Physical and functional association of c-Src and adhesion and degranulation promoting adaptor protein (ADAP) in osteoclastogenesis in vitro.
c-Src plays a crucial role in osteoclastogenesis. In this study, we searched for c-Src-binding proteins using a combination of pull-down assays and mass spectrometric analysis, and identified the association of adhesion and degranulation promoting adaptor protein (ADAP) with c-Src in RAW264 cells and osteoclast precursors prepared from bone marrow cells. The kinase activity and the SH2 domain of c-Src were required for this association and Tyr807 in the extreme carboxyl terminus of ADAP was identified as a major recognition site. ADAP was found to be expressed in cells at the prefusion stage and localized mainly in the leading edge of lamellipodia and in pseudopodia. Tyrosine phosphorylation of ADAP was induced in an integrin-dependent manner, and the level was Src kinase-dependent. ADAP-knockdown RAW264 cells showed retarded migration and formed few multinucleated cells. Cas, known to be phosphorylated by c-Src, was identified as a major tyrosine-phosphorylated protein in differentiating RAW264 cells and the phosphorylation appeared to be decreased in ADAP-knockdown cells. ADAP thus may play an important role as a partner of c-Src for cell migration and progression to the multinucleated cell stage in osteoclastogenesis in vitro.
Koga
Shintaro
S
Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma, Nara 630-0101, Japan.
Yogo
Keiichiro
K
Yoshikawa
Kana
K
Samori
Hidetomo
H
Goto
Masashi
M
Uchida
Toshihiro
T
Ishida
Norihiro
N
Takeya
Tatsuo
T
D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000502:physiology*; D000818:Animals; D000081247:CSK Tyrosine-Protein Kinase; D002448:Cell Adhesion / Q000502:physiology; D015550:Cell Degranulation / Q000502:physiology*; D002454:Cell Differentiation / Q000502:physiology*; D002460:Cell Line; D002465:Cell Movement / Q000502:physiology; D050719:Crk-Associated Substrate Protein; D005260:Female; D020869:Gene Expression Profiling; D006801:Humans; D051379:Mice; D041681:NIH 3T3 Cells; D010010:Osteoclasts / Q000166:cytology / Q000502:physiology*; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000502:physiology*; D011506:Proteins / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D050717:Retinoblastoma-Like Protein p130; D013234:Stem Cells / Q000166:cytology / Q000502:physiology; D014443:Tyrosine / Q000235:genetics / Q000378:metabolism; D019061:src-Family Kinases
D048868:Adaptor Proteins, Signal Transducing; C493841:BCAR1 protein, human; C493842:Bcar1 protein, mouse; D050719:Crk-Associated Substrate Protein; C491468:Fyb protein, mouse; D011506:Proteins; D034741:RNA, Small Interfering; D050717:Retinoblastoma-Like Protein p130; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; D000081247:CSK Tyrosine-Protein Kinase; D019061:src-Family Kinases; C000631346:CSK protein, human