Dimeric Plant RhoGAPs Are Regulated by Its CRIB Effector Motif to Stimulate a Sequential GTP Hydrolysis

Schaefer, Antje; Miertzschke, Mandy; Berken, Antje; Wittinghofer, Alfred

doi:10.1016/j.jmb.2011.06.033

Cited by 18 publications

(7 citation statements)

References 51 publications

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“…The mean FRET efficiency between CFP-ROPGAP3 and YFP-ROP11 (18%) or YFP-ROP11 G17V (39%) was significantly higher than that between CFP-ROPGAP3 and YFP-ROP11 T22N (−0.2%) (Figure S3B ). These data suggested that ROPGAP3 specifically interacted with GTP-ROP11 in vivo , a result consistent with previous reports that ROPGAPs interact through their Cdc42/Rac interactive binding motif (CRIB, an effector-binding domain) and GAP domain with the active form of ROPs to inactivate them 25 , 26 . We therefore concluded that ROPGAP3 was not part of the feedback reaction and acted simply as a GAP in vivo .…”

Section: Resultssupporting

confidence: 91%

A Rho-based reaction-diffusion system governs cell wall patterning in metaxylem vessels

Nagashima

Tsugawa

Mochizuki

et al. 2018

Sci Rep

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Rho GTPases play crucial roles in cell polarity and pattern formation. ROPs, Rho of plant GTPases, are widely involved in cell wall patterning in plants, yet the molecular mechanism underlying their action remains unknown. Arabidopsis ROP11 is locally activated to form plasma membrane domains, which direct formation of cell wall pits in metaxylem vessel cells through interaction with cortical microtubules. Here, we show that the pattern formation of cell wall pits is governed by ROP activation via a reaction-diffusion mechanism. Genetic analysis and reconstructive assays revealed that ROPGEF4/7 and ROPGAP3/4, which encode ROP activators and inactivators, respectively, regulated the formation of ROP-activated domains; these in turn determined the pattern of cell wall pits. Mathematical modelling showed that ROP-activation cycle generated ROP domains by reaction-diffusion mechanism. The model predicted that a positive feedback and slow diffusion of ROP11-ROPGEF4 complex were required to generate ROP-activated domains. ROPGEF4 formed a dimer that interacted with activated ROP11 in vivo, which could provide positive feedback for ROP activation. ROPGEF4 was highly stable on the plasma membrane and inhibited ROP11 diffusion. Our study indicated that ROP-based reaction-diffusion system self-organizes ROP-activated domains, thereby determines the pit pattern of metaxylem vessels.

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Section: Resultssupporting

confidence: 91%

A Rho-based reaction-diffusion system governs cell wall patterning in metaxylem vessels

Nagashima

Tsugawa

Mochizuki

et al. 2018

Sci Rep

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“…The invariant Arg797 of the HMHA1 RhoGAP domain which may represent the catalytic Arg residue (Arg finger) is orientated into the active site of RhoA, close to AlFx and the nucleotide phosphates (Figure 8B). The catalytic Arg residue neutralizes the developing charge during the GTP hydrolysis and thus stabilizes the transition state [31]–[33]. The highly conserved Gln residue in the switch II region of Rho GTPases (Gln63 in RhoA, Gln61 in Rac1/Cdc42) is required for an efficient GAP-catalyzed GTP hydrolysis as well, since it coordinates the attacking water for the GTP cleavage [31]–[33].…”

Section: Resultsmentioning

confidence: 99%

The Human Minor Histocompatibility Antigen1 Is a RhoGAP

et al. 2013

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The human minor Histocompatibility Antigen HMHA-1 is a major target of immune responses after allogeneic stem cell transplantation applied for the treatment of leukemia and solid tumors. The restriction of its expression to hematopoietic cells and many solid tumors raised questions regarding its cellular functions. Sequence analysis of the HMHA-1 encoding HMHA1 protein revealed the presence of a possible C-terminal RhoGTPase Activating Protein (GAP) domain and an N-terminal BAR domain. Rho-family GTPases, including Rac1, Cdc42, and RhoA are key regulators of the actin cytoskeleton and control cell spreading and migration. RhoGTPase activity is under tight control as aberrant signaling can lead to pathology, including inflammation and cancer. Whereas Guanine nucleotide Exchange Factors (GEFs) mediate the exchange of GDP for GTP resulting in RhoGTPase activation, GAPs catalyze the low intrinsic GTPase activity of active RhoGTPases, resulting in inactivation. Here we identify the HMHA1 protein as a novel RhoGAP. We show that HMHA1 constructs, lacking the N-terminal region, negatively regulate the actin cytoskeleton as well as cell spreading. Furthermore, we show that HMHA1 regulates RhoGTPase activity in vitro and in vivo. Finally, we demonstrate that the HMHA1 N-terminal BAR domain is auto-inhibitory as HMHA1 mutants lacking this region, but not full-length HMHA1, showed GAP activity towards RhoGTPases. In conclusion, this study shows that HMHA1 acts as a RhoGAP to regulate GTPase activity, cytoskeletal remodeling and cell spreading, which are crucial functions in normal hematopoietic and cancer cells.

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“…FEBRUARY 2020 CANCER DISCOVERY | 303 column (Agilent 1100). GDP and GTP were separated on a C18 column (Agilent) with 100 mmol/L potassium-phosphate pH 6.5, 10 mmol/L tert-butyl-ammonium-bromide, and 7.5% acetonitrile as mobile phase (65). The concentration of nonhydrolyzed GTP was plotted against time.…”

Section: Research Articlementioning

confidence: 99%

Gain-of-Function RHOA Mutations Promote Focal Adhesion Kinase Activation and Dependency in Diffuse Gastric Cancer

et al. 2020

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Diffuse gastric cancer (DGC) is a lethal malignancy lacking effective systemic therapy. Among the most provocative recent results in DGC has been that of highly recurrent missense mutations in the GTPase RHOA. The function of these mutations has remained unresolved. We demonstrate that RHOA Y42C , the most common RHOA mutation in DGC, is a gain-offunction oncogenic mutant, and that expression of RHOA Y42C with inactivation of the canonical tumor suppressor Cdh1 induces metastatic DGC in a mouse model. Biochemically, RHOA Y42C exhibits impaired GTP hydrolysis and enhances interaction with its effector ROCK. RHOA Y42C mutation and Cdh1 loss induce actin/cytoskeletal rearrangements and activity of focal adhesion kinase (FAK), which activates YAP-TAZ, PI3K-AKT, and β-catenin. RHOA Y42C murine models were sensitive to FAK inhibition and to combined YAP and PI3K pathway blockade. These results, coupled with sensitivity to FAK inhibition in patient-derived DGC cell lines, nominate FAK as a novel target for these cancers. SIGNIFICANCE:The functional signifi cance of recurrent RHOA mutations in DGC has remained unresolved. Through biochemical studies and mouse modeling of the hotspot RHOA Y42C mutation, we establish that these mutations are activating, detail their effects upon cell signaling, and defi ne how RHOA-mediated FAK activation imparts sensitivity to pharmacologic FAK inhibitors.

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Dimeric Plant RhoGAPs Are Regulated by Its CRIB Effector Motif to Stimulate a Sequential GTP Hydrolysis

Cited by 18 publications

References 51 publications

A Rho-based reaction-diffusion system governs cell wall patterning in metaxylem vessels

A Rho-based reaction-diffusion system governs cell wall patterning in metaxylem vessels

The Human Minor Histocompatibility Antigen1 Is a RhoGAP

Gain-of-Function RHOA Mutations Promote Focal Adhesion Kinase Activation and Dependency in Diffuse Gastric Cancer

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