Identification of tyrosine residues on ELMO1 that are phosphorylated by the Src-family kinase Hck.

The SH2 and SH31 domains of Src-family tyrosine kinases play important roles in substrate recognition (1–4). In Src-family kinases, two intramolecular interactions regulate enzymatic activity: (i) an interaction between the SH2 domain and the C-terminal tail, and (ii) an interaction between the SH3 domain and a polyproline type II helix in the SH2-kinase linker region (5–7). Ligands for the SH2 and SH3 domains can disrupt the autoinhibitory interactions and stimulate Src kinase activity (8–10). Many Src kinase substrates possess SH3 or SH2 ligands; in this way, enzyme activation is coupled to substrate phosphorylation (4, 11–13). Numerous substrates that bind to the SH3 domain of Src-family kinases have been reported. Well-studied examples of this class of Src-kinase substrates include p130Cas (14), FAK (15), and AFAP-110 (16). Hematopoietic cell kinase (Hck) is a Src kinase that has been implicated in a variety of signaling pathways in granulocytic and monocytic cells. Hck plays an important role in phagocytosis and lysosome–phagosome fusion; macrophages from Hck-deficient mice show impaired phagocytosis (17). To identify novel components of Hck signaling pathways, we previously carried out a mass spectrometry based search for proteins from U937 monocytic cells that bind to the SH3 domain of Hck. We identified approximately 25 potential binding partners for Hck (18). One of them, ELMO1, is the subject of the present study. ELMO1 is the mammalian orthologue of the Caenorhabditis elegans gene, ced-12. ELMO1/ced-12 was identified as an upstream regulator of Rac that functions genetically at the same step as Dock180/ced-5 in the engulfment of apoptotic cells and in cell migration (19). Overexpression of mammalian ELMO1 rescues the distal tip cell migration defect of the ced-12 mutant C. elegans (19). In mammalian fibroblasts, the ELMO1/Crk/Dock180 complex functions upstream of Rac during phagocytosis and cell migration. Thus, coexpression of ELMO1 with CrkII and Dock180 in LR73 fibroblasts promotes phagocytosis, and causes localization of ELMO1 to the membrane ruffles that are formed. Expression of ELMO1 augments the interaction between Dock180 and Rac. The Dock180–ELMO1 complex functions as an unconventional two-component exchange factor for Rac, and ELMO1 directly participates in the stimulation of Rac GEF activity and Rac GTP loading (20, 21). ELMO1 has no discernible catalytic domain, but at its C-terminus it possesses a pleckstrin homology (PH) domain, a putative leucine zipper motif, and a proline-rich motif (19, 22). The C-terminal 100 amino acids of ELMO1 are essential for the interaction with Dock180 (20), while the N-terminus of ELMO1 is important for mediating cell migration via the ELMO1–Dock180 complex (23). We previously showed that ELMO1 binds directly to the Hck SH3 domain via a polyproline-rich motif. We also demonstrated that coexpression of Hck and ELMO1 in mammalian cells resulted in tyrosine phosphorylation of ELMO1 (18). In this report, we have mapped the sites of tyrosine phosphorylation, and we report that phosphorylation has the potential to modulate the ELMO/Dock/CrkII signaling pathway that leads to Rac activation.