Phosphorylation-dependent interactions between ADAM15 cytoplasmic domain and Src family protein-tyrosine kinases

The adamalysins (ADAMs) are transmembrane glycoproteins involved in cell adhesion and proteolytic ectodomain processing of cytokines and adhesion molecules. Many ADAM cytoplasmic domains are proline-rich and have potential phosphorylation sites. We show here that the cytoplasmic domain of ADAM15, metargidin, can interact specifically with Src family protein-tyrosine kinases (PTKs) and the adaptor protein Grb2 in hematopoietic cells (Jurkat, THP-1, U937, and K562 cell lines). Src homology 3 domains from several Src family PTKs including Lck, Fyn, Abl, and Src associate with ADAM15 in vitro. Dephosphorylation of cell extracts resulted in decreased association of ADAM15 with Src family PTK SH3 domains, indicating that phosphorylation influences ADAM15 interactions with its binding partners. This was confirmed in vitro for Hck, Lck, and Grb2, which showed enhanced association with tyrosine-phosphorylated glutathione S-transferase-ADAM15 cytoplasmic domain compared with unphosphorylated protein. In contrast, binding of MAD2 to ADAM15 was slightly reduced by phosphorylation of the ADAM. Immunoprecipitation of ADAM15 from Jurkat cells confirmed the association with Lck in vivo, and upon PMA stimulation, the phosphorylation level of ADAM15 was increased. Cotransfection of ADAM15 and Hck showed Hck-dependent phosphorylation of ADAM15 in vivo. Hck, and to a lesser extent Lck, phosphorylated the ADAM15 cytoplasmic domain in vitro in immune complex kinase assays. Binding of ADAM15 cytoplasmic domain to Hck and Lck was also shown by Far Western analysis. In contrast to Hck, Lck activity was not required for binding to ADAM15, as shown by treatment of cells with PP1. Deletion and point mutation analysis of the ADAM15 cytoplasmic domain confirmed the importance of the proline-rich motifs for Grb2 and Lck binding and indicated the regulatory nature of Tyr(715) and Tyr(735). These data demonstrate selective, phosphorylation-dependent interactions of ADAM15 with Src family PTKs and Grb2, which highlight the potential for integration of ADAM functions and cellular signaling.