Breast cancer-associated gene 3 (BCA3) is a novel Rac1-interacting protein.
BCA3 was identified in a yeast two-hybrid screen as a novel Rac1-interacting partner in osteoclasts. BCA3 binds directly to Rac and, in vivo, binds GTP-Rac but not GDP-Rac. Perinuclear co-localization of BCA3 and Rac1 is observed in CSF-1-treated osteoclasts. Overexpression of BCA3 attenuates CSF-1-induced cell spreading. We conclude that BCA3 regulates CSF-1-dependent Rac activation.


BACKGROUND
Rac1, a ubiquitously expressed GTPase, is a mediator of colony-stimulating factor 1 (CSF-1)-dependent actin remodeling in osteoclasts. Because the role of Rac in osteoclasts has not been fully defined, we undertook a yeast two-hybrid screen to identify Rac-interacting partners in these cells.


METHODS
A yeast two-hybrid screen was undertaken using a cDNA library prepared from osteoclast-like cells as prey and either native Rac1 or constitutively active Rac1 (Q61L) as bait. Radiolabeled breast cancer-associated gene 3 (BCA3) protein constructs were generated in vitro using rabbit reticulate lysates and used in vitro binding assays with Rac1. In vivo binding was assessed using myc-tagged Rac1(Q61L) and HA-tagged BCA3. PBD pull-down assays were used to determine if GTP-loaded Rac1 preferentially bound BCA3. Co-localization of Rac1 and BCA3 in osteoclasts was assessed using confocal immunofluorescence. The functional significance of the BCA3-Rac1 interaction was assessed by examining the effect of overexpressing BCA3 in RAW 264.7 cells on the subsequent spreading response to CSF-1.


RESULTS
One of three positive clones from the wildtype Rac1 screen and all three positive clones from the Rac1(Q61L) screen encoded the same protein, BCA3. BCA3 expression in osteoclasts was confirmed by RT-PCR and immunocytochemistry. BCA3 bound directly to Rac1 in vitro. Deletional analysis indicated that amino acids 76-125 in BCA3 are important for its ability to bind Rac. In vivo association of the two proteins was shown by co-immunoprecipitation of BCA3 and Rac1. Only GTP-bound-Rac but not GDP-bound Rac could interact with BCA3 in vivo. Confocal immunocytochemistry showed perinuclear co-localization of BCA3 and Rac1 in CSF-1-treated neonatal rat osteoclasts but not in resting osteoclasts. Overexpression of BCA3 markedly attenuated the spreading response to CSF-1 in RAW 264.7 cells.


CONCLUSIONS
These data establish that BCA3 is a novel Rac1-interacting protein and suggest that it may influence the ability of Rac1 to remodel the actin cytoskeleton.
Yu
Kuan-ping
KP
Department of Medicine, Yale School of Medicine, New Haven, CT 06520-8020, USA. kuan-ping.yu@yale.edu
Itokawa
Takashi
T
Zhu
Mei-ling
ML
Syam
Sujata
S
Seth
Arun
A
Insogna
Karl
K
D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D017931:DNA Primers / Q000235:genetics; D005260:Female; D006801:Humans; D066298:In Vitro Techniques; D051379:Mice; D009363:Neoplasm Proteins / Q000235:genetics* / Q000378:metabolism*; D009687:Nuclear Proteins / Q000235:genetics* / Q000378:metabolism*; D010010:Osteoclasts / Q000378:metabolism*; D011485:Protein Binding; D011817:Rabbits; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D020798:Two-Hybrid System Techniques; D020830:rac1 GTP-Binding Protein / Q000235:genetics / Q000378:metabolism*
C471621:AKIP1 protein, human; D048868:Adaptor Proteins, Signal Transducing; D017931:DNA Primers; D009363:Neoplasm Proteins; D009687:Nuclear Proteins; D011994:Recombinant Proteins; D020830:rac1 GTP-Binding Protein