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) domaincontaining, 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 prolinerich 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. 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) domaincontaining, 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 prolinerich 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. Cells undergo dynamic changes as part of their adaptation and response to stimuli. These include their abilities to proliferate, differentiate, or execute death. Many of these processes are controlled by a series of signaling events relayed via a cascade of molecular interaction that are normally associated with the enzymatic or structural modifications of target proteins. Furthermore, there exist various checkpoints that serve to fine-tune the amplitude, duration, as well as the integration of such circuitry response. One of the relatively well characterized signaling circuits in eukaryotic system is the Ras small GTP-binding protein (GTPase) superfamily (1Etienne-Manneville S. Hall A. Nature. 2002; 420: 629-635Crossref PubMed Scopus (3839) Google Scholar, 2Bar-Sagi D. Hall A. Cell. 2000; 103: 227-238Abstract Full Text Full Text PDF PubMed Scopus (702) Google Scholar, 3Mackay D.J. Hall A. J. Biol. Chem. 1998; 273: 20685-20688Abstract Full Text Full Text PDF PubMed Scopus (567) Google Scholar) that binds and slowly hydrolyzes GTP to GDP, which is still bound to the proteins. The GTP-bound form assumes an active conformation that allows interaction with downstream effectors, thus the “on-switch,” whereas its conversion to the GDP-bound form keeps the proteins in an “off-switch” mode and renders the GTPase inactive. The balance of these two forms determines the final execution of the pathway. This is regulated by two other important classes of proteins, one that helps enhance its GTPase activity, termed GTPase-activating proteins (GAPs) 1The abbreviations used are: GAPs, GTPase-activating proteins; EST, expressed sequence tag; HA, hemagglutinin; ANOVA, analysis of variance; LD, longest diameter; SD, shortest diameter; IP, immunoprecipitation; IB, immunoblotting; FL, full-length; GFP, green fluorescent protein; GST, glutathione S-transferase; WCL, whole cell lysate.1The abbreviations used are: GAPs, GTPase-activating proteins; EST, expressed sequence tag; HA, hemagglutinin; ANOVA, analysis of variance; LD, longest diameter; SD, shortest diameter; IP, immunoprecipitation; IB, immunoblotting; FL, full-length; GFP, green fluorescent protein; GST, glutathione S-transferase; WCL, whole cell lysate. and the other, termed guanine nucleotide exchange factors (GEFs) that activate the protein by catalyzing its exchange of GDP for GTP. Many members of the small GTPases have already been identified, and they can further be subdivided into various families or subfamilies according to the similarities in their primary sequences. Members from different families exhibit diverse functions ranging from the control of intracellular trafficking to cytoskeletal rearrangements and cell cycle progression. The degree of specificity is further extended to even closely related members within the same families. For example, in the Rho family, the Cdc42 plays an important role in the formation of filopodia, whereas RhoA and Rac1 activation results in the formation of stress fibers and membrane ruffles respectively (4Hall A. Science. 1998; 279: 509-514Crossref PubMed Scopus (5216) Google Scholar). In addition, there is a hierarchy of network in certain cell types where activation of one member can affect the activity of another. For example, activation of Cdc42 leads to filopodia formation, which could in turn activate Rac1 (5Kozma R. Ahmed S. Best A. Lim L. Mol. Cell Biol. 1995; 15: 1942-1952Crossref PubMed Scopus (881) Google Scholar, 6Nobes C.D. Hall A. Biochem. Soc. Trans. 1995; 23: 456-459Crossref PubMed Scopus (302) Google Scholar), whereas Rac1 activation leads to inactivation of RhoA in NIH3T3 resulting in the epithelioid phenotype (7Sander E.E. ten Klooster J.P. van Delft S. van der Kammen R.A. Collard J.G. J. Cell Biol. 1999; 147: 1009-1022Crossref PubMed Scopus (737) Google Scholar, 8Evers E.E. Zondag G.C. Malliri A. Price L.S. ten Klooster J.P. van der Kammen R.A Collard J.G. Eur. J. Cancer. 2000; 36: 1269-1274Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar, 9Zondag G.C. Evers E.E. ten Klooster J.P. Janssen L. van der Kammen R.A. Collard J.G. J. Cell Biol. 2000; 149: 775-782Crossref PubMed Scopus (251) Google Scholar). 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Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar), a proline-rich and a GAP We that BP-GAP1 differentially RhoA, Cdc42, and Rac1 signaling by a that required the BCH and GAP expressed in human cell MCF7 BPGAP1 induced cell that required its GAP activity as well as the BCH but not the proline-rich However, the proline-rich region was required for cell migration the changes induced by GAP and BCH These results the by different domains of BPGAP1 in cell dynamics and that changes in cell morphology is a but not the for cell it the of other as Our findings also the to functions of protein domains in various RhoGAP families in to have a of their functions and novel proteins GAP the sequence of the RhoGAP of was used as sequence in the the sequence as well as human and of the was in the sequence of BPGAP1 and the of was from MCF7 the according to the of was to the with with for at in a of of was by the to the sequence at at 50 at and final at These and the and to their The and into a epitope-tagged or of and Cell to BPGAP1 was and its various domains from the in a and for For each and in the for in For of was to region of whereas was by as J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). was used as for the of the used of and for molecular and as J. Scholar). the of BPGAP1 and Cdc42GAP in various and the was was the from various cell or from various from a and for the as of the to for BPGAP1 and The of BPGAP1 to that BCH region that the The was used to the of The results in at least two with of to Cell and human human and in with and from and at in a in whereas human and in Cells at in or with or of indicated according to the and or with in of 50 50 and a of The as whole cell or used in with various proteins as J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). in and by with in with various for and for as J. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). with a system proteins with by For and proteins, with by For the morphology of was a fluorescent the for as J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). of proteins In by in a multiple protein and the of with and at for with of proteins with glutathione in the by In GTPase activity with the to the of from GTP by Cdc42, RhoA, or Rac1 of these and Cdc42GAP are from A. in the of control or or its For these used a G. 2000; PubMed Scopus Google Scholar) with In of or mutant proteins a of was in a with of of of nucleotide and of The was in the to at of was to of GST, or proteins and for at the multiple an at the of small GTPase was to the The at was In GTPase and Cdc42, or RhoA was by to the of S. C.L. J. R.A. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar) or J. 1999; PubMed Scopus Google Scholar) by In cell small GTPases or with or without BPGAP1 with of or with for at with a of and and Cdc42, or RhoA was by cell also for the of expressed Cdc42, RhoA, and BPGAP1 for For of Rho GTPases, the and from with for or with or with and the associated proteins and with RhoA, or to the of Cell with control or BPGAP1 and the longest and shortest that the of and to each J. Cell Biol. 2003; PubMed Scopus Google Scholar). The cell and the of the cell also as and the and from each with at least different cells. was of with the Cell of to was with a as 2002; PubMed Scopus Google Scholar). The of the was with as a MCF7 with different or mutants at a of in of with The was with of for at in the that not the with and the that to the of the with and for each The as the of with was of with the GTPase-activating novel GTPase-activating proteins (GAPs) in the human genome and to an how they regulate various processes their various protein and the with the well characterized GAP of as the We have identified in classes of proteins that the GAP with other signaling protein of them include the Rho guanine nucleotide exchange and the Rho One of these classes is by members that the of the Cdc42GAP protein. are by the at the of the identified BNIP-2 and Cdc42GAP that in the BNIP-2 family Lim Lim J. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar) and a well GAP at its in these two domains is a proline-rich the predicted from one of the and used in to the from human MCF7 cells. one sequence of was identified which for a protein that from by The protein also at the from two proteins from the same human and to not identified the for such in thus Many classes of have been identified from the human genome and However, they are not related to each other as each one different types and of other associated protein provide to the of GAP with its to family of proteins (for BCH and Cdc42GAP-like with their We further to how one novel member identified BPGAP1 processes via these protein are to the for other by and the longest is that these could be from of the same with to human BPGAP1 was also identified from the genome by or with BPGAP1 at various the BPGAP1 a sequence at the but a Cdc42GAP the degree of or for the BCH and GAP with domains in other proteins. The BCH of BPGAP1 is closely related to that of Cdc42GAP its GAP also shares the degree of with that of Cdc42GAP BPGAP1 an at indicated by an This in other is known as an and to be for as a D. R.A. Biol. 1998; PubMed Scopus Google Scholar, Biol. 1998; PubMed Scopus Google Scholar, R.A. 2002; PubMed Scopus Google Scholar). In addition, BPGAP1 in the proline-rich which is to identified in and proteins could one for the or domains S. 2002; PubMed Scopus Google Scholar, Cell. 2000; 103: Full Text Full Text PDF PubMed Scopus Google Scholar), the of which to be BPGAP1 and with an into the of the of BPGAP1 and the of the various protein domains it human cell in the of and for the of In to Cdc42GAP was to of such as and the of BPGAP1 BPGAP1 can be in the and not For and various as epitope-tagged that of proteins that the proline-rich or GAP Cells with these and for their by as and that and mutants expressed well and for or We that BCH a novel protein interaction Lim Lim J. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). BPGAP1 could with other BCH domain-containing proteins, the to with of the Cdc42GAP or or their BCH BPGAP1 in and by that BPGAP1 was a target of Cdc42GAP or its BCH but it failed to with BNIP-2 or its BCH in that the BCH of BPGAP1 was BNIP-2 or its BCH could form a heterophilic with Furthermore, the interaction with Cdc42GAP or its BCH was These results that BPGAP1 can form a heterophilic with Cdc42GAP it can with these the that the GAP without the BCH not to any of the further these the Cdc42GAP and BNIP-2 expressed as proteins in the and to as and BNIP-2 by BPGAP1 and its BCH could also be with with is that and BNIP-2 could also be with BPGAP1 These results that BPGAP1 could with other BCH domain-containing proteins in and in vivo. BPGAP1 RhoA, Cdc42, and Rac1 via Their BCH and GAP the GAP that the BPGAP1 was predicted to and confer the GTP of certain Rho in and in vivo GTPase activity For the in GTPase activity of Cdc42, or Rac1 with GTP and the of GTP in the or of BPGAP1 or its mutants the G. 2000; PubMed Scopus Google Scholar) as and that BPGAP1 GTPase activity of Cdc42 and RhoA in by and In contrast, it activation Rac1 GTPase These via its GAP as by the same of activation from the the GAP and the of from the that the but of the GAP Furthermore, such activation was a at the the of such activity in how the activity of the Rho GTPases the could be regulated by BPGAP1 in vivo. Cells with Cdc42, RhoA, or Rac1 with the control or The activity of these GTPases in vivo was by their of to the as and that in RhoA to its was in the of In contrast, the of Cdc42 and Rac1 to their and at they to be increased These results that BPGAP1 its GAP activity RhoA, but not Cdc42 or Rac1 in vivo. closely the of Rho GTPases to the and GAP domains of that Cdc42, despite their in the whole cell of could be by the RhoA was not bound by BPGAP1 its was by with was the that RhoA could be with BPGAP1 and of interaction was further increased RhoA was also to the Cdc42, and Rac1 interacted strongly with BPGAP1 in the or co-immunoprecipitation that BPGAP1 could with Cdc42GAP strongly the there still the that of these Rho GTPases with BPGAP1 via results that BPGAP1 RhoA, but not Cdc42 or Rac1 in vivo and that it still its to form a with Cdc42 and Rac1. BPGAP1 via BCH and GAP further the of BPGAP1 interaction via its different to their in one of the key by Rho GTPases, the control of cell We to how BPGAP1 affect the morphology as well as the migration of the target and to changes in cell morphology is and to cell We to the human MCF7 as the of its in of its cell morphology and it is with cell MCF7 with of or the for as and that control with of an and with of the the of BPGAP1 was to be in the and it induced and as in the further such and to the dynamics without to the for with green fluorescent protein of BPGAP1 and its various mutants to of the cell dynamics could be by certain types of changes in cell morphology and their and MCF7 the control in and of them with However, induced pseudopodia in of the a that was normally by of from the of the cell by the but without proline-rich or BCH and proline-rich pseudopodia and of the with the cell In the of the GAP in the was to and pseudopodia. This was further by the GAP mutant that in the to that induced by the or In contrast, of the proline-rich region of BPGAP1 not affect the formation of by the cells. that the formation of induced by BPGAP1 was not the of cell or as various for cell also as and These include the and the that the of and to each other, the of the cell and also the of The results that with the BPGAP1 or mutants induced changes without changes in the cell the longest or the shortest that there was cell or they the of pseudopodia. these results that BPGAP1 pseudopodia formation via the BCH and GAP domains of the proline-rich of pseudopodia by BPGAP1 was also in and not of RhoA but of of pseudopodia required its GAP and the BCH and that in vivo BPGAP1 inactivated the RhoA but not Cdc42 and that such formation of pseudopodia inactivation of RhoA but not the inactivation of Cdc42 or Rac1. Furthermore, activation of RhoA could such whereas activation of Cdc42 or Rac1 changes by these two with BPGAP1 with the constitutively or dominant negative mutants of the RhoA, Cdc42, or Rac1. studies that of or constitutively active mutant of RhoA the formation of pseudopodia with still However, its dominant negative mutant not affect any of the These results are with the that GAP pseudopodia and its or its mutant in the pseudopodia and with the of the inactivation of RhoA was the of stress fibers by for the in BPGAP1 not In contrast, or mutant Cdc42 of cell that with whereas the negative mutant of Cdc42 the of BPGAP1 for of the Rac1 of the despite the of However, present with its active mutants in and the for a in these there various of at the to the from cells. Cdc42 the Rac1 could the formation of with still in certain cells. In to the of the induction by active Rac1 and Rac1 mutant was with the the BCH or the GAP of BPGAP1 by that BCH with the Rac1 in with whereas the GAP but with of These changes provide evidence that BCH and GAP are in the regulation of cell dynamics other downstream of Cdc42 and Rac1 without the GTPase activity of Cdc42 or Rac1 BPGAP1 Cell via of BCH and GAP with the Proline-rich BPGAP1 changes to cell it a key as to the that such in cell morphology are associated with cell as in and in E.E. Zondag G.C. Malliri A. Price L.S. ten Klooster J.P. van der Kammen R.A Collard J.G. Eur. J. Cancer. 2000; 36: 1269-1274Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar, J. Cell PubMed Google Scholar). We to induction of pseudopodia in MCF7 for their to cell migration and to was to cell at least within the of BPGAP1 Cells with the control or or of BPGAP1 or the for their to the as and that of the with the BPGAP1 to the the control whereas or domains not any increase in their migration despite the induction of pseudopodia with and These results that BCH or GAP domains despite their is not to cell the despite the BCH and GAP domains and the to failed to confer any increase in the cell These results that cell changes induced by BPGAP1 the BCH and GAP are required but still not for cell required further of via the proline-rich The of the BCH GAP and proline-rich sequence in cell dynamics is The studies the and of a novel protein that plays an important role in cell and functions of its protein BCH proline-rich region, and the GAP in or as a whole protein in and in vivo We that BCH and GAP domains induced and that to cell migration to its proline-rich The of the BCH and GAP domains of BPGAP1 in different of pseudopodia is and it different BPGAP1 functions as a GAP for RhoA in and in it also Cdc42 and Rac1. These the that despite not the Cdc42 and Rac1 could still be in BPGAP1 results that pseudopodia formation could be by the dominant negative mutants of Cdc42 and inhibited by dominant negative mutants of Rac1 the However, it was by the mutant of RhoA RhoA activation inhibited formation of pseudopodia whereas Cdc42 or Rac1 the cell changes in the with Rac1 Furthermore, could be associated with the and with an as by BCH or GAP In it is that Cdc42 Rac1 could be to BPGAP1 for other to as to further the signaling in pseudopodia. is to These results strongly the of BPGAP1 in differentially pathways of Rho GTPases that could have important the cell migration in The where BPGAP1 cell dynamics via two domains of BCH and GAP that are to each other also a novel and mode of regulation for GAP proteins. at that such of BCH with GAP domains are also in proteins from various eukaryotic the but not in have and characterized at least one member of the in that it could a such as the control of cell morphology and cell as in the However, of various and there exist at least closely related to to be to be how such sequence the of these GAP proteins have been identified to regulate cell morphology but is known the of cell morphology to cell migration via their protein domains in BPGAP1 an for such an With multiple signaling BPGAP1 is to target different classes of signaling and thus could a role in the integration of signaling results that cell changes induced by BPGAP1 the BCH and GAP domains are required but still not for cell an from the proline-rich region that the control of cell to the changes that the This of is different from other was the GAP For example, of the GAP RhoA activity, the formation of membrane and enhanced Mol. Biol. Cell. PubMed Scopus Google Scholar). GAP for enhanced cell via a J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar) but also a the its RhoGAP to cell of the other domains S. A. J. Zheng Y. Mol. Cell. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). In addition, the RhoGAP of could an cell and of Y. Y. Y. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). is also to that the proline-rich region of BPGAP1 is to identified in and and could one for S. 2002; PubMed Scopus Google Scholar) or Cell. 2000; 103: Full Text Full Text PDF PubMed Scopus Google Scholar) domains and protein are members of the family proteins that are associated with cell and and dynamic membrane R. J. Cell. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, Cell. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). the of the proline-rich region and the of a for domain-containing proteins, the that and other processes a but for This the of BPGAP1 in the and regulation of small GTPase signaling in cell dynamics We and for and Lim for in
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