Cdc42 Regulates the Par-6 PDZ Domain through an Allosteric CRIB-PDZ Transition
160
Citation
50
Reference
10
Related Paper
Citation Trend
Keywords:
PDZ domain
CDC42
Small GTPase
PDZ proteins usually contain multiple protein-protein interaction domains and act as molecular scaffolds that are important for the generation and maintenance of cell polarity and cell signaling. Here, we identify and characterize TIP-1 as an atypical PDZ protein that is composed almost entirely of a single PDZ domain and functions as a negative regulator of PDZ-based scaffolding. We found that TIP-1 competes with the basolateral membrane mLin-7/CASK complex for interaction with the potassium channel Kir 2.3 in model renal epithelia. Consequently, polarized plasma membrane expression of Kir 2.3 is disrupted resulting in pronounced endosomal targeting of the channel, similar to the phenotype observed for mutant Kir 2.3 channels lacking the PDZ-binding motif. TIP-1 is ubiquitously expressed, raising the possibility that TIP-1 may play a similar role in regulating the expression of other membrane proteins containing a type I PDZ ligand.
PDZ domain
CASK
Cell polarity
Transport protein
HEK 293 cells
Guanylate kinase
Cite
Citations (47)
PDZK1 (also known as CAP70, NHERF3, or NaPi-Cap1) is a scaffolding protein composed of four PDZ (Post-Synaptic Density-95, Discs Large, Zonula Occludens-1) domains followed by a short carboxyl-terminal tail. This scaffold acts as a mediator of localization and expression levels of multiple receptors in the kidney, liver, and endothelium. Here, we characterize the self-association properties of the protein. PDZK1 can undergo modest homodimerization in vivo and in vitro through self-association involving its third PDZ domain. In addition, the tail of PDZK1 interacts in an intramolecular fashion with the first PDZ domain, but this interaction does not contribute to dimer formation. The interaction between the tail of PDZK1 and its first PDZ domain induces the protein to adopt a more compact conformation. A head-to-tail association has also been reported for EBP50/NHERF1, a two-PDZ domain member of the same scaffolding protein family as PDZK1, and shown to regulate binding of target proteins to the EBP50 PDZ domains. As opposed to EBP50, the association of PDZK1 with specific ligands for its PDZ domains is unaffected by the intramolecular association, establishing a different mode of interaction among these two members of the same scaffolding family. However, the tail of PDZK1 interacts with the PDZ domains of EBP50, and this interaction is negatively regulated by the intramolecular association of PDZK1. Thus, we have uncovered a regulated association between the two PDZ-containing scaffolding molecules, PDZK1 and EBP50.
PDZ domain
Cite
Citations (46)
PDZ domain
Cite
Citations (45)
PSD-95 is a member of the membrane-associated guanylate kinase class of proteins that forms scaffolding interactions with partner proteins, including ion and receptor channels. PSD-95 is directly implicated in modulating the electrical responses of excitable cells. The first two PSD-95/disks large/zona occludens (PDZ) domains of PSD-95 have been shown to be the key component in the formation of channel clusters. We report crystal structures of this dual domain in both apo- and ligand-bound form: thermodynamic analysis of the ligand association and small-angle x-ray scattering of the dual domain in the absence and presence of ligands. These experiments reveal that the ligated double domain forms a three-dimensional scaffold that can be described by a space group. The concentration of the components in this study is comparable with those found in compartments of excitable cells such as the postsynaptic density and juxtaparanodes of Ranvier. These in vitro experiments inform the basis of the scaffolding function of PSD-95 and provide a detailed model for scaffold formation by the PDZ domains of PSD-95.
PDZ domain
Guanylate kinase
Postsynaptic density
Cite
Citations (11)
Here we used RNA interference and examined possible redundancy amongst Rho GTPases in their mitotic role. Chromosome misalignment is induced significantly in HeLa cells by Cdc42 depletion and not by depletion of either one or all of the other four Cdc42‐like GTPases (TC10, TCL, Wrch1 or Wrch2), four Rac‐like GTPases or three Rho‐like GTPases. Notably, combined depletion of Cdc42 and all of the other four Cdc42‐like GTPases significantly enhances chromosomal misalignment. These observations suggest that Cdc42 is the primary GTPase functioning during mitosis but that the other four Cdc42‐like GTPases can also assume the mitotic role in its absence.
CDC42
Cite
Citations (35)
PDZ domain
Visual phototransduction
Transduction (biophysics)
Cell Signaling
Cite
Citations (18)
단백질-단백질 결합은 수용체 단백질, 효소, 세포 골격 단백질의 세포내 위치 결정 및 기능 조절에 중요한 역할을 한다. Postsynaptic density-95/disks large/zonula occludens-1 (PDZ) 도메인을 가진 단백질들은 시냅스 가소성, 신경세포 성장과 분화뿐만 아니라 많은 질병의 병태생리에 중요하게 관여하는 scaffold 단백질로 작용한다. Multi-PDZ domain protein 1 (MUPP1)은 13개 PDZ 도메인을 가지는 단백질로서 세포막 수용체 군집화, 신호전달 복합체 구성, 세포 골격 조정에 대한 매개 역할을 하는 것으로 알려지고 있지만 MUPP1의 세포 내 기능은 아직 명확히 밝혀지지 않았다. 본 연구에서 MUPP1의 아미노 말단 PDZ 도메인과 결합하는 새로운 단백질을 규명하기 위하여 효모 two-hybrid 방법을 이용하였고 Wdpcp (전에 Fritz로 알려짐)이 MUPP1과 결합하는 것을 확인하였다. Wdpcp는 planar cell polarity (PCP) effector로서 세포 이동과 섬모형성에 관여하는 것으로 알려져 있다. Wdpcp는 MUPP1의 첫 번째 PDZ 도메인과 결합하지만, 다른 PDZ 도메인과는 결합하지 않았다. 또한 MUPP1와 Wdpcp의 결합에서 Wdpcp의 C-말단부위가 결합에 필수적임을 효모 two-hybrid 방법으로 확인하였다. 이러한 단백질간 결합은 glutathione S-transferase (GST) pull-down assay, 공동면역침강, HEK-293T 세포에서의 발현위치를 통하여 추가적으로 확인하였다. 이러한 결과들은, MUPP1과 Wdpcp 결합은 세포내 액틴 다이내믹스(dynamics)와 세포이동 조절에 역할을 할 가능성을 시사한다. Protein-protein interactions regulate the subcellular localization and function of receptors, enzymes, and cytoskeletal proteins. Proteins containing the postsynaptic density-95/disks large/zonula occludens-1 (PDZ) domain have potential to act as scaffolding proteins and play a pivotal role in various processes, such as synaptic plasticity, neural guidance, and development, as well as in the pathophysiology of many diseases. Multi-PDZ domain protein 1 (MUPP1), which has 13 PDZ domains, has a scaffolding function in the clustering of surface receptors, organization of signaling complexes, and coordination of cytoskeletal dynamics. However, the cellular function of MUPP1 has not been fully elucidated. In the present study, a yeast two-hybrid system was used to identify proteins that interacted with the N-terminal PDZ domain of MUPP1. The results revealed an interaction between MUPP1 and Wdpcp (formerly known as Fritz). Wdpcp was identified as a planar cell polarity (PCP) effector, which is known to have a role in collective cell migration and cilia formation. Wdpcp bound to the PDZ1 domain but not to other PDZ domains of MUPP1. The C-terminal end of Wdpcp was essential for the interaction with MUPP1 in the yeast two-hybrid assay. This interaction was further confirmed in a glutathione S-transferase (GST) pull-down assay. When coexpressed in HEK-293T cells, Wdpcp was coimmunoprecipitated with MUPP1. In addition, MUPP1 colocalized with Wdpcp at the same subcellular region in cells. Collectively, these results suggest that the MUPP1-Wdpcp interaction could modulate actin cytoskeleton dynamics and polarized cell migration.
PDZ domain
Postsynaptic density
HEK 293 cells
Cell polarity
Cite
Citations (0)
Cdc42, a member of the Rho family of GTPases, has been shown to play a role in cell adhesion, cytoskeletal arrangement, phagocytosis and cell motility and migration, in addition to a host of other diverse biological processes. The function of Rho-family GTPases in disease pathogenesis has been well established and identification of small, cell permeable molecules that selectively and reversibly regulate Rho GTPases is of high scientific and potentially therapeutic interest. There has been limited success in identifying inhibitors that specifically interact with small Rho family GTPases. The identified probe, ML141 (CID-2950007), is demonstrated to be a potent, selective and reversible non-competitive inhibitor of Cdc42 GTPase suitable for in vitro assays, with low micromolar potency and selectivity against other members of the Rho family of GTPases (Rac1, Rab2, Rab7). Given the highly complementary nature of the function of the Rho family GTPases, Cdc42 selective inhibitors such as those reported here should help untangle the roles of the proteins in this family.
CDC42
PAK1
Cite
Citations (57)
Regulation of protein interaction domains is required for cellular signaling dynamics. Here, we show that the PDZ protein interaction domain from the cell polarity protein Par-6 is regulated by the Rho GTPase Cdc42. Cdc42 binds to a CRIB domain adjacent to the PDZ domain, increasing the affinity of the Par-6 PDZ for its carboxy-terminal ligand by approximately 13-fold. Par-6 PDZ regulation is required for function as mutational disruption of Cdc42-Par-6 PDZ coupling leads to inactivation of Par-6 in polarized MDCK epithelial cells. Structural analysis reveals that the free PDZ domain has several deviations from the canonical PDZ conformation that account for its low ligand affinity. Regulation results from a Cdc42-induced conformational transition in the CRIB-PDZ module that causes the PDZ to assume a canonical, high-affinity PDZ conformation. The coupled CRIB and PDZ architecture of Par-6 reveals how simple binding domains can be combined to yield complex regulation. PMID: 15023337 Funding information This work was supported by: NIGMS NIH HHS, United States Grant ID: R01 GM068032-05 NIGMS NIH HHS, United States Grant ID: R01 GM068032-01 NIGMS NIH HHS, United States Grant ID: R01 GM068032-03 NIGMS NIH HHS, United States Grant ID: R01 GM068032-02 NIGMS NIH HHS, United States Grant ID: R01 GM068032-04
PDZ domain
CDC42
Cite
Citations (0)
The Rho family GTPases are tightly regulated between the active GTP-bound state and the inactive GDP-bound state in a variety of signal transduction processes. Here the Rho family members Cdc42, Rac2, and RhoA were found to form reversible homodimers in both the GTP- and the GDP-bound states. The homophilic interaction of Cdc42 and Rac2, but not RhoA, in the GTP-bound state, caused a significant stimulation of the intrinsic GTPase activity, i.e. the activated form of Cdc42 and Rac2 acts as GTPase-activating proteins toward Cdc42-GTP or Rac2-GTP. The dimerization of the GTPases appeared to be mediated by the carboxyl-terminal polybasic domain, and the specific GTPase-activating effects of Cdc42 and Rac2 were also attributed to the structural determinant(s) in the same region of the molecules. Moreover, similar to the case of Cdc42 and Cdc42GAP interaction, Cdc42-GDP interacted with tetrafluoroaluminate and Cdc42-GTPγS (guanosine 5′-3-O-(thio)triphosphate) to form a transition state complex of the GTPase-activating reaction in which the carboxyl-terminal determinant(s) of the GTPγS-bound Cdc42 plays a critical role. These results provide a rationale for the fast rate of intrinsic GTP hydrolysis by Cdc42 and Rac and suggest that dimerization may play a role in the negative regulation of specific Rho family GTPases mediated by the carboxyl-terminal polybasic domain. The Rho family GTPases are tightly regulated between the active GTP-bound state and the inactive GDP-bound state in a variety of signal transduction processes. Here the Rho family members Cdc42, Rac2, and RhoA were found to form reversible homodimers in both the GTP- and the GDP-bound states. The homophilic interaction of Cdc42 and Rac2, but not RhoA, in the GTP-bound state, caused a significant stimulation of the intrinsic GTPase activity, i.e. the activated form of Cdc42 and Rac2 acts as GTPase-activating proteins toward Cdc42-GTP or Rac2-GTP. The dimerization of the GTPases appeared to be mediated by the carboxyl-terminal polybasic domain, and the specific GTPase-activating effects of Cdc42 and Rac2 were also attributed to the structural determinant(s) in the same region of the molecules. Moreover, similar to the case of Cdc42 and Cdc42GAP interaction, Cdc42-GDP interacted with tetrafluoroaluminate and Cdc42-GTPγS (guanosine 5′-3-O-(thio)triphosphate) to form a transition state complex of the GTPase-activating reaction in which the carboxyl-terminal determinant(s) of the GTPγS-bound Cdc42 plays a critical role. These results provide a rationale for the fast rate of intrinsic GTP hydrolysis by Cdc42 and Rac and suggest that dimerization may play a role in the negative regulation of specific Rho family GTPases mediated by the carboxyl-terminal polybasic domain. GTPase-activating protein glutathione S-transferase 2′(3′)-O-(N-methylanthraniloyl)-GDP 2-amino-6-mercapto-7-methylpurine ribonucleoside guanosine 5′-3-O-(thio)triphosphate. Members of the Rho family GTPases of the Ras superfamily including Rho, Rac, and Cdc42 are involved in intracellular signaling events that cause reorganization of actin-cytoskeleton (1Tapon N. Hall A. Curr. Opin. Cell Biol. 1997; 9: 86-94Crossref PubMed Scopus (698) Google Scholar), focal adhesion formation (2Ridley A.J. Curr. Biol. 1996; 6: 1256-1264Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar), transcription activation (3Hill C.S. Wynne J. Treisman R. Cell. 1995; 81: 1159-1170Abstract Full Text PDF PubMed Scopus (1207) Google Scholar, 4Perona R. Montaner S. Saniger L. Sanchez P.I. Bravo R. Lacal J.C. Genes Dev. 1997; 11: 463-475Crossref PubMed Scopus (537) Google Scholar), cell cycle progression (5Olson M.F. Ashworth A. Hall A. Science. 1995; 269: 1270-1272Crossref PubMed Scopus (1059) Google Scholar), endocytosis (6Lamaze C. Chuang T.H. Terlecky L.J. Bokoch G.M. Schmid S.L. Nature. 1996; 382: 177-179Crossref PubMed Scopus (331) Google Scholar, 7Schmalzing G. Richter H.P. Hansen A. Schwarz W. Just I. Aktories K. J. Cell Biol. 1995; 130: 1319-1332Crossref PubMed Scopus (96) Google Scholar), and exocytosis (8O'Sullivan A.J. Brown A.M. Freeman H.N.M. Gomperts B.D. Mol. Biol. Cell. 1996; 7: 397-408Crossref PubMed Scopus (52) Google Scholar, 9Norman J.C. Price L.S. Ridley A.J. Koffer A. Mol. Biol. Cell. 1996; 7: 1429-1442Crossref PubMed Scopus (112) Google Scholar). When activated by upstream signals such as growth factors and phospholipids, these proteins are converted to the active GTP-bound form from the inactive GDP-bound form (10Cerione R.A. Zheng Y. Curr. Opin. Cell Biol. 1996; 8: 216-222Crossref PubMed Scopus (466) Google Scholar) to interact with specific effector targets that mediate their biological functions (11Hall A. Science. 1998; 279: 509-513Crossref PubMed Scopus (5230) Google Scholar, 12Van Aelst L. D'Souza-Schorey C. Genes Dev. 1997; 11: 2295-3011Crossref PubMed Scopus (2101) Google Scholar). The signals transduced by the small GTPases are transient; the GTP-bound Rho family proteins are capable of cleaving the phosphodiester bond linking β-phosphate of GTP to its γ-phosphate via the intrinsic GTP-hydrolysis machinery of the GTPases, a process that can be further accelerated by the GTPase-activating proteins (GAPs)1 (13Lamarche N. Hall A. Trends Genet. 1994; 10: 436-440Abstract Full Text PDF PubMed Scopus (210) Google Scholar). The effective cycling of these GTPases between the GTP-bound and the GDP-bound states is critical to their biological functions; mutations in the regulators of the GTPases or in the GTPases themselves effecting their GTP-binding/GTPase activities may result in transformation (10Cerione R.A. Zheng Y. Curr. Opin. Cell Biol. 1996; 8: 216-222Crossref PubMed Scopus (466) Google Scholar, 14Lin R. Bagrodia S. Cerione R.A. Manor D. Curr. Biol. 1997; 7: 794-797Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar,15Michiels F. Habets G.M. Stam J.C. van der Kammen R.A. Collard J.G. Nature. 1995; 375: 338-340Crossref PubMed Scopus (509) Google Scholar), morphogenetic changes (16Luo L. Liao Y.J. Jan L.Y. Jan Y.N. Genes Dev. 1994; 8: 1787-1792Crossref PubMed Scopus (819) Google Scholar, 17Eaton S. Wepf R. Simons K. J. Cell Biol. 1996; 135: 1277-1289Crossref PubMed Scopus (189) Google Scholar, 18Murphy A.M. Montell D.J. J. Cell Biol. 1996; 133: 617-630Crossref PubMed Scopus (167) Google Scholar), or developmental disorder (19Luo L. Hensch T.K. Ackerman L. Barbel S. Jan L.Y. Jan Y.N. Nature. 1996; 379: 837-840Crossref PubMed Scopus (397) Google Scholar,20Pasteris N.G. Cadle A. Logie L.J. Porteous M.E.M. Schwartz C.E. Stevenson R.E. Glover T.W. Wilroy R.S. Gorski J.L. Cell. 1994; 79: 669-678Abstract Full Text PDF PubMed Scopus (270) Google Scholar). The mechanism of negative regulation of Rho family GTPases has been a subject of intensive investigation. The Rho GAPs, which share a homologous RhoGAP domain spanning ∼180 amino acids (13Lamarche N. Hall A. Trends Genet. 1994; 10: 436-440Abstract Full Text PDF PubMed Scopus (210) Google Scholar), preferentially interact with the GTP-bound form of Rho proteins, resulting in a significant stimulation of the intrinsic GTPase activity. Recent mutagenesis (21Li R. Zhang B. Zheng Y. J. Biol. Chem. 1997; 272: 32830-32835Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar) and kinetic studies (22Zhang B. Wang Z.-X. Zheng Y. J. Biol. Chem. 1997; 272: 21999-22007Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 23Zhang B. Chernoff J. Zheng Y. J. Biol. Chem. 1998; 273: 8776-8782Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 24Zhang B. Zheng Y. Biochemistry. 1998; 37: 5249-5257Crossref PubMed Scopus (56) Google Scholar), and the x-ray crystallography study of Rho and Rho GAP complex (25Rittinger K. Walker P. Eccleston J.F. Nurmahomed K. Owen D. Laue E. Gamblin S.J. Smerdon S.J. Nature. 1997; 388: 693-697Crossref PubMed Scopus (223) Google Scholar, 26Rittinger K. Walker P.A. Eccleston J.F. Smerdon S.J. Gamblin S.J. Nature. 1997; 389: 758-762Crossref PubMed Scopus (355) Google Scholar) indicate that the effect of GAPs is exerted directly at the stage of GTP cleavage by Rho proteins. The GDP-bound Rho was found to form a stable complex with aluminum tetrafluoride and GAP and thereby to stabilize a transition state of the GAP reaction (26Rittinger K. Walker P.A. Eccleston J.F. Smerdon S.J. Gamblin S.J. Nature. 1997; 389: 758-762Crossref PubMed Scopus (355) Google Scholar), further suggesting that Rho GAP contributes directly to Rho GTP-hydrolysis by supplying essential determinants to the catalytic core of the GTPase. Although Rho GAP shares no sequence homology to RasGAP or Gα of heterotrimeric G-proteins, this feature of formation of a transition state of G-protein-GAP complex is apparently conserved across the panel of Ras superfamily members (27Mittal R. Ahmadian M.R. Goody R.S. Wittinghofer A. Science. 1996; 273: 115-117Crossref PubMed Scopus (193) Google Scholar, 28Ahmadian M.R. Mittal R. Hall A. Wittinghofer A. FEBS Lett. 1997; 408: 315-318Crossref PubMed Scopus (65) Google Scholar) and is similar to the case of Gα (29Bourne H.R. Nature. 1997; 389: 673-675Crossref PubMed Scopus (68) Google Scholar, 30Sprang S.R. Science. 1997; 277: 329-330Crossref PubMed Scopus (16) Google Scholar, 31Noel J.P. Nat. Struct. Biol. 1997; 4: 677-680Crossref PubMed Scopus (17) Google Scholar), raising the possibility of a divergent structural evolution that utilizes multiple opportunities to negatively regulate the GTPase activity of respective GTP-binding proteins. In the absence of GAP stimulation, certain members of the Rho family,i.e. Cdc42 and Rac, have been known to contain significantly higher intrinsic GTPase activity than others (e.g. RhoA and TC10) and Ras (22Zhang B. Wang Z.-X. Zheng Y. J. Biol. Chem. 1997; 272: 21999-22007Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 23Zhang B. Chernoff J. Zheng Y. J. Biol. Chem. 1998; 273: 8776-8782Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 24Zhang B. Zheng Y. Biochemistry. 1998; 37: 5249-5257Crossref PubMed Scopus (56) Google Scholar). It was unclear how the fast rate of intrinsic GTP hydrolysis by Cdc42 and Rac come to arise and how this special property of the GTPases may contribute to their mechanism of regulation. In the present study, we found that the Rho family proteins Cdc42, Rac2, and RhoA were capable of forming reversible homodimers. The homodimer formation by Cdc42 and Rac2, but not RhoA, at the GTP-bound state, caused a marked activation of intrinsic GTPase activity. Furthermore, the specific GTPase-activating effects of Cdc42-GTP and Rac2-GTP because of the homophilic interaction were dependent upon the structural determinant(s) in the carboxyl-terminal polybasic domain of the GTPases that was required for the formation of a transition state complex of the GTPase-activating reaction. Our findings provide a rationale for the fast rate of intrinsic GTP-hydrolysis by Cdc42 and Rac and suggest that dimerization may play a role in the negative regulation of specific Rho family GTPases mediated by the carboxyl-terminal polybasic domain. GDP, GTP, GTPγS, and bacterial purine nucleoside phosphorylase were from Sigma. 2-Amino-6-mercapto-7-methylpurine ribonucleoside (MESG) and 2′(3′)-O-(N-methylanthraniloyl) (mant)-GDP were synthesized as described previously (32Webb M.R. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4884-4887Crossref PubMed Scopus (492) Google Scholar, 33Hiratsuka T. Biochim. Biophys. Acta. 1983; 742: 496-508Crossref PubMed Scopus (396) Google Scholar). Recombinant human Cdc42, RhoA, Rac2, and N-Ras were expressed in Escherichia coli as His6-tagged fusions using the pET expression system (22Zhang B. Wang Z.-X. Zheng Y. J. Biol. Chem. 1997; 272: 21999-22007Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 23Zhang B. Chernoff J. Zheng Y. J. Biol. Chem. 1998; 273: 8776-8782Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 24Zhang B. Zheng Y. Biochemistry. 1998; 37: 5249-5257Crossref PubMed Scopus (56) Google Scholar) and as GST fusions using the pGEX expression vector (22Zhang B. Wang Z.-X. Zheng Y. J. Biol. Chem. 1997; 272: 21999-22007Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 23Zhang B. Chernoff J. Zheng Y. J. Biol. Chem. 1998; 273: 8776-8782Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 24Zhang B. Zheng Y. Biochemistry. 1998; 37: 5249-5257Crossref PubMed Scopus (56) Google Scholar). The GAP domain of Cdc42GAP containing amino acids 205–439 was expressed as a glutathione-S-transferase (GST)-fusion using the pGEX-KG vector as described (22Zhang B. Wang Z.-X. Zheng Y. J. Biol. Chem. 1997; 272: 21999-22007Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 23Zhang B. Chernoff J. Zheng Y. J. Biol. Chem. 1998; 273: 8776-8782Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 24Zhang B. Zheng Y. Biochemistry. 1998; 37: 5249-5257Crossref PubMed Scopus (56) Google Scholar). The post-translationally modified Cdc42 was obtained in an insect expression system (34Zheng Y. Fischer D.J. Santos M.F. Tigyi G. Pasteris N.G. Gorski J.L. Xu Y. J. Biol. Chem. 1996; 271: 33169-33172Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). Thrombin cleavage after affinity purification allowed removal of the N-terminal tagged sequences (22Zhang B. Wang Z.-X. Zheng Y. J. Biol. Chem. 1997; 272: 21999-22007Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). The carboxyl-terminal truncation mutations of Cdc42 (C-7) and RhoA (Rho-8) were generated by the polymerase chain reaction-based amplification technique using Pfu polymerase (Stratagene) (35Li R. Zheng Y. J. Biol. Chem. 1997; 272: 4671-4679Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar) with primers that contained a stop codon at amino acid residues 185 (C-7) and 186 (Rho-8), respectively. The sequences of mutagenized cDNA were confirmed by automated sequencing prior to protein expression by the pET expression system. GTP-hydrolysis of the small GTPases were measured by the MESG/phosphorylase system monitoring the free γPi release as described for the cases of Cdc42, Rac1, and RhoA (22Zhang B. Wang Z.-X. Zheng Y. J. Biol. Chem. 1997; 272: 21999-22007Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 23Zhang B. Chernoff J. Zheng Y. J. Biol. Chem. 1998; 273: 8776-8782Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 24Zhang B. Zheng Y. Biochemistry. 1998; 37: 5249-5257Crossref PubMed Scopus (56) Google Scholar). Single turnover GTPase reactions were initiated by the addition of MgCl2 to a final concentration of 5 mM. Data were fitted into a single exponential to derive the apparent rate constant K app. For measurements of GTPase-activating reactions, 5–50 μl of stock solution containing the indicated amount of GAP (Cdc42GAP) or the GTPγS-bound G-proteins were added together with MgCl2 to the reaction mixtures. A control experiment in which the substrate GTPases were omitted was carried out in each independent measurement to provide a background. The concentration of Pi in the reaction solution was calculated by a factor of extinction coefficient ε360 nm = 11,000 m−1cm−1 at pH 7.6 from the absorbance change resulting from the phosphorylase coupling reactions (32Webb M.R. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4884-4887Crossref PubMed Scopus (492) Google Scholar) and was used to determine the effective concentrations of the small G-proteins after one round of single turnover reaction. A Superdex 200HR 10/30 gel filtration column (Amersham Pharmacia Biotech) coupled to a Bio-Rad biologic liquid chromatography system was used to analyze the homophilic interactions of the small GTPases. Purified samples (200 μl) of the respective GTPases in the GDP or GTPγS-bound form at the indicated concentrations were injected to the column, and an elution buffer containing 50 mm HEPES, pH 7.6, 5 mmMgCl2, 150 mm NaCl, and 1 mmdithiothreitol was applied at a flow rate of 0.5 ml/min. The elution profiles were monitored by UV absorption. Complex formation between the immobilized GST-fusion G-proteins and the endogenous G-proteins of NIH 3T3 cell lysates or purified recombinant G-proteins was carried out following previously established protocols (34Zheng Y. Fischer D.J. Santos M.F. Tigyi G. Pasteris N.G. Gorski J.L. Xu Y. J. Biol. Chem. 1996; 271: 33169-33172Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 36Zheng Y. Bagrodia S. Cerione R.A. J. Biol. Chem. 1994; 269: 18727-18730Abstract Full Text PDF PubMed Google Scholar). The GDP or GTPγS preloaded cell lysates containing the endogenous G-proteins or the His6-tagged recombinant G-proteins were mixed with the glutathione-agarose beads bound to various GST-G-proteins at either the GDP or GTPγS-bound state in the presence or absence of the indicated amount of AlCl3 and NaF for 20 min at 4 °C, and the coprecipitates of the agarose beads were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis or Coomassie Blue staining. The fluorescence emission spectra of mantGDP-bound small GTPases were monitored with the excitation wavelength at 360 nm using an SLM-Aminco Series 2 luminescence spectrometer similar to that previously described (27Mittal R. Ahmadian M.R. Goody R.S. Wittinghofer A. Science. 1996; 273: 115-117Crossref PubMed Scopus (193) Google Scholar). 150 μm AlCl3 and 15 mm NaF and/or various concentrations of Cdc42-GTPγS (or C-7-GTPγS) were titrated into a 0.6-ml sample containing the mantGDP-bound GTPase in 50 mm HEPES, pH 7.6, 150 mm NaCl, and 5 mm MgCl2. It has been known that the Ras proteins have slow intrinsic GTPase reaction rates (37Bourne H.R. Sanders D.A. McCormick F. Nature. 1991; 349: 117-127Crossref PubMed Scopus (2698) Google Scholar). The apparent rate was determined to be 0.022 min−1 for N-Ras by an assay measuring the time course of γPi release from Ras-GTP, and it did not seem to be affected by increasing concentrations of Ras (Fig. 1 A). RhoA behaved similarly to Ras in this aspect, whereas the apparent rates of GTP-hydrolysis of Cdc42 and Rac2 were found to increase significantly with increasing concentrations of the G-proteins bound to GTP (Fig. 1 A). Titration of Cdc42-GTPγS to Cdc42-GTP under single turnover conditions resulted in a significant increase of the initial rate of the GTPase reaction, similar to the effect of the addition of Cdc42GAP, whereas addition of Cdc42-GDP had no detectable effect (Fig. 1,B and C), indicating that the activated form of Cdc42 presents a GAP activity toward Cdc42. This apparent GTPase-activating effect by Cdc42-GTPγS was not affected by isoprenylation modification since Cdc42 isolated from an insect cell expression system (*Cdc42) had a similar behavior (Fig. 1 C). Similar observations were also made for Rac2 (Fig. 1 C). In contrast, the GTPγS-bound RhoA lacked the ability to stimulate the GTPase activity of RhoA-GTP (Fig. 1 C). Therefore, it appears that the Rho family proteins Cdc42 and Rac2, but not RhoA, possess self-stimulatory GAP activity when in the activated state. The self-stimulatory multiple turnover GTPase reactions (Fig. 1 A) may provide a rationale for the previously observed fast rate of intrinsic GTP-hydrolysis by recombinant Cdc42 and Rac (22Zhang B. Wang Z.-X. Zheng Y. J. Biol. Chem. 1997; 272: 21999-22007Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 23Zhang B. Chernoff J. Zheng Y. J. Biol. Chem. 1998; 273: 8776-8782Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 24Zhang B. Zheng Y. Biochemistry. 1998; 37: 5249-5257Crossref PubMed Scopus (56) Google Scholar). To examine the apparent homophilic interaction between molecules of the small GTPases, recombinant Cdc42 and Rac2, as well as the controls RhoA and N-Ras, were subjected to gel filtration chromatography analysis. Aside from the ∼25-kDa elution peak expected for the monomeric form of the proteins, an absorption peak at ∼50 kDa corresponding to the dimer position was observed in the chromatography profiles of all three Rho family GTPases, Cdc42, Rac2, and RhoA (Figs. 2 A). The homodimer formation was slightly affected by replacement of bound GDP with GTPγS, which resulted in a minor shift of the overall population of the GTPases from dimers to monomers (Figs. 2 A). A concentration-dependent breakdown of preformed dimers of Cdc42-GTPγ35S was detected from the traces of the radioactive GTPγ35S-bound Cdc42 eluents (Fig. 2 A), indicating that the dimer formation is a reversible process. The estimated dissociation constant is at ∼2 μm for (Cdc42-GTPγS)2 based upon the concentration-dependent absorption peak distributions. The property of dimer formation appeared to be specific to the Rho family members, because N-Ras at either nucleotide binding state remained exclusively monomeric (Fig. 2 A). These results indicate that the Rho family GTPases Cdc42, Rac2, and RhoA can form reversible homodimers at the GTP-bound state with micromolar binding affinity. To assess whether the homophilic interaction may apply to native Rho GTPases of mammalian cells, a complex-formation assay was performed using the immobilized GST-Cdc42, GST-RhoA, or GST-Ras as bait and NIH 3T3 cell lysates as the source for endogenous small GTPases. Western blot analysis of the coprecipitates of the GST fusions revealed that GST-Cdc42-GDP and GST-Cdc42-GTPγS were capable of binding to the endogenous Cdc42-GDP and Cdc42-GTPγS, respectively, but with much reduced affinity to Cdc42 of the different nucleotide binding states (Fig. 2 B). Neither GST-RhoA-GDP, GST-RhoA-GTPγS, GST-Ras-GDP, nor GST-Ras-GTPγS interacted with Cdc42 at a detectable level (Fig. 2 B) even though both the GST-RhoA and GST-Ras were able to bind to guanine nucleotides and to interact with their respective regulatory proteins (data not shown). Thus, endogenous Cdc42 of the mammalian cells is capable of forming a homophilic complex. The carboxyl-terminal polybasic domain of the Rho family proteins, spanning the last eight amino acid residues before the CAAXisoprenylation sequences (Fig. 3 A), was suspected to have a role in homodimer formation because of its positively charged exposure on the surface of the molecules (38Wei Y. Zhang Y. Derewenda U. Liu X. Minor W. Nakamoto R.K. Somlyo A.V. Somlyo A.P. Derewenda Z.S. Nat. Struct. Biol. 1997; 9: 699-702Crossref Scopus (156) Google Scholar, 39Feltham J.L. Dotsch V. Raza S. Manor D. Cerione R.A. Sutcliffe M.J. Wagner G. Oswald R.E. Biochemistry. 1997; 36: 8755-8766Crossref PubMed Scopus (79) Google Scholar). Mutants of Cdc42 with deletion of the last seven residues of the C terminus (C-7) and of RhoA with the carboxyl-terminal eight residues truncated (Rho-8) were found to be present only in monomeric form (Fig. 3 B). These deletion mutations did not seem to affect the tertiary folding of the proteins and maintained the full capability to interact with Rho GAP (25Rittinger K. Walker P. Eccleston J.F. Nurmahomed K. Owen D. Laue E. Gamblin S.J. Smerdon S.J. Nature. 1997; 388: 693-697Crossref PubMed Scopus (223) Google Scholar, 26Rittinger K. Walker P.A. Eccleston J.F. Smerdon S.J. Gamblin S.J. Nature. 1997; 389: 758-762Crossref PubMed Scopus (355) Google Scholar). C-7 lost the dose dependence of the intrinsic GTPase activity of the wild-type protein (Fig. 3 C), which was consistent with its inability to catalyze the GTPase reaction of itself or wild-type Cdc42 when bound to GTPγS (Fig. 3 D). However, C-7 remained a good substrate for the GAP reaction catalyzed by wild-type Cdc42 bound to GTPγS (Fig. 3 D), suggesting that determinant(s) in the polybasic domain of Cdc42 contributes to the catalytic GAP reaction. We conclude that the carboxyl-terminal polybasic domain of the Rho family proteins mediate their homodimer formation and that structural determinants within the polybasic domain play a critical role in the self-stimulatory GAP reactions of Cdc42 and Rac2. Interactions between Rho proteins and Rho GAP and between Ras and RasGAP have been found to undergo a transition state of the respective GAP reactions, which can be mimicked by the ternary complex of the respective G-proteins at the GDP-bound state, AlF4−, and the respective GAPs (27Mittal R. Ahmadian M.R. Goody R.S. Wittinghofer A. Science. 1996; 273: 115-117Crossref PubMed Scopus (193) Google Scholar, 28Ahmadian M.R. Mittal R. Hall A. Wittinghofer A. FEBS Lett. 1997; 408: 315-318Crossref PubMed Scopus (65) Google Scholar, 40Hoffman G.R. Nassar N. Oswald R.E. Cerione R.A. J. Biol. Chem. 1998; 273: 4392-4399Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). To determine whether the self-stimulatory GAP reaction of the Rho family homodimers, i.e. (Cdc42-GTP)2 and (Rac2-GTP)2, may be through a mechanism similar to that of GAP activations of Ras and Cdc42, a fluorescence assay originally designed to detect the formation of the ternary transition state complex of Ras-GDP, AlF4−, and GAP for the Ras-RasGAP reaction (27Mittal R. Ahmadian M.R. Goody R.S. Wittinghofer A. Science. 1996; 273: 115-117Crossref PubMed Scopus (193) Google Scholar) was employed to examine the case of Cdc42 homodimers. The addition of AlF4− or Cdc42-GTPγS alone did not change the emission spectrum of Cdc42 bound to the fluorescent GDP-analog mantGDP, whereas the spectrum was changed both in maximum absorption wavelength (from 448 to 430 nm) and in intensity (a 100% increase at 448 nm) when AlF4− and Cdc42-GTPγS were added together to Cdc42-mantGDP (Fig. 4 A). This change in Cdc42-mantGDP fluorescence indicated a conformational change around the GTPase active site, which attributes to the formation of an analog of the GTP-bound transition state of the GTPase reaction involving Cdc42-GDP, AlF4−, and Cdc42-GTPγS, as were the cases for Ras-GDP, AlF4−, and RasGAP (27Mittal R. Ahmadian M.R. Goody R.S. Wittinghofer A. Science. 1996; 273: 115-117Crossref PubMed Scopus (193) Google Scholar) and for Cdc42-GDP, AlF4−, and Cdc42GAP (40Hoffman G.R. Nassar N. Oswald R.E. Cerione R.A. J. Biol. Chem. 1998; 273: 4392-4399Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Moreover, stoichiometric amounts of Cdc42-GTPγS to Cdc42-mantGDP were needed for this effect (Fig. 4 B). The effective dissociation constant (K d) of the transition state complex Cdc42-mantGDP-AlF4−-Cdc42-GTPγS was calculated at 5.8 μm after the background signals were subtracted. When the C-7 mutant of Cdc42 was examined under similar conditions, it became clear that it had lost the ability to form the transition state complex with Cdc42-mantGDP and AlF4− when bound to GTPγS (Fig. 4 B). RhoA, which is capable of forming homodimers through the carboxyl-terminal polybasic domain (Fig. 2 A) but lacks the self-stimulatory GAP activity (Fig. 1 C), was found unable to form the transition state complex (data not shown). To further visualize the AlF4−-induced ternary complex formation between the inactive and active forms of Cdc42, a pull-down assay using the agarose-immobilized GST-Cdc42 and recombinant His6-tagged Cdc42 was carried out. As shown in Fig. 4 C, whereas both GST-Cdc42-GDP and GST-Cdc42-GTPγS were able to pull down a significant amount of His6-Cdc42 of the same nucleotide binding states, presumably because of the formation of dimer complex, GST-Cdc42-GDP were only able to marginally complex with His6-Cdc42-GTPγS. However, the ability of complex formation between Cdc42-GDP and Cdc42-GTPγS was significantly enhanced when AlF4− was included in the incubation mixture, indicating the formation of Cdc42-GDP-AlF4−-Cdc42-GTPγS complex. These results provide evidence that the homodimer formation of specific Rho family GTPase, i.e. Cdc42 and Rac2, in the GTP-bound form, undergoes a similar transition state like that of the Ras or Rho proteins interacting with GAPs. From both the biochemical and structural analysis of G-protein-GAP interactions, it has become evident that evolutionarily divergent structures, e.g. RasGAP, RhoGAP, and a built-in domain of Gαi, may utilize a similar mechanism in the course of down-regulation of respective G-proteins (29Bourne H.R. Nature. 1997; 389: 673-675Crossref PubMed Scopus (68) Google Scholar, 30Sprang S.R. Science. 1997; 277: 329-330Crossref PubMed Scopus (16) Google Scholar, 31Noel J.P. Nat. Struct. Biol. 1997; 4: 677-680Crossref PubMed Scopus (17) Google Scholar). One common feature of the GTPase-activating reactions is the progression from the ground state (GTP-bound state) to the transition state (GDP-AlF4−-bound state), which appears to be critical for the activation of GTPase activity. Another is the direct participation of critical amino acids from the respective GAP molecules in the GTP hydrolysis reaction to stabilize the transition state conformation of the GTPases. The findings reported in this study, that the Rho family members Cdc42 and Rac2 in the GTP-bound form forms a similar transition state complex in the homodimers and that one molecule of the dimer supplies specific structural determinant(s) to the GTPase core of the other molecule and thereby stabilizes the transition state conformation of the GTPase-activating reaction, may provide yet another example of evolutional convergence of the mechanism of negative regulations of GTPases. The carboxyl-terminal region of Rho family proteins represents the most divergent sequences in the family. Most members contain a stretch of two to six poly-lysine and/or arginine residues immediately before the isoprenylation CAAX sequence. Not unlike the similar structure of K-Ras, this polybasic domain of S. cerevisiaeCdc42 has been suggested to have a role in membrane attachment (41Davis C.R. Richman T.J. Deliduka S.B. Blaisdell J.O. Collins C.C. Johnson D.I. J. Biol. Chem. 1998; 273: 849-858Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar) and, in the case of human Cdc42, may be involved in interaction with acidic phospholipids such as phosphatidylinositol 4,5-bisphosphate (42Zheng Y. Glaven J.A. Wu W.J. Cerione R.A. J. Biol. Chem. 1996; 271: 23815-23819Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). Our observation of the dimer-formation and the GTPase-activating capability of wild-type and C-terminal mutant (C-7) of Cdc42 further suggest that the C-terminal region of certain Rho family proteins is essential for dimerization and self-stimulatory GAP activity and may be involved in a non-symmetric homodimer configuration: one molecule of Cdc42 contributes the C-terminal domain containing the binding and catalytic determinant(s) (such as a critical arginine residue) to function as a GAP, whereas the other molecule may act as a substrate to interact with this "GAP" through a distinct region of the molecule such as the GTP-binding core. Based upon studies of the carboxyl-terminal hypervariable region of Ras, it was expected that the C-terminal domain might not contain definable tertiary structure (37Bourne H.R. Sanders D.A. McCormick F. Nature. 1991; 349: 117-127Crossref PubMed Scopus (2698) Google Scholar). Our findings that the carboxyl-terminal polybasic domain can act only as a GAP when bound to GTP in the homophilic interaction of Cdc42 and Rac2 and that synthetic polypeptides corresponding to the domain sequences are capable of competing with the homodimer formation yet contain no GAP activity 2B. Zhang and Y. Zheng, unpublished observations. indicate that the conformation of this region of Rho proteins is sensitive to the nucleotide binding state of the GTPases. Moreover, all Rho family proteins examined containing the polybasic sequences at the carboxyl termini, including the additional mammalian members Rac1, RhoC, and RhoG, have been found capable of forming homodimers or higher oligomers, whereas others lacking polybasic residues in the region,e.g. TC10 and RhoB, are in monomeric form,2suggesting that the polybasic nature of the domain is critical in mediating homophilic interactions of specific Rho family GTPases. The fact that RhoA is found to form homodimers but yet is unable to stimulate the GTPase activity in the homophilic interaction further implies that additional unique structural determinant(s) is required to elicit the GTPase-activating effect. Whether the observed homodimer formation and self-stimulatory GTPase-activating activity of Cdc42 and Rac2 bear physiological significance remains unclear. Given that these Rho family proteins are expected to assemble directly or indirectly to large molecular complexes in cells (1Tapon N. Hall A. Curr. Opin. Cell Biol. 1997; 9: 86-94Crossref PubMed Scopus (698) Google Scholar, 2Ridley A.J. Curr. Biol. 1996; 6: 1256-1264Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar, 11Hall A. Science. 1998; 279: 509-513Crossref PubMed Scopus (5230) Google Scholar) and the potential dual roles as both activator and down-regulator of their own signals of the GTPases at the activated state, it will be a particularly challenging issue to address. Although the K cat values of the self-stimulatory GAP reaction of the GTP-bound homodimers may be an order of magnitude lower than that catalyzed by Rho GAP,2the observed K d of the Cdc42-GTP homodimers and the effective K d of the transition state complex are comparable with that of Cdc42-RhoGAP interaction (∼2 μm) and Ras-RasGAP interaction (∼1.3 μm), respectively (22Zhang B. Wang Z.-X. Zheng Y. J. Biol. Chem. 1997; 272: 21999-22007Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 27Mittal R. Ahmadian M.R. Goody R.S. Wittinghofer A. Science. 1996; 273: 115-117Crossref PubMed Scopus (193) Google Scholar). The relatively high abundance and ubiquitous distribution of Rho family proteins also make it an attractive possibility that the homophilic interaction may have a role in the regulation of these small GTPases. Future work to examine the structural determinant(s) of the carboxyl-terminal polybasic domain of Cdc42 and Rac2 involved in the dimer formation and catalysis and the site(s) of the GTPase hydrolysis core of the substrate required for the interaction should provide additional insight for the mechanism of dimer formation.
CDC42
Rac GTP-Binding Proteins
Small GTPase
Cite
Citations (83)