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

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.

Intracellular Processing of Metalloprotease Disintegrin ADAM12

“…Antibodies-Rabbit anti-ADAM12 and anti-ADAM9 antibodies were as described previously (45). Mouse anti-KDEL monoclonal antibody was obtained from Stressgen Biotech Corp. (Victoria, British Columbia, Canada).…”

“…The expression of full-length mouse ADAM12 (amino acids 1-903), a truncated form of ADAM12 extending from the disintegrin to the cytoplasmic domain (amino acids 425-903, ADAM12(⌬1-424)), and a similarly truncated form of ADAM9 (amino acids 417-845, ADAM9(⌬1-416)) was carried out as described (45). The mutation L73P in the prodomain of ADAM12 has been identified to be responsible for the lack of proteolytic processing of ADAM12, as observed in our previous work (45)(46)(47). ADAM12(⌬1-424,⌬C) (amino acids 425-845) contains a 58-amino acid truncation at the C terminus in addition to the N-terminal truncation (Fig.…”

Anchoring Junctions As Drug Targets: Role in Contraceptive Development

“…On the other hand, the cytoplasmic tails are divergent and contain several potential phosphorylation sites, as well as binding sites for Src homology region 3 domain-containing proteins (Seals and Courtneidge, 2003), which may ultimately regulate the ability of an ADAM to cleave a specific substrate. It is noteworthy that ADAMs 9 (Weskamp et al, 1996), 12 (Kang et al, 2000;Suzuki et al, 2000), 13 (Cousin et al, 2000), and 15 (Poghosyan et al, 2002) associate with c-Src. ADAM activity is limited by a family of protease inhibitors known as tissue inhibitor of metalloproteases (Handsley and Edwards, 2005;Huovila et al, 2005;Malemud, 2006).…”

The Differentiation of Skeletal Muscle Cells Involves a Protein-tyrosine Phosphatase-α-mediated C-Src Signaling Pathway

“…In the case of neuronal cell differentiation, distinct signals mediated by both ras and Src were shown to be required (62). In skeletal muscle C2C12 cells, a transmembrane disintegrin and metalloprotease termed ADAM12 was described recently to be up-regulated during differentiation (63,64) and to be associated via its proline-rich cytoplasmic domains with the SH3 domain of Src (65). This suggests involvement in recruitment or localization of Src to the cytoskeleton as ADAM12 binds to ␣-actinin-1 (65) and ␣-actinin-2 (66).…”