Identification of Tyrosine Residues on ELMO1 That Are Phosphorylated by the Src-Family Kinase Hck
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- 作者：Noriko Yokoyama ; Colin D. deBakker ; Francesca Zappacosta ; Michael J. Huddleston ; Roland S. Annan ; Kodi S. Ravichandran ; and W. Todd Miller
- 刊名：Biochemistry
- 出版年：2005
- 出版时间：June 21, 2005
- 年：2005
- 卷：44
- 期：24
- 页码：8841 - 8849
- 全文大小：396K
- 年卷期：v.44,no.24(June 21, 2005)
- ISSN：1520-4995
文摘
The SH3 and SH2 domains of hematopoietic cell kinase (Hck) play important roles in substratetargeting. To identify new components of Hck signaling pathways, we identified proteins that bind to theSH3 domain of Hck (Scott et al. (2002) J. Biol. Chem. 277, 28238). One such protein was ELMO1, themammalian orthologue of the Caenorhabditis elegans gene, ced-12. ELMO1 is an 
80-kD proteincontaining a PH domain and a C-terminal Pro-rich sequence. In C. elegans, ced-12 is required for theengulfment 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 ELMO1binds directly to the Hck SH3 domain and is phosphorylated by Hck. In this study, we used massspectrometry to identify the following sites of ELMO1 phosphorylation: Tyr 18, Tyr 216, Tyr 511, Tyr395, and Tyr 720. Mutant forms of ELMO1 lacking these sites were defective in their ability to promotephagocytosis and migration in fibroblasts. Single tyrosine mutations showed that Tyr 511 is particularlyimportant in mediating these biological effects. These mutants displayed comparable binding to Dock180and Crk as wild-type ELMO1, but gave a lowered activation of Rac. The data suggest that Src familykinase mediated tyrosine phosphorylation of ELMO1 might represent an important regulatory mechanismthat controls signaling through the ELMO1/Crk/Dock180 pathway.
80-kD proteincontaining a PH domain and a C-terminal Pro-rich sequence. In C. elegans, ced-12 is required for theengulfment 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 ELMO1binds directly to the Hck SH3 domain and is phosphorylated by Hck. In this study, we used massspectrometry to identify the following sites of ELMO1 phosphorylation: Tyr 18, Tyr 216, Tyr 511, Tyr395, and Tyr 720. Mutant forms of ELMO1 lacking these sites were defective in their ability to promotephagocytosis and migration in fibroblasts. Single tyrosine mutations showed that Tyr 511 is particularlyimportant in mediating these biological effects. These mutants displayed comparable binding to Dock180and Crk as wild-type ELMO1, but gave a lowered activation of Rac. The data suggest that Src familykinase mediated tyrosine phosphorylation of ELMO1 might represent an important regulatory mechanismthat controls signaling through the ELMO1/Crk/Dock180 pathway.