Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 2000 Dec 15;352(Pt 3):883–892.

Metalloprotease-disintegrin ADAM 12 binds to the SH3 domain of Src and activates Src tyrosine kinase in C2C12 cells.

Q Kang 1, Y Cao 1, A Zolkiewska 1
PMCID: PMC1221530  PMID: 11104699

Abstract

ADAM 12, a member of the ADAM (protein containing a disintegrin and metalloprotease) family of metalloprotease-disintegrins, has been implicated in the differentiation and fusion of skeletal myoblasts, and its expression is dramatically up-regulated in many cancer cells. While the extracellular portion of ADAM 12 contains an active metalloprotease and a cell-adhesion domain, the function of the cytoplasmic portion is much less clear. In this paper, we show that the cytoplasmic tail of ADAM 12 mediates interactions with the non-receptor protein tyrosine kinase Src. The interaction is direct, specific, and involves the N-terminal proline-rich region in the cytoplasmic tail of ADAM 12 and the Src homology 3 (SH3) domain of Src. ADAM 12 and Src co-immunoprecipitate from transfected C2C12 cells, suggesting that the two proteins form a complex in vivo. Co-expression of Src and ADAM 12, but not ADAM 9, in C2C12 cells results in activation of the recombinant Src. Moreover, endogenous ADAM 12 associates with and activates endogenous Src in differentiating C2C12 cells. These results indicate that ADAM 12 may mediate adhesion-induced signalling during myoblast differentiation.

Full Text

The Full Text of this article is available as a PDF (305.7 KB).

Selected References

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

  1. Abe E., Mocharla H., Yamate T., Taguchi Y., Manolagas S. C. Meltrin-alpha, a fusion protein involved in multinucleated giant cell and osteoclast formation. Calcif Tissue Int. 1999 Jun;64(6):508–515. doi: 10.1007/s002239900641. [DOI] [PubMed] [Google Scholar]
  2. Alexandropoulos K., Cheng G., Baltimore D. Proline-rich sequences that bind to Src homology 3 domains with individual specificities. Proc Natl Acad Sci U S A. 1995 Apr 11;92(8):3110–3114. doi: 10.1073/pnas.92.8.3110. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Almeida E. A., Huovila A. P., Sutherland A. E., Stephens L. E., Calarco P. G., Shaw L. M., Mercurio A. M., Sonnenberg A., Primakoff P., Myles D. G. Mouse egg integrin alpha 6 beta 1 functions as a sperm receptor. Cell. 1995 Jun 30;81(7):1095–1104. doi: 10.1016/s0092-8674(05)80014-5. [DOI] [PubMed] [Google Scholar]
  4. Bigler D., Takahashi Y., Chen M. S., Almeida E. A., Osbourne L., White J. M. Sequence-specific interaction between the disintegrin domain of mouse ADAM 2 (fertilin beta) and murine eggs. Role of the alpha(6) integrin subunit. J Biol Chem. 2000 Apr 21;275(16):11576–11584. doi: 10.1074/jbc.275.16.11576. [DOI] [PubMed] [Google Scholar]
  5. Brown M. T., Cooper J. A. Regulation, substrates and functions of src. Biochim Biophys Acta. 1996 Jun 7;1287(2-3):121–149. doi: 10.1016/0304-419x(96)00003-0. [DOI] [PubMed] [Google Scholar]
  6. Cal S., Freije J. M., López J. M., Takada Y., López-Otín C. ADAM 23/MDC3, a human disintegrin that promotes cell adhesion via interaction with the alphavbeta3 integrin through an RGD-independent mechanism. Mol Biol Cell. 2000 Apr;11(4):1457–1469. doi: 10.1091/mbc.11.4.1457. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Castellani L., Reedy M. C., Gauzzi M. C., Provenzano C., Alemà S., Falcone G. Maintenance of the differentiated state in skeletal muscle: activation of v-Src disrupts sarcomeres in quail myotubes. J Cell Biol. 1995 Aug;130(4):871–885. doi: 10.1083/jcb.130.4.871. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Castellani L., Reedy M., Airey J. A., Gallo R., Ciotti M. T., Falcone G., Alemà S. Remodeling of cytoskeleton and triads following activation of v-Src tyrosine kinase in quail myotubes. J Cell Sci. 1996 Jun;109(Pt 6):1335–1346. doi: 10.1242/jcs.109.6.1335. [DOI] [PubMed] [Google Scholar]
  9. Evans J. P., Kopf G. S., Schultz R. M. Characterization of the binding of recombinant mouse sperm fertilin beta subunit to mouse eggs: evidence for adhesive activity via an egg beta1 integrin-mediated interaction. Dev Biol. 1997 Jul 1;187(1):79–93. doi: 10.1006/dbio.1997.8611. [DOI] [PubMed] [Google Scholar]
  10. Evans J. P., Schultz R. M., Kopf G. S. Characterization of the binding of recombinant mouse sperm fertilin alpha subunit to mouse eggs: evidence for function as a cell adhesion molecule in sperm-egg binding. Dev Biol. 1997 Jul 1;187(1):94–106. doi: 10.1006/dbio.1997.8612. [DOI] [PubMed] [Google Scholar]
  11. Falcone G., Alemà S., Tatò F. Transcription of muscle-specific genes is repressed by reactivation of pp60v-src in postmitotic quail myotubes. Mol Cell Biol. 1991 Jun;11(6):3331–3338. doi: 10.1128/mcb.11.6.3331. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Feng S., Chen J. K., Yu H., Simon J. A., Schreiber S. L. Two binding orientations for peptides to the Src SH3 domain: development of a general model for SH3-ligand interactions. Science. 1994 Nov 18;266(5188):1241–1247. doi: 10.1126/science.7526465. [DOI] [PubMed] [Google Scholar]
  13. Fuhrer C., Hall Z. W. Functional interaction of Src family kinases with the acetylcholine receptor in C2 myotubes. J Biol Chem. 1996 Dec 13;271(50):32474–32481. doi: 10.1074/jbc.271.50.32474. [DOI] [PubMed] [Google Scholar]
  14. Galbiati F., Volonte D., Engelman J. A., Scherer P. E., Lisanti M. P. Targeted down-regulation of caveolin-3 is sufficient to inhibit myotube formation in differentiating C2C12 myoblasts. Transient activation of p38 mitogen-activated protein kinase is required for induction of caveolin-3 expression and subsequent myotube formation. J Biol Chem. 1999 Oct 15;274(42):30315–30321. doi: 10.1074/jbc.274.42.30315. [DOI] [PubMed] [Google Scholar]
  15. Galliano M. F., Huet C., Frygelius J., Polgren A., Wewer U. M., Engvall E. Binding of ADAM12, a marker of skeletal muscle regeneration, to the muscle-specific actin-binding protein, alpha -actinin-2, is required for myoblast fusion. J Biol Chem. 2000 May 5;275(18):13933–13939. doi: 10.1074/jbc.275.18.13933. [DOI] [PubMed] [Google Scholar]
  16. Gilpin B. J., Loechel F., Mattei M. G., Engvall E., Albrechtsen R., Wewer U. M. A novel, secreted form of human ADAM 12 (meltrin alpha) provokes myogenesis in vivo. J Biol Chem. 1998 Jan 2;273(1):157–166. doi: 10.1074/jbc.273.1.157. [DOI] [PubMed] [Google Scholar]
  17. Gonfloni S., Weijland A., Kretzschmar J., Superti-Furga G. Crosstalk between the catalytic and regulatory domains allows bidirectional regulation of Src. Nat Struct Biol. 2000 Apr;7(4):281–286. doi: 10.1038/74041. [DOI] [PubMed] [Google Scholar]
  18. Gutwein P., Oleszewski M., Mechtersheimer S., Agmon-Levin N., Krauss K., Altevogt P. Role of Src kinases in the ADAM-mediated release of L1 adhesion molecule from human tumor cells. J Biol Chem. 2000 May 19;275(20):15490–15497. doi: 10.1074/jbc.275.20.15490. [DOI] [PubMed] [Google Scholar]
  19. Howard L., Nelson K. K., Maciewicz R. A., Blobel C. P. Interaction of the metalloprotease disintegrins MDC9 and MDC15 with two SH3 domain-containing proteins, endophilin I and SH3PX1. J Biol Chem. 1999 Oct 29;274(44):31693–31699. doi: 10.1074/jbc.274.44.31693. [DOI] [PubMed] [Google Scholar]
  20. Hubbard S. R., Mohammadi M., Schlessinger J. Autoregulatory mechanisms in protein-tyrosine kinases. J Biol Chem. 1998 May 15;273(20):11987–11990. doi: 10.1074/jbc.273.20.11987. [DOI] [PubMed] [Google Scholar]
  21. Iba K., Albrechtsen R., Gilpin B. J., Loechel F., Wewer U. M. Cysteine-rich domain of human ADAM 12 (meltrin alpha) supports tumor cell adhesion. Am J Pathol. 1999 May;154(5):1489–1501. doi: 10.1016/s0002-9440(10)65403-x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Iba K., Albrechtsen R., Gilpin B., Fröhlich C., Loechel F., Zolkiewska A., Ishiguro K., Kojima T., Liu W., Langford J. K. The cysteine-rich domain of human ADAM 12 supports cell adhesion through syndecans and triggers signaling events that lead to beta1 integrin-dependent cell spreading. J Cell Biol. 2000 May 29;149(5):1143–1156. doi: 10.1083/jcb.149.5.1143. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Inoue D., Reid M., Lum L., Krätzschmar J., Weskamp G., Myung Y. M., Baron R., Blobel C. P. Cloning and initial characterization of mouse meltrin beta and analysis of the expression of four metalloprotease-disintegrins in bone cells. J Biol Chem. 1998 Feb 13;273(7):4180–4187. doi: 10.1074/jbc.273.7.4180. [DOI] [PubMed] [Google Scholar]
  24. Knudsen B. S., Zheng J., Feller S. M., Mayer J. P., Burrell S. K., Cowburn D., Hanafusa H. Affinity and specificity requirements for the first Src homology 3 domain of the Crk proteins. EMBO J. 1995 May 15;14(10):2191–2198. doi: 10.1002/j.1460-2075.1995.tb07213.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Kurisaki T., Masuda A., Osumi N., Nabeshima Y., Fujisawa-Sehara A. Spatially- and temporally-restricted expression of meltrin alpha (ADAM12) and beta (ADAM19) in mouse embryo. Mech Dev. 1998 May;73(2):211–215. doi: 10.1016/s0925-4773(98)00043-4. [DOI] [PubMed] [Google Scholar]
  26. Loechel F., Gilpin B. J., Engvall E., Albrechtsen R., Wewer U. M. Human ADAM 12 (meltrin alpha) is an active metalloprotease. J Biol Chem. 1998 Jul 3;273(27):16993–16997. doi: 10.1074/jbc.273.27.16993. [DOI] [PubMed] [Google Scholar]
  27. Mayer B. J., Eck M. J. SH3 domains. Minding your p's and q's. Curr Biol. 1995 Apr 1;5(4):364–367. doi: 10.1016/s0960-9822(95)00073-x. [DOI] [PubMed] [Google Scholar]
  28. Mohamed A. S., Swope S. L. Phosphorylation and cytoskeletal anchoring of the acetylcholine receptor by Src class protein-tyrosine kinases. Activation by rapsyn. J Biol Chem. 1999 Jul 16;274(29):20529–20539. doi: 10.1074/jbc.274.29.20529. [DOI] [PubMed] [Google Scholar]
  29. Musacchio A., Wilmanns M., Saraste M. Structure and function of the SH3 domain. Prog Biophys Mol Biol. 1994;61(3):283–297. doi: 10.1016/0079-6107(94)90003-5. [DOI] [PubMed] [Google Scholar]
  30. Nath D., Slocombe P. M., Stephens P. E., Warn A., Hutchinson G. R., Yamada K. M., Docherty A. J., Murphy G. Interaction of metargidin (ADAM-15) with alphavbeta3 and alpha5beta1 integrins on different haemopoietic cells. J Cell Sci. 1999 Feb;112(Pt 4):579–587. doi: 10.1242/jcs.112.4.579. [DOI] [PubMed] [Google Scholar]
  31. Nath D., Slocombe P. M., Webster A., Stephens P. E., Docherty A. J., Murphy G. Meltrin gamma(ADAM-9) mediates cellular adhesion through alpha(6)beta(1 )integrin, leading to a marked induction of fibroblast cell motility. J Cell Sci. 2000 Jun;113(Pt 12):2319–2328. doi: 10.1242/jcs.113.12.2319. [DOI] [PubMed] [Google Scholar]
  32. Nelson K. K., Schlöndorff J., Blobel C. P. Evidence for an interaction of the metalloprotease-disintegrin tumour necrosis factor alpha convertase (TACE) with mitotic arrest deficient 2 (MAD2), and of the metalloprotease-disintegrin MDC9 with a novel MAD2-related protein, MAD2beta. Biochem J. 1999 Nov 1;343(Pt 3):673–680. [PMC free article] [PubMed] [Google Scholar]
  33. Rickles R. J., Botfield M. C., Zhou X. M., Henry P. A., Brugge J. S., Zoller M. J. Phage display selection of ligand residues important for Src homology 3 domain binding specificity. Proc Natl Acad Sci U S A. 1995 Nov 21;92(24):10909–10913. doi: 10.1073/pnas.92.24.10909. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Schlöndorff J., Blobel C. P. Metalloprotease-disintegrins: modular proteins capable of promoting cell-cell interactions and triggering signals by protein-ectodomain shedding. J Cell Sci. 1999 Nov;112(Pt 21):3603–3617. doi: 10.1242/jcs.112.21.3603. [DOI] [PubMed] [Google Scholar]
  35. Schneider M. D., Olson E. N. Control of myogenic differentiation by cellular oncogenes. Mol Neurobiol. 1988 Spring;2(1):1–39. doi: 10.1007/BF02935631. [DOI] [PubMed] [Google Scholar]
  36. Sicheri F., Kuriyan J. Structures of Src-family tyrosine kinases. Curr Opin Struct Biol. 1997 Dec;7(6):777–785. doi: 10.1016/s0959-440x(97)80146-7. [DOI] [PubMed] [Google Scholar]
  37. Song K. S., Scherer P. E., Tang Z., Okamoto T., Li S., Chafel M., Chu C., Kohtz D. S., Lisanti M. P. Expression of caveolin-3 in skeletal, cardiac, and smooth muscle cells. Caveolin-3 is a component of the sarcolemma and co-fractionates with dystrophin and dystrophin-associated glycoproteins. J Biol Chem. 1996 Jun 21;271(25):15160–15165. doi: 10.1074/jbc.271.25.15160. [DOI] [PubMed] [Google Scholar]
  38. Sparks A. B., Rider J. E., Hoffman N. G., Fowlkes D. M., Quillam L. A., Kay B. K. Distinct ligand preferences of Src homology 3 domains from Src, Yes, Abl, Cortactin, p53bp2, PLCgamma, Crk, and Grb2. Proc Natl Acad Sci U S A. 1996 Feb 20;93(4):1540–1544. doi: 10.1073/pnas.93.4.1540. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Thomas S. M., Brugge J. S. Cellular functions regulated by Src family kinases. Annu Rev Cell Dev Biol. 1997;13:513–609. doi: 10.1146/annurev.cellbio.13.1.513. [DOI] [PubMed] [Google Scholar]
  40. Turner A. J., Hooper N. M. Role for ADAM-family proteinases as membrane protein secretases. Biochem Soc Trans. 1999 Feb;27(2):255–259. doi: 10.1042/bst0270255. [DOI] [PubMed] [Google Scholar]
  41. Weng Z., Rickles R. J., Feng S., Richard S., Shaw A. S., Schreiber S. L., Brugge J. S. Structure-function analysis of SH3 domains: SH3 binding specificity altered by single amino acid substitutions. Mol Cell Biol. 1995 Oct;15(10):5627–5634. doi: 10.1128/mcb.15.10.5627. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Weskamp G., Krätzschmar J., Reid M. S., Blobel C. P. MDC9, a widely expressed cellular disintegrin containing cytoplasmic SH3 ligand domains. J Cell Biol. 1996 Feb;132(4):717–726. doi: 10.1083/jcb.132.4.717. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Wolfsberg T. G., White J. M. ADAMs in fertilization and development. Dev Biol. 1996 Dec 15;180(2):389–401. doi: 10.1006/dbio.1996.0313. [DOI] [PubMed] [Google Scholar]
  44. Xu W., Harrison S. C., Eck M. J. Three-dimensional structure of the tyrosine kinase c-Src. Nature. 1997 Feb 13;385(6617):595–602. doi: 10.1038/385595a0. [DOI] [PubMed] [Google Scholar]
  45. Yagami-Hiromasa T., Sato T., Kurisaki T., Kamijo K., Nabeshima Y., Fujisawa-Sehara A. A metalloprotease-disintegrin participating in myoblast fusion. Nature. 1995 Oct 19;377(6550):652–656. doi: 10.1038/377652a0. [DOI] [PubMed] [Google Scholar]
  46. Yoon H., Boettiger D. Expression of v-src alters the expression of myogenic regulatory factor genes. Oncogene. 1994 Mar;9(3):801–807. [PubMed] [Google Scholar]
  47. Zhang X. P., Kamata T., Yokoyama K., Puzon-McLaughlin W., Takada Y. Specific interaction of the recombinant disintegrin-like domain of MDC-15 (metargidin, ADAM-15) with integrin alphavbeta3. J Biol Chem. 1998 Mar 27;273(13):7345–7350. doi: 10.1074/jbc.273.13.7345. [DOI] [PubMed] [Google Scholar]
  48. Zhu X., Bansal N. P., Evans J. P. Identification of key functional amino acids of the mouse fertilin beta (ADAM2) disintegrin loop for cell-cell adhesion during fertilization. J Biol Chem. 2000 Mar 17;275(11):7677–7683. doi: 10.1074/jbc.275.11.7677. [DOI] [PubMed] [Google Scholar]
  49. Zolkiewska A. Disintegrin-like/cysteine-rich region of ADAM 12 is an active cell adhesion domain. Exp Cell Res. 1999 Nov 1;252(2):423–431. doi: 10.1006/excr.1999.4632. [DOI] [PubMed] [Google Scholar]

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

RESOURCES