed] [Google Scholar]
  39. 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]
  40. Wahl M. I., Nishibe S., Suh P. G., Rhee S. G., Carpenter G. Epidermal growth factor stimulates tyrosine phosphorylation of phospholipase C-II independently of receptor internalization and extracellular calcium. Proc Natl Acad Sci U S A. 1989 Mar;86(5):1568–1572. doi: 10.1073/pnas.86.5.1568. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Wahl M. I., Olashaw N. E., Nishibe S., Rhee S. G., Pledger W. J., Carpenter G. Platelet-derived growth factor induces rapid and sustained tyrosine phosphorylation of phospholipase C-gamma in quiescent BALB/c 3T3 cells. Mol Cell Biol. 1989 Jul;9(7):2934–2943. doi: 10.1128/mcb.9.7.2934-2943.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Zippel R., Morello L., Brambilla R., Comoglio P. M., Alberghina L., Sturani E. Inhibition of phosphotyrosine phosphatases reveals candidate substrates of the PDGF receptor kinase. Eur J Cell Biol. 1989 Dec;50(2):428–434. [PubMed] [Google Scholar]
  43. Zippel R., Sturani E., Toschi L., Naldini L., Alberghina L., Comoglio P. M. In vivo phosphorylation and dephosphorylation of the platelet-derived growth factor receptor studied by immunoblot analysis with phosphotyrosine antibodies. Biochim Biophys Acta. 1986 Mar 19;881(1):54–61. doi: 10.1016/0304-4165(86)90096-6. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES