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. 2003 Apr;23(7):2476-88.
doi: 10.1128/MCB.23.7.2476-2488.2003.

GGAPs, a new family of bifunctional GTP-binding and GTPase-activating proteins

Chunzhi Xia  1 , Wenbin Ma, Lewis Joe Stafford, Chengyu Liu, Liming Gong, James F Martin, Mingyao Liu
Affiliations

GGAPs, a new family of bifunctional GTP-binding and GTPase-activating proteins

Chunzhi Xia et al. Mol Cell Biol. 2003 Apr.

Abstract

G proteins are molecular switches that control a wide variety of physiological functions, including neurotransmission, transcriptional activation, cell migration, cell growth. and proliferation. The ability of GTPases to participate in signaling events is determined by the ratio of GTP-bound to GDP-bound forms in the cell. All known GTPases exist in an inactive (GDP-bound) and an active (GTP-bound) conformation, which are catalyzed by guanine nucleotide exchange factors and GTPase-activating proteins (GAPs), respectively. In this study, we identified and characterized a new family of bifunctional GTP-binding and GTPase-activating proteins, named GGAP. GGAPs contain an N-terminal Ras homology domain, called the G domain, followed by a pleckstrin homology (PH) domain, a C-terminal GAP domain, and a tandem ankyrin (ANK) repeat domain. Expression analysis indicates that this new family of proteins has distinct cell localization, tissue distribution, and even message sizes. GTPase assays demonstrate that GGAPs have high GTPase activity through direct intramolecular interaction of the N-terminal G domain and the C-terminal GAP domain. In the absence of the GAP domain, the N-terminal G domain has very low activity, suggesting a new model of GGAP protein regulation via intramolecular interaction like the multidomain protein kinases. Overexpression of GGAPs leads to changes in cell morphology and activation of gene transcription.

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Figures

FIG.1.
FIG.1.
Sequence alignment of GGAP1, GGAP2, and MRIP1 (GenBank accession numbers: AY033765 for GGAP1, AF384128 for GGAP2, and AF359283 for MRIP1). Identical amino acids are indicated by an asterisk. GGAP1 shares approximately 50 and 70% sequence homology with GGAP2 and MRIP1 at the amino acid level, respectively. A database search identified two cDNAs, KIAA1099 and KIAA0167, that are the same as GGAP1 and GGAP2 (25).
FIG. 2.
FIG. 2.
Domain structure and sequence comparison of GGAP proteins. (A) The three GGAP proteins share the same domain structure with an N-terminal GTPase domain, a PH domain, followed by a C-terminal GAP domain, and an ANK repeat domain. (B) The N-termini of the GGAPs share sequence and motif homology with Ras family of G proteins (12, 32). Residues in boldface type indicate conserved consensus motifs in the proteins. Dark residues are unique insertions in the sequences. (C) Sequence comparison of the C-terminal GAP domain with Arf GAP (20, 36). (D) Sequence homology of ANK domains between GGAP family of proteins and other ANK repeat domain proteins. Residues represent consensus conserved ANK repeat amino acids and nonconserved amino acids. (E) Rooted phylogenetic tree of GGAP family proteins and Ras family of proteins. Nucleotide sequences of the above proteins were obtained from the GenBank. After being multiply aligned using the ClustalW program available at Biology workbench, the obtained multiple alignments were then used to construct rooted phylogenetic tree using ClustalW program and then viewing with DRAWGRAM program (http://workbench.sdsc.edu) as described by Li and Gouy (30).
FIG. 2.
FIG. 2.
Domain structure and sequence comparison of GGAP proteins. (A) The three GGAP proteins share the same domain structure with an N-terminal GTPase domain, a PH domain, followed by a C-terminal GAP domain, and an ANK repeat domain. (B) The N-termini of the GGAPs share sequence and motif homology with Ras family of G proteins (12, 32). Residues in boldface type indicate conserved consensus motifs in the proteins. Dark residues are unique insertions in the sequences. (C) Sequence comparison of the C-terminal GAP domain with Arf GAP (20, 36). (D) Sequence homology of ANK domains between GGAP family of proteins and other ANK repeat domain proteins. Residues represent consensus conserved ANK repeat amino acids and nonconserved amino acids. (E) Rooted phylogenetic tree of GGAP family proteins and Ras family of proteins. Nucleotide sequences of the above proteins were obtained from the GenBank. After being multiply aligned using the ClustalW program available at Biology workbench, the obtained multiple alignments were then used to construct rooted phylogenetic tree using ClustalW program and then viewing with DRAWGRAM program (http://workbench.sdsc.edu) as described by Li and Gouy (30).
FIG. 3.
FIG. 3.
Expression of GGAP1 and GGAP2 in human tissues and mouse embryo. (A) Human multitissue Northern blot hybridized with a probe derived from N-terminal domains of GGAP1 and GGAP2, respectively. For GGAP1, two message RNAs (∼5 and 8 kb) were detected in most of the human tissues for GGAP1 while a different splicing form was detected in periphery blood leukocytes (PBL). GGAP2 is highly expressed in brain. Different sizes of transcripts were detected in excitable tissues (brain, heart, and smooth muscle [S. muscle]) compared to immune tissues (thymus, spleen, and PBL). S. intestine, small intestine. (B) Expression of GGAP1 in 12.5-day mouse embryo. Whole-mount in situ hybridization shows GGAP1 is highly expressed in forebrain, middle brain, and neural tubes during embryo development (arrows). Whole-mount in situ hybridization, sectioning and staining of tissue sections were performed as described elsewhere (33).
FIG.4.
FIG.4.
Intracellular expression and localization of GGAP1 and GGAP2. (A) GGAP1 is expressed in the cytoplasm, possibly in ER and Golgi apparatus in COS-7 cells. Flag-tagged GGAP1 was stained with a specific anti-Flag M2 monoclonal antibody. (a) Expression of GGAP1 in the cytosol, possibly with intracellular membrane structures, such as ER and Golgi apparatus. (b) Actin staining with Texas red-labeled phalloidin. (c) Nuclear staining of COS-7 cells with DAPI (4′,6′-diamidino-2-phenylindole). (d) The merger picture of panels a, b, and c, showing expression of GGAP1, actin, and nucleus in the cells. (B) Expression and localization of GGAP2 in cytosol and nucleus in COS-7 cells. (a) GGAP2 expression in both cytosol and nucleus. (b and c) actin and nuclear staining, respectively. (d) Merger picture showing GGAP2 expressed in both cytoplasm and nucleus. Fluorescent images of cells were captured on a charge-coupled device camera mounted on Olympus inverted research microscope using Ultraview imaging software.
FIG. 5.
FIG. 5.
GTP-binding and GTPase activities of GGAP1 and GGAP2. (A) GGAP1 and GGAP2 bind to [α-32P]GTP. Purified His-GGAP1-NT (1 to 292), His-GGAP1-CT (467 to 804), His-GGAP2-NT (1 to 294), His-GGAP2-CT (390 to 826), and Ni2+ beads (control) were incubated with [α-32P]GTP in the absence or presence of excess unlabeled GTP (10 mM). After extensive washing, bound radioactivity was counted in a scintillation counter. Error bars, standard deviations. (B) Enzymatic activity of GGAP1 and GGAP2. Immunopurified Flag-tagged GGAP1 and GGAP2 (0.5 μg) was incubated with [α-32P]GTP for 1 h at 30°C. The extent of GTP hydrolysis was assessed by thin layer chromatography. Flag-tagged C-terminal domains of GGAP1 and GGAP2 (0. 5 μg) were used as negative control. The eluted GTP and GDP were separated by thin layer chromatography on polyethyleneimine-celluose plates (J. T. Baker). (C) Dissociation of GDP from GGAPs. Squares present data for GGAP1 and circles represent data for GGAP2. A 2 μM concentration of [3H]GDP was incubated with the purified GST-G domains (2 μg) of GGAP1 and GGAP2 at 25°C for 60 min. The dissociation reaction were initiated by adding 2 mM unlabeled GDP to the incubation mixtures (160 μl), at the indicated time intervals, aliquots of 20 μl were withdrawn from the reaction mixture, the remaining G-protein-bound radionucleotides were quantitated by scintillation counting.
FIG. 6.
FIG. 6.
Activation of G domains of GGAP1 and GGAP2 by the C-terminal GAP domains. (A) GTPase assays of the N-terminal G domain in the absence or presence of the C-terminal GAP domain of GGAP1 and GGAP2, respectively. Purified His-tagged proteins (GGAP1 and GGAP2, 0.5 μg) were rinsed twice with ice-cold loading buffer followed by resuspension in a 50-μl reaction buffer (20 mM Tris-HCl [pH 8.0], 10 mM MgCl2, 10 mM DTT). The proteins were incubated with [α-32P]GTP (0.1 μM) for 30 min at 30°C with or without the addition of the C-terminal GAP domains (1 μg each) of GGAP1 and GGAP2, respectively. The extent of GTP hydrolysis was assessed by thin layer chromatography. The radioactivity of GTP and GDP was quantitated, and the percentages of GTP/[GTP+GDP] are shown in the bottom. (B and C) Time-dependent activation of G domains by the GAP domains of GGAP1 and GGAP2, respectively. Purified His-tagged G domains (1 μg) were assayed for their activities in the presence of the purified GAP domain (1.5 μg) at indicated time (0, 10, 30, and 60 min). Guanine nucleotides hydrolyzed by the G domains in the presence of GAP domains were separated by thin layer chromatography and detected by autoradiography.
FIG. 7.
FIG. 7.
Direct interaction of the N-terminal GTPase domain with the C-terminal GAP domain of GGAPs. (A) Flag-tagged GAP domains of GGAP1 and GGAP2, respectively, by coimmunoprecipitation. (B) His-tagged GAP domains interact with the 35S-labeled N-terminal GTPase domain of GGAP1 or GGAP2 by protein pull down assay. (C) Model of intramolecular interaction and activation of GGAPs. In the inactive status, the N-terminal GTPase domain interacts with the C-terminal GAP domain, resulting in a protein with high GTPase activity and in the GDP-bound inactive status. Activation or binding of GGAP proteins with other proteins will disrupt the intramolecular interaction of the GTPase domain and the GAP domain. Therefore, the GGAP proteins will have low GTPase activity and exist in the GTP-bound active status.
FIG. 8.
FIG. 8.
GGAP1 synergistically activates the Ras-mediated mitogen-activated protein kinase signaling pathway at the serum response element (SRE) with signals that activate TCF (such as SAP1 and Elk1). (A) Activation of the c-fos SRE by GGAP1, GGAP2, and their N- and C-terminal domains, respectively. (B and C) Effects of GGAP1 and GGAP2 on SAP1- and Elk1-mediated transcriptional activation. Cells were transfected with luciferase reporter plasmids controlled by SRE, SAP1, and Elk1, respectively, together with the expression plasmids encoding LacZ (control), GGAP1, GGAP2, and their N- and C-terminal domains as indicated. Data shown are average of three qualitatively similar independent experiments with standard deviations (error bars).
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References

    1. Aghazadeh, B., W. E. Lowry, X. Y. Huang, and M. K. Rosen. 2000. Structural basis for relief of autoinhibition of the Dbl homology domain of proto-oncogene Vav by tyrosine phosphorylation. Cell 102:625-633. - PubMed
    1. Bagrodia, S., and R. A. Cerione. 1999. Pak to the future. Trends Cell Biol. 9:350-355. - PubMed
    1. Bar-Sagi, D., and A. Hall. 2000. Ras and Rho GTPases: a family reunion. Cell 103:227-238. - PubMed
    1. Berman, D. M., T. M. Wilkie, and A. G. Gilman. 1996. GAIP and RGS4 are GTPase-activating proteins for the Gi subfamily of G protein alpha subunits. Cell 86:445-452. - PubMed
    1. Bollag, G., and F. McCormick. 1995. Intrinsic and GTPase-activating protein-stimulated Ras GTPase assays. Methods Enzymol. 255:161-170. - PubMed

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