tailieunhanh - Báo cáo khoa học: Identification and functional characterization of an Src homology domain 3 domain-binding site on Cbl

Cbl is an adaptor protein and ubiquitin ligase that binds and is phosphory-lated by the nonreceptor tyrosine kinase Src. We previously showed that the primary interaction between Src and Cbl is mediated by the Src homology domain 3 (SH3) of Src binding to proline-rich sequences of Cbl. | ễFEBS Journal Identification and functional characterization of an Src homology domain 3 domain-binding site on Cbl Archana Sanjay t Tsuyoshi Miyazaki t Cecile Itzstein Enkhtsetseg Purev William C. Horne and Roland Baron Departments of Orthopedics Rehabilitation and Cell Biology Yale University Schoolof Medicine New Haven CT USA Keywords bone resorption Cbl osteoclast SH3 Src Correspondence R. Baron Department of Orthopedics Rehabilitation Yale University Schoolof Medicine PO Box 208044 New Haven CT USA Fax 1 203 785 2744 Tel 1 203 785 5986 E-mail Present address TDepartment of Anatomy CellBiology Temple University Schoolof Medicine Philadelphia PA USA Department of Orthopedic Surgery Faculty of Medicine University of Tokyo Japan These authors contributed equally to this work Received 9 March 2006 revised 10 October 2006 accepted 12 October 2006 doi Cbl is an adaptor protein and ubiquitin ligase that binds and is phosphorylated by the nonreceptor tyrosine kinase Src. We previously showed that the primary interaction between Src and Cbl is mediated by the Src homology domain 3 SH3 of Src binding to proline-rich sequences of Cbl. The peptide Cbl RDLPPPPPPDRP 540-551 which corresponds to residues 540-551 of Cbl inhibited the binding of a GST-Src SH3 fusion protein to Cbl whereas RDLAPPAPPPDR 540-551 did not suggesting that Src binds to this site on Cbl in a class I orientation. Mutating prolines 543-548 reduced Src binding to the Cbl 479-636 fragment significantly more than mutating the prolines in the PPVPPR 494-499 motif which was previously reported to bind Src SH3. Mutating Cbl prolines 543-548 to alanines substantially reduced Src binding to Cbl Src-induced phosphorylation of Cbl and the inhibition of Src kinase activity by Cbl. Expressing the mutated Cbl in osteoclasts induced a moderate reduction in bone-resorbing activity and increased amounts of Src protein. In contrast disabling the tyrosine .