Concerted regulation of cell dynamics by BNIP-2 and Cdc42GAP homology/Sec14p-like, proline-rich, and GTPase-activating protein domains of a novel Rho GTPase-activating protein, BPGAP1.
RhoA, Cdc42, and Rac1 are small GTPases that regulate cytoskeletal reorganization leading to changes in cell morphology and cell motility. Their signaling pathways are activated by guanine nucleotide exchange factors and inactivated by GTPase-activating proteins (GAPs). We have identified a novel RhoGAP, BPGAP1 (for BNIP-2 and Cdc42GAP Homology (BCH) domain-containing, Proline-rich and Cdc42GAP-like protein subtype-1), that is ubiquitously expressed and shares 54% sequence identity to Cdc42GAP/p50RhoGAP. BP-GAP1 selectively enhanced RhoA GTPase activity in vivo although it also interacted strongly with Cdc42 and Rac1. "Pull-down" and co-immunoprecipitation studies indicated that it formed homophilic or heterophilic complexes with other BCH domain-containing proteins. Fluorescence studies of epitope-tagged BPGAP1 revealed that it induced pseudopodia and increased migration of MCF7 cells. Formation of pseudopodia required its BCH and GAP domains but not the proline-rich region, and was differentially inhibited by coexpression of the constitutively active mutant of RhoA, or dominant negative mutants of Cdc42 and Rac1. However, the mutant without the proline-rich region failed to confer any increase in cell migration despite the induction of pseudopodia. Our findings provide evidence that cell morphology changes and migration are coordinated via multiple domains in BPGAP1 and present a novel mode of regulation for cell dynamics by a RhoGAP protein.
Shang
Xun
X
Cell Signaling and Developmental Biology Laboratory, Department of Biological Sciences, The National University of Singapore, 14 Science Drive 4, Singapore 117543, Republic of Singapore.
Zhou
Yi Ting
YT
Low
Boon Chuan
BC
D000595:Amino Acid Sequence; D001483:Base Sequence; D015153:Blotting, Western; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D002460:Cell Line; D045744:Cell Line, Tumor; D002465:Cell Movement; D018076:DNA, Complementary / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes / Q000737:chemistry; D020558:GTP Phosphohydrolases / Q000737:chemistry / Q000378:metabolism; D020690:GTPase-Activating Proteins / Q000737:chemistry* / Q000502:physiology*; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D008969:Molecular Sequence Data; D049294:Phospholipid Transfer Proteins; D011233:Precipitin Tests; D011392:Proline / Q000737:chemistry; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D011518:Proto-Oncogene Proteins / Q000378:metabolism / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012097:Repressor Proteins / Q000378:metabolism / Q000502:physiology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D029701:Saccharomyces cerevisiae Proteins; D017386:Sequence Homology, Amino Acid; D014162:Transfection; D020764:cdc42 GTP-Binding Protein / Q000378:metabolism / Q000502:physiology*
C479829:ARHGAP8 protein, human; C499184:BNIP2 protein, human; D002352:Carrier Proteins; D018076:DNA, Complementary; D000939:Epitopes; D020690:GTPase-Activating Proteins; D008565:Membrane Proteins; D049294:Phospholipid Transfer Proteins; D020033:Protein Isoforms; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; C118289:SEC24 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D011392:Proline; D005982:Glutathione Transferase; D020558:GTP Phosphohydrolases; D020764:cdc42 GTP-Binding Protein