Role of Src Kinases in the ADAM-mediated Release of L1 Adhesion Molecule from Human Tumor Cells

Abstract

The ectodomain of certain transmembrane molecules can be released by proteolysis, and the solubilized antigens often exert important biological functions. We demonstrated before that the L1 adhesion molecule is shed from the cell surface. Here we show that L1 release in AR breast carcinoma cells is mediated by a member of the disintegrin metalloproteinase (ADAM) family of proteinases. Up-regulation of L1 shedding by phorbol ester or pervanadate involved distinct mechanisms. Pervanadate induced shedding and rounding-up of cells from the substrate, which was blocked by the Src kinase inhibitor PP2. Tyr phosphorylation of the L1 cytoplasmic tail and the Src kinase Fyn was observed following pervanadate treatment. Up-regulation of L1 release and activation of Fyn occurred also when cells were detached by EDTA suggesting that the regulation of L1 shedding by this pathway was linked to cell morphology and adhesion. The phorbol 12-myristate 13-acetate-induced shedding was inhibited by the protein kinase C inhibitor bisindolylmaleimide I and by PD98059, a specific inhibitor of the mitogen-activated protein kinase pathway. Soluble L1 binds to the proteoglycan neurocan and in bound form could support integrin-mediated cell adhesion and migration. We propose that the release of cell-associated adhesion molecules such as L1 may be relevant to promote cell migration.Many transmembrane proteins can undergo cleavage and release of their ectodomain into the medium (for review see Refs. 1-3). These proteins are diverse in structure and function and comprise molecules such as TNF-␣ 1 (4 -8), FasL (9), interleukin-6 receptor (10, 11), L-selectin (12-14), pro-TGF-␣, and the ␤-amyloid precursor protein (15-17). Although there is evidence for a physiological function of many released molecules, a general significance for ectodomain shedding is still disputed (2). Recently, members of the ADAM metalloproteinase family, which are membrane proteins composed of a disintegrin and metalloproteinase domain, were shown to be important in ectodomain release (for review see Refs. 3 and 18). TACE/ADAM17 mediates the membrane release of TNF-␣, Lselectin, TGF-␣ (8), and TRANCE (tumor necrosis factor-related activation-induced cytokine), a TNF family member involved in osteoclastogenesis and dendritic cell survival (19). ADAM10/Kuz has been described as an ␣-secretase for the cleavage of ␤-amyloid precursor protein (20), and ADAM9 is involved in the phorbol ester-induced ectodomain shedding of membrane-anchored heparin-binding EGF-like growth factor (HB-EGF) (21, 22). Certain released molecules can be cleaved by more than one enzyme, and some enzymes can cleave more than one substrate. For example, TNF-␣ cleavage can also be mediated by ADAM10 (23), and ␣-secretase activity for ␤-amyloid precursor protein has been attributed to TACE/ADAM17 (24) and ADAM9 (25). It is not known how the protease(s) select their substrate, because consensus cleavage sites have not been identified. In addition to the proteolytic function, some members of the ADAM famil...

Membrane-Type 1 Matrix Metalloproteinase Stimulates Cell Migration through Epidermal Growth Factor Receptor Transactivation

“…It has been reported that G protein a subunits, such as G ai , directly stimulate Src activity (60), which can lead to MMP-dependent release of EGFR ligands (61). For example, Src activation has been shown to regulate the ADAM-dependent shedding of the L1 adhesion molecule (62). Moreover, several ADAMs have proline-rich Src-homology (SH3) binding domains in their cytoplasmic tails, which can allow interaction with Src (44).…”

The Proprotein Convertase PC5A and a Metalloprotease Are Involved in the Proteolytic Processing of the Neural Adhesion Molecule L1

“…Cleavage within this domain by plasmin reduces multimerization and RGD-independent integrin binding (21). The 180-and 50-kDa fragments result from membrane-proximal cleavage of the membrane-spanning 200-and 80-kDa L1 forms, respectively, by a metalloprotease, most likely of the ADAM (a disintegrin and metalloprotease) family (27,28). This cleavage step has been proposed to be required for cell migration (28).…”

“…The 180-and 50-kDa fragments result from membrane-proximal cleavage of the membrane-spanning 200-and 80-kDa L1 forms, respectively, by a metalloprotease, most likely of the ADAM (a disintegrin and metalloprotease) family (27,28). This cleavage step has been proposed to be required for cell migration (28). Because specific proteolytic processing of L1 is important for regulation of neuronal migration and neurite outgrowth, we have searched for the proteases responsible for cleaving L1 at the two sites and investigated some of the structural and functional consequences of this proteolytic cleavage.…”

Characterization of Growth Factor-induced Serine Phosphorylation of Tumor Necrosis Factor-α Converting Enzyme and of an Alternatively Translated Polypeptide

“…In contrast, little is as yet known about natural inducers and signaling pathways that activate ectodomain shedding under physiological conditions. Recent studies (32)(33)(34)(35)(36) have uncovered the roles of mitogen-activated protein (MAP) kinase signaling pathways in the activation of ectodomain shedding. Thus, growth factors and activated tyrosine kinase receptors rapidly induce ectodomain shedding of TGF-␣ and other transmembrane proteins through induction of the Erk MAP kinase signaling pathway without the need for new protein synthesis (32).…”