Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 2000 Mar 1;346(Pt 2):359–367.

Stimulation of cleavage of membrane proteins by calmodulin inhibitors.

E Díaz-Rodríguez 1, A Esparís-Ogando 1, J C Montero 1, L Yuste 1, A Pandiella 1
PMCID: PMC1220861  PMID: 10677354

Abstract

The ectodomain of several membrane-bound proteins can be shed by proteolytic cleavage. The activity of the proteases involved in shedding is highly regulated by several intracellular second messenger pathways, such as protein kinase C (PKC) and intracellular Ca(2+). Recently, the shedding of the adhesion molecule L-selectin has been shown to be regulated by the interaction of calmodulin (CaM) with the cytosolic tail of L-selectin. Prevention of CaM-L-selectin interaction by CaM inhibitors or mutation of a CaM binding site in L-selectin induced L-selectin ectodomain shedding. Whether this action of CaM inhibitors also affects other membrane-bound proteins is not known. In the present paper we show that CaM inhibitors also stimulate the cleavage of several other transmembrane proteins, such as the membrane-bound growth factor precursors pro-transforming growth factor-alpha and pro-neuregulin-alpha2c, the receptor tyrosine kinase, TrkA, and the beta-amyloid precursor protein. Cleavage induced by CaM inhibitors was a rapid event, and resulted from the activation of a mechanism that was independent of PKC or intracellular Ca(2+) increases, but was highly sensitive to hydroxamic acid-based metalloprotease inhibitors. Mutational analysis of the intracellular domain of the TrkA receptor indicated that CaM inhibitors may stimulate membrane-protein ectodomain cleavage by mechanisms independent of CaM-substrate interaction.

Full Text

The Full Text of this article is available as a PDF (276.0 KB).

Selected References

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

  1. Black R. A., Rauch C. T., Kozlosky C. J., Peschon J. J., Slack J. L., Wolfson M. F., Castner B. J., Stocking K. L., Reddy P., Srinivasan S. A metalloproteinase disintegrin that releases tumour-necrosis factor-alpha from cells. Nature. 1997 Feb 20;385(6618):729–733. doi: 10.1038/385729a0. [DOI] [PubMed] [Google Scholar]
  2. Black R. A., White J. M. ADAMs: focus on the protease domain. Curr Opin Cell Biol. 1998 Oct;10(5):654–659. doi: 10.1016/s0955-0674(98)80042-2. [DOI] [PubMed] [Google Scholar]
  3. Blobel C. P. Metalloprotease-disintegrins: links to cell adhesion and cleavage of TNF alpha and Notch. Cell. 1997 Aug 22;90(4):589–592. doi: 10.1016/s0092-8674(00)80519-x. [DOI] [PubMed] [Google Scholar]
  4. Bosenberg M. W., Pandiella A., Massagué J. The cytoplasmic carboxy-terminal amino acid specifies cleavage of membrane TGF alpha into soluble growth factor. Cell. 1992 Dec 24;71(7):1157–1165. doi: 10.1016/s0092-8674(05)80064-9. [DOI] [PubMed] [Google Scholar]
  5. Brakebusch C., Varfolomeev E. E., Batkin M., Wallach D. Structural requirements for inducible shedding of the p55 tumor necrosis factor receptor. J Biol Chem. 1994 Dec 23;269(51):32488–32496. [PubMed] [Google Scholar]
  6. Briley G. P., Hissong M. A., Chiu M. L., Lee D. C. The carboxyl-terminal valine residues of proTGF alpha are required for its efficient maturation and intracellular routing. Mol Biol Cell. 1997 Aug;8(8):1619–1631. doi: 10.1091/mbc.8.8.1619. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Bringman T. S., Lindquist P. B., Derynck R. Different transforming growth factor-alpha species are derived from a glycosylated and palmitoylated transmembrane precursor. Cell. 1987 Feb 13;48(3):429–440. doi: 10.1016/0092-8674(87)90194-2. [DOI] [PubMed] [Google Scholar]
  8. Buxbaum J. D., Liu K. N., Luo Y., Slack J. L., Stocking K. L., Peschon J. J., Johnson R. S., Castner B. J., Cerretti D. P., Black R. A. Evidence that tumor necrosis factor alpha converting enzyme is involved in regulated alpha-secretase cleavage of the Alzheimer amyloid protein precursor. J Biol Chem. 1998 Oct 23;273(43):27765–27767. doi: 10.1074/jbc.273.43.27765. [DOI] [PubMed] [Google Scholar]
  9. Cabrera N., Díaz-Rodríguez E., Becker E., Martín-Zanca D., Pandiella A. TrkA receptor ectodomain cleavage generates a tyrosine-phosphorylated cell-associated fragment. J Cell Biol. 1996 Feb;132(3):427–436. doi: 10.1083/jcb.132.3.427. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Chakravarthy B., Morley P., Whitfield J. Ca2+-calmodulin and protein kinase Cs: a hypothetical synthesis of their conflicting convergences on shared substrate domains. Trends Neurosci. 1999 Jan;22(1):12–16. doi: 10.1016/s0166-2236(98)01288-0. [DOI] [PubMed] [Google Scholar]
  11. Downing J. R., Roussel M. F., Sherr C. J. Ligand and protein kinase C downmodulate the colony-stimulating factor 1 receptor by independent mechanisms. Mol Cell Biol. 1989 Jul;9(7):2890–2896. doi: 10.1128/mcb.9.7.2890. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Díaz-Rodríguez E., Cabrera N., Esparís-Ogando A., Montero J. C., Pandiella A. Cleavage of the TrkA neurotrophin receptor by multiple metalloproteases generates signalling-competent truncated forms. Eur J Neurosci. 1999 Apr;11(4):1421–1430. doi: 10.1046/j.1460-9568.1999.00552.x. [DOI] [PubMed] [Google Scholar]
  13. Ehlers M. R., Riordan J. F. Membrane proteins with soluble counterparts: role of proteolysis in the release of transmembrane proteins. Biochemistry. 1991 Oct 22;30(42):10065–10074. doi: 10.1021/bi00106a001. [DOI] [PubMed] [Google Scholar]
  14. Esparís-Ogando A., Díaz-Rodríguez E., Pandiella A. Signalling-competent truncated forms of ErbB2 in breast cancer cells: differential regulation by protein kinase C and phosphatidylinositol 3-kinase. Biochem J. 1999 Dec 1;344(Pt 2):339–348. [PMC free article] [PubMed] [Google Scholar]
  15. Gearing A. J., Beckett P., Christodoulou M., Churchill M., Clements J., Davidson A. H., Drummond A. H., Galloway W. A., Gilbert R., Gordon J. L. Processing of tumour necrosis factor-alpha precursor by metalloproteinases. Nature. 1994 Aug 18;370(6490):555–557. doi: 10.1038/370555a0. [DOI] [PubMed] [Google Scholar]
  16. Hooper N. M., Karran E. H., Turner A. J. Membrane protein secretases. Biochem J. 1997 Jan 15;321(Pt 2):265–279. doi: 10.1042/bj3210265. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Izumi Y., Hirata M., Hasuwa H., Iwamoto R., Umata T., Miyado K., Tamai Y., Kurisaki T., Sehara-Fujisawa A., Ohno S. A metalloprotease-disintegrin, MDC9/meltrin-gamma/ADAM9 and PKCdelta are involved in TPA-induced ectodomain shedding of membrane-anchored heparin-binding EGF-like growth factor. EMBO J. 1998 Dec 15;17(24):7260–7272. doi: 10.1093/emboj/17.24.7260. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. James P., Vorherr T., Carafoli E. Calmodulin-binding domains: just two faced or multi-faceted? Trends Biochem Sci. 1995 Jan;20(1):38–42. doi: 10.1016/s0968-0004(00)88949-5. [DOI] [PubMed] [Google Scholar]
  19. Kahn J., Walcheck B., Migaki G. I., Jutila M. A., Kishimoto T. K. Calmodulin regulates L-selectin adhesion molecule expression and function through a protease-dependent mechanism. Cell. 1998 Mar 20;92(6):809–818. doi: 10.1016/s0092-8674(00)81408-7. [DOI] [PubMed] [Google Scholar]
  20. Kishimoto T. K., Jutila M. A., Berg E. L., Butcher E. C. Neutrophil Mac-1 and MEL-14 adhesion proteins inversely regulated by chemotactic factors. Science. 1989 Sep 15;245(4923):1238–1241. doi: 10.1126/science.2551036. [DOI] [PubMed] [Google Scholar]
  21. Massagué J., Pandiella A. Membrane-anchored growth factors. Annu Rev Biochem. 1993;62:515–541. doi: 10.1146/annurev.bi.62.070193.002503. [DOI] [PubMed] [Google Scholar]
  22. McDermott M. F., Aksentijevich I., Galon J., McDermott E. M., Ogunkolade B. W., Centola M., Mansfield E., Gadina M., Karenko L., Pettersson T. Germline mutations in the extracellular domains of the 55 kDa TNF receptor, TNFR1, define a family of dominantly inherited autoinflammatory syndromes. Cell. 1999 Apr 2;97(1):133–144. doi: 10.1016/s0092-8674(00)80721-7. [DOI] [PubMed] [Google Scholar]
  23. McGeehan G. M., Becherer J. D., Bast R. C., Jr, Boyer C. M., Champion B., Connolly K. M., Conway J. G., Furdon P., Karp S., Kidao S. Regulation of tumour necrosis factor-alpha processing by a metalloproteinase inhibitor. Nature. 1994 Aug 18;370(6490):558–561. doi: 10.1038/370558a0. [DOI] [PubMed] [Google Scholar]
  24. Mohler K. M., Sleath P. R., Fitzner J. N., Cerretti D. P., Alderson M., Kerwar S. S., Torrance D. S., Otten-Evans C., Greenstreet T., Weerawarna K. Protection against a lethal dose of endotoxin by an inhibitor of tumour necrosis factor processing. Nature. 1994 Jul 21;370(6486):218–220. doi: 10.1038/370218a0. [DOI] [PubMed] [Google Scholar]
  25. Moss M. L., Jin S. L., Milla M. E., Bickett D. M., Burkhart W., Carter H. L., Chen W. J., Clay W. C., Didsbury J. R., Hassler D. Cloning of a disintegrin metalloproteinase that processes precursor tumour-necrosis factor-alpha. Nature. 1997 Feb 20;385(6618):733–736. doi: 10.1038/385733a0. [DOI] [PubMed] [Google Scholar]
  26. Nishizuka Y. Intracellular signaling by hydrolysis of phospholipids and activation of protein kinase C. Science. 1992 Oct 23;258(5082):607–614. doi: 10.1126/science.1411571. [DOI] [PubMed] [Google Scholar]
  27. Pandiella A., Magni M., Lovisolo D., Meldolesi J. The effect of epidermal growth factor on membrane potential. Rapid hyperpolarization followed by persistent fluctuations. J Biol Chem. 1989 Aug 5;264(22):12914–12921. [PubMed] [Google Scholar]
  28. Pandiella A., Magni M., Meldolesi J. Plasma membrane hyperpolarization and [Ca2+]i increase induced by fibroblast growth factor in NIH-3T3 fibroblasts: resemblance to early signals generated by platelet-derived growth factor. Biochem Biophys Res Commun. 1989 Sep 29;163(3):1325–1331. doi: 10.1016/0006-291x(89)91123-6. [DOI] [PubMed] [Google Scholar]
  29. Pandiella A., Massagué J. Cleavage of the membrane precursor for transforming growth factor alpha is a regulated process. Proc Natl Acad Sci U S A. 1991 Mar 1;88(5):1726–1730. doi: 10.1073/pnas.88.5.1726. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Pandiella A., Massagué J. Multiple signals activate cleavage of the membrane transforming growth factor-alpha precursor. J Biol Chem. 1991 Mar 25;266(9):5769–5773. [PubMed] [Google Scholar]
  31. Peschon J. J., Slack J. L., Reddy P., Stocking K. L., Sunnarborg S. W., Lee D. C., Russell W. E., Castner B. J., Johnson R. S., Fitzner J. N. An essential role for ectodomain shedding in mammalian development. Science. 1998 Nov 13;282(5392):1281–1284. doi: 10.1126/science.282.5392.1281. [DOI] [PubMed] [Google Scholar]
  32. Porteu F., Nathan C. Shedding of tumor necrosis factor receptors by activated human neutrophils. J Exp Med. 1990 Aug 1;172(2):599–607. doi: 10.1084/jem.172.2.599. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Prat M., Crepaldi T., Gandino L., Giordano S., Longati P., Comoglio P. C-terminal truncated forms of Met, the hepatocyte growth factor receptor. Mol Cell Biol. 1991 Dec;11(12):5954–5962. doi: 10.1128/mcb.11.12.5954. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Rhoads A. R., Friedberg F. Sequence motifs for calmodulin recognition. FASEB J. 1997 Apr;11(5):331–340. doi: 10.1096/fasebj.11.5.9141499. [DOI] [PubMed] [Google Scholar]
  35. Sadhukhan R., Santhamma K. R., Reddy P., Peschon J. J., Black R. A., Sen I. Unaltered cleavage and secretion of angiotensin-converting enzyme in tumor necrosis factor-alpha-converting enzyme-deficient mice. J Biol Chem. 1999 Apr 9;274(15):10511–10516. doi: 10.1074/jbc.274.15.10511. [DOI] [PubMed] [Google Scholar]
  36. Shum L., Reeves S. A., Kuo A. C., Fromer E. S., Derynck R. Association of the transmembrane TGF-alpha precursor with a protein kinase complex. J Cell Biol. 1994 May;125(4):903–916. doi: 10.1083/jcb.125.4.903. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Teixidó J., Gilmore R., Lee D. C., Massagué J. Integral membrane glycoprotein properties of the prohormone pro-transforming growth factor-alpha. 1987 Apr 30-May 6Nature. 326(6116):883–885. doi: 10.1038/326883a0. [DOI] [PubMed] [Google Scholar]
  38. Van Eldik L. J., Wolchok S. R. Conditions for reproducible detection of calmodulin and S100 beta in immunoblots. Biochem Biophys Res Commun. 1984 Nov 14;124(3):752–759. doi: 10.1016/0006-291x(84)91022-2. [DOI] [PubMed] [Google Scholar]
  39. Vecchi M., Baulida J., Carpenter G. Selective cleavage of the heregulin receptor ErbB-4 by protein kinase C activation. J Biol Chem. 1996 Aug 2;271(31):18989–18995. doi: 10.1074/jbc.271.31.18989. [DOI] [PubMed] [Google Scholar]
  40. Vecchi M., Rudolph-Owen L. A., Brown C. L., Dempsey P. J., Carpenter G. Tyrosine phosphorylation and proteolysis. Pervanadate-induced, metalloprotease-dependent cleavage of the ErbB-4 receptor and amphiregulin. J Biol Chem. 1998 Aug 7;273(32):20589–20595. doi: 10.1074/jbc.273.32.20589. [DOI] [PubMed] [Google Scholar]
  41. Wright C. D., Hoffman M. D. Comparison of the roles of calmodulin and protein kinase C in activation of the human neutrophil respiratory burst. Biochem Biophys Res Commun. 1987 Jan 15;142(1):53–62. doi: 10.1016/0006-291x(87)90450-5. [DOI] [PubMed] [Google Scholar]
  42. Yee N. S., Hsiau C. W., Serve H., Vosseller K., Besmer P. Mechanism of down-regulation of c-kit receptor. Roles of receptor tyrosine kinase, phosphatidylinositol 3'-kinase, and protein kinase C. J Biol Chem. 1994 Dec 16;269(50):31991–31998. [PubMed] [Google Scholar]

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

RESOURCES