Identification of tyrosine residues on ELMO1 that are phosphorylated by the Src-family kinase Hck.
The SH3 and SH2 domains of hematopoietic cell kinase (Hck) play important roles in substrate targeting. To identify new components of Hck signaling pathways, we identified proteins that bind to the SH3 domain of Hck (Scott et al. (2002) J. Biol. Chem. 277, 28238). One such protein was ELMO1, the mammalian orthologue of the Caenorhabditis elegans gene, ced-12. ELMO1 is an approximately 80-kD protein containing a PH domain and a C-terminal Pro-rich sequence. In C. elegans, ced-12 is required for the engulfment of dying cells and for cell migration. In mammalian fibroblasts, ELMO1 binds to Dock180, and functions upstream of Rac during phagocytosis and cell migration. We previously showed that ELMO1 binds directly to the Hck SH3 domain and is phosphorylated by Hck. In this study, we used mass spectrometry to identify the following sites of ELMO1 phosphorylation: Tyr 18, Tyr 216, Tyr 511, Tyr 395, and Tyr 720. Mutant forms of ELMO1 lacking these sites were defective in their ability to promote phagocytosis and migration in fibroblasts. Single tyrosine mutations showed that Tyr 511 is particularly important in mediating these biological effects. These mutants displayed comparable binding to Dock180 and Crk as wild-type ELMO1, but gave a lowered activation of Rac. The data suggest that Src family kinase mediated tyrosine phosphorylation of ELMO1 might represent an important regulatory mechanism that controls signaling through the ELMO1/Crk/Dock180 pathway.
Yokoyama
Noriko
N
Department of Physiology and Biophysics, School of Medicine, State University of New York at Stony Brook, Stony Brook, New York 11794-8661, USA.
deBakker
Colin D
CD
Zappacosta
Francesca
F
Huddleston
Michael J
MJ
Annan
Roland S
RS
Ravichandran
Kodi S
KS
Miller
W Todd
WT
D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry* / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D002460:Cell Line; D002522:Chlorocebus aethiops; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry; D010449:Peptide Mapping; D010587:Phagocytosis; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000737:chemistry / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000737:chemistry / Q000378:metabolism*; D051572:Proto-Oncogene Proteins c-hck; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014443:Tyrosine
D048868:Adaptor Proteins, Signal Transducing; C465214:ELMO1 protein, human; D010446:Peptide Fragments; D011518:Proto-Oncogene Proteins; D011994:Recombinant Proteins; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; C496631:HCK protein, human; D051572:Proto-Oncogene Proteins c-hck