Guanosine Triphosphatase-activating Protein Research Articles

CYK4 inhibits Rac1-dependent PAK1 and ARHGEF7 effector pathways during cytokinesis

In mitosis, animal cells lose their adhesion to the surrounding surfaces and become rounded. During mitotic exit, they reestablish these adhesions and at the same time physically contract and divide. How these competing processes are spatially segregated at the cell cortex remains mysterious. To address this question, we define the specific effector pathways used by RhoA and Rac1 in mitotic cells. We demonstrate that the MKlp1-CYK4 centralspindlin complex is a guanosine triphosphatase-activating protein (GAP) for Rac1 and not RhoA and that CYK4 negatively regulated Rac1 activity at the cell equator in anaphase. Cells expressing a CYK4 GAP mutant had defects in cytokinesis and showed elevated staining for the cell adhesion marker vinculin. These defects could be rescued by depletion of ARHGEF7 and p21-activated kinase, Rac1-specific effector proteins required for cell adhesion. Based on these findings, we propose that CYK4 GAP activity is required during anaphase to inhibit Rac1-dependent effector pathways associated with control of cell spreading and adhesion.

Matrix rigidity controls endothelial differentiation and morphogenesis of cardiac precursors.

Tissue development and regeneration involve tightly coordinated and integrated processes: selective proliferation of resident stem and precursor cells, differentiation into target somatic cell type, and spatial morphological organization. The role of the mechanical environment in the coordination of these processes is poorly understood. We show that multipotent cells derived from native cardiac tissue continually monitored cell substratum rigidity and showed enhanced proliferation, endothelial differentiation, and morphogenesis when the cell substratum rigidity closely matched that of myocardium. Mechanoregulation of these diverse processes required p190RhoGAP, a guanosine triphosphatase-activating protein for RhoA, acting through RhoA-dependent and -independent mechanisms. Natural or induced decreases in the abundance of p190RhoGAP triggered a series of developmental events by coupling cell-cell and cell-substratum interactions to genetic circuits controlling differentiation.

TBC1D14 regulates autophagosome formation via Rab11- and ULK1-positive recycling endosomes

Autophagy is a bulk degradation process characterized by the formation of double membrane vesicles called autophagosomes. The exact molecular mechanism of autophagosome formation and the origin of the autophagosomal membrane remain unclear. We screened 38 human Tre-2/Bub2/Cdc16 domain-containing Rab guanosine triphosphatase-activating proteins (GAPs) and identified 11 negative regulators of starvation-induced autophagy. One of these putative RabGAPs, TBC1D14, colocalizes and interacts with the autophagy kinase ULK1. Overexpressed TBC1D14 tubulates ULK1-positive recycling endosomes (REs), impairing their function and inhibiting autophagosome formation. TBC1D14 binds activated Rab11 but is not a GAP for Rab11, and loss of Rab11 prevents TBC1D14-induced tubulation of REs. Furthermore, Rab11 is required for autophagosome formation. ULK1 and Atg9 are found on Rab11- and transferrin (Tfn) receptor (TfnR)-positive recycling endosomes. Amino acid starvation causes TBC1D14 to relocalize from REs to the Golgi complex, whereas TfnR and Tfn localize to forming autophagosomes, which are ULK1 and LC3 positive. Thus, TBC1D14- and Rab11-dependent vesicular transport from REs contributes to and regulates starvation-induced autophagy.

Ras signaling pathways mediate NGF-induced enhancement of excitability of small-diameter capsaicin-sensitive sensory neurons from wildtype but not Nf1+/− mice

Nerve growth factor (NGF) activates multiple downstream effectors, including Ras, phosphoinositide-3 kinase, and sphingomyelins. However, pathway mediating the NGF-induced augmentation of sensory neuronal excitability remains largely unknown. We previously reported that small-diameter sensory neurons with a heterozygous mutation of the Nf1 gene ( Nf1+/−) exhibited increased excitability. The protein product of the Nf1 gene is neurofibromin, a guanosine triphosphatase-activating protein (GAP) for p21ras (Ras) that accelerates the conversion of active Ras-GTP to inactive Ras-GDP. Thus, Nf1+/− cells have augmented basal and stimulated Ras activity. To investigate whether NGF-induced increases in excitability of small-diameter sensory neurons are dependent on Ras signaling, an antibody that blocks the activation of Ras, Y13-259, was perfused into the cell. Under these conditions, the enhanced excitability produced by NGF was suppressed in wildtype neurons but the excitability of Nf1+/− neurons was unaltered. In addition, expression of a dominant-negative form of Ras abolished the ability of NGF to increase the excitability of small-diameter sensory neurons. These results demonstrate that NGF enhances excitability of small-diameter sensory neurons in a Ras-dependent manner while the consequences of decreased expression of neurofibromin cannot be restored by blocking Ras signaling; suggesting that Ras-initiated signaling pathways can regulate both transcriptional and posttranslational control of ion channels important in neuronal excitability.

OATL1, a novel autophagosome-resident Rab33B-GAP, regulates autophagosomal maturation

Macroautophagy is a bulk degradation system conserved in all eukaryotic cells. A ubiquitin-like protein, Atg8, and its homologues are essential for autophagosome formation and act as a landmark for selective autophagy of aggregated proteins and damaged organelles. In this study, we report evidence demonstrating that OATL1, a putative Rab guanosine triphosphatase-activating protein (GAP), is a novel binding partner of Atg8 homologues in mammalian cells. OATL1 is recruited to isolation membranes and autophagosomes through direct interaction with Atg8 homologues and is involved in the fusion between autophagosomes and lysosomes through its GAP activity. We further provide evidence that Rab33B, an Atg16L1-binding protein, is a target substrate of OATL1 and is involved in the fusion between autophagosomes and lysosomes, the same as OATL1. Because both its GAP activity and its Atg8 homologue-binding activity are required for OATL1 to function, we propose a model that OATL1 uses Atg8 homologues as a scaffold to exert its GAP activity and to regulate autophagosomal maturation.

The F-BAR domain of SRGP-1 facilitates cell-cell adhesion during C. elegans morphogenesis.

Robust cell-cell adhesion is critical for tissue integrity and morphogenesis, yet little is known about the molecular mechanisms controlling cell-cell junction architecture and strength. We discovered that SRGP-1 is a novel component of cell-cell junctions in Caenorhabditis elegans, localizing via its F-BAR (Bin1, Amphiphysin, and RVS167) domain and a flanking 200-amino acid sequence. SRGP-1 activity promotes an increase in membrane dynamics at nascent cell-cell contacts and the rapid formation of new junctions; in addition, srgp-1 loss of function is lethal in embryos with compromised cadherin-catenin complexes. Conversely, excess SRGP-1 activity leads to outward bending and projections of junctions. The C-terminal half of SRGP-1 interacts with the N-terminal F-BAR domain and negatively regulates its activity. Significantly, in vivo structure-function analysis establishes a role for the F-BAR domain in promoting rapid and robust cell adhesion during embryonic closure events, independent of the Rho guanosine triphosphatase-activating protein domain. These studies establish a new role for this conserved protein family in modulating cell-cell adhesion.

The Cdc42-selective GAP Rich regulates postsynaptic development and retrograde BMP transsynaptic signaling

Retrograde bone morphogenetic protein signaling mediated by the Glass bottom boat (Gbb) ligand modulates structural and functional synaptogenesis at the Drosophila melanogaster neuromuscular junction. However, the molecular mechanisms regulating postsynaptic Gbb release are poorly understood. In this study, we show that Drosophila Rich (dRich), a conserved Cdc42-selective guanosine triphosphatase-activating protein (GAP), inhibits the Cdc42-Wsp pathway to stimulate postsynaptic Gbb release. Loss of dRich causes synaptic undergrowth and strongly impairs neurotransmitter release. These presynaptic defects are rescued by targeted postsynaptic expression of wild-type dRich but not a GAP-deficient mutant. dRich inhibits the postsynaptic localization of the Cdc42 effector Wsp (Drosophila orthologue of mammalian Wiskott-Aldrich syndrome protein, WASp), and manifestation of synaptogenesis defects in drich mutants requires Wsp signaling. In addition, dRich regulates postsynaptic organization independently of Cdc42. Importantly, dRich increases Gbb release and elevates presynaptic phosphorylated Mad levels. We propose that dRich coordinates the Gbb-dependent modulation of synaptic growth and function with postsynaptic development.

PI3K Signaling Through the Dual GTPase–Activating Protein ARAP3 Is Essential for Developmental Angiogenesis

One function of phosphoinositide 3-kinase α (PI3Kα), which generates the lipid second messenger phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P(3)], is its regulation of angiogenesis in the developing embryo and in pathological situations. ARAP3 is a PtdIns(3,4,5)P(3)- and Rap-activated guanosine triphosphatase (GTPase)-activating protein (GAP) for the small GTPases RhoA and Arf6. Here, we show that deleting Arap3 in the mouse caused embryonic death in mid-gestation due to an endothelial cell-autonomous defect in sprouting angiogenesis. Explants taken at a developmental stage at which no defect was yet present reproduced this phenotype ex vivo, demonstrating that the defect was not secondary to hypoxia, placental defects, or organ failure. In addition, knock-in mice expressing an ARAP3 point mutant that cannot be activated by PtdIns(3,4,5)P(3) had angiogenesis defects similar to those of Arap3(-/-) embryos. Our work delineates a previously unknown signaling pathway that controls angiogenesis immediately downstream of PI3Kα through ARAP3 to the Rho and Arf family of small GTPases.

Science Signaling Podcast: 26 October 2010

Disrupting activity of the guanosine triphosphatase-activating protein ARAP3 is a potential strategy for anti-angiogenic therapy.

Decreased expression of spine-associated Rap guanosine triphosphatase-activating protein (SPAR) in glutamate-treated primary hippocampal neurons

Spine-associated Rap guanosine triphosphatase-activating protein (SPAR) is an important regulator of activity-dependent remodeling of synapses. It is also critically involved in both mature dendritic spine formation and the maintenance of spine maturity. Glutamate is a major neurotransmitter of the brain, and is involved in all aspects of cognitive function, as it is the primary transmitter utilized by the cortical and hippocampal pyramidal neurons. Glutamate has also been associated with neuronal dendritic spine damage. The precise molecular mechanisms underlying dendritic spine damage following glutamate-induced neurotoxicity remain unknown. In the current study, we measured mRNA and protein expression levels of SPAR and serum-inducible kinase (SNK) in primary hippocampal neurons following glutamate treatment. Expression of SPAR and SNK was altered by glutamate treatment, indicating that the SPAR and SNK signaling pathways may be involved in the damage to dendritic spines in hippocampal neurons following excitotoxicity induced by glutamate.

Augmented Sodium Currents Contribute to the Enhanced Excitability of Small Diameter Capsaicin-Sensitive Sensory Neurons Isolated From Nf1+/− Mice

Neurofibromin, the product of the Nf1 gene, is a guanosine triphosphatase activating protein (GAP) for p21ras (Ras) that accelerates conversion of active Ras-GTP to inactive Ras-GDP. Sensory neurons with reduced levels of neurofibromin likely have augmented Ras-GTP activity. We reported previously that sensory neurons isolated from a mouse model with a heterozygous mutation of the Nf1 gene (Nf1+/⁻) exhibited greater excitability compared with wild-type mice. To determine the mechanism giving rise to the augmented excitability, differences in specific membrane currents were examined. Consistent with the enhanced excitability of Nf1+/⁻ neurons, peak current densities of both tetrodotoxin-resistant sodium current (TTX-R I(Na)) and TTX-sensitive (TTX-S) I(Na) were significantly larger in Nf1+/⁻ than in wild-type neurons. Although the voltages for half-maximal activation (V(0.5)) were not different, there was a significant depolarizing shift in the V(0.5) for steady-state inactivation of both TTX-R and TTX-S I(Na) in Nf1+/⁻ neurons. In addition, levels of persistent I(Na) were significantly larger in Nf1+/⁻ neurons. Neither delayed rectifier nor A-type potassium currents were altered in Nf1+/⁻ neurons. These results demonstrate that enhanced production of action potentials in Nf1+/⁻ neurons results, in part, from larger current densities and a depolarized voltage dependence of steady-state inactivation for I(Na) that potentially leads to a greater availability of sodium channels at voltages near the firing threshold for the action potential.

CHN1 mutations are not a common cause of sporadic Duane's retraction syndrome

<i>CHN1</i> mutations are not a common cause of sporadic Duane's retraction syndrome

Deletion of the Tumor Suppressor Gene NF1 Is An Alternative Mechanism for Aberrant Activation of the RAS Pathway and Is Found in 11% of Acute Myeloid Leukemia.

Abstract 401The NF1 gene encodes neurofibromin 1, a Ras-specific guanosine triphosphatase-activating protein (GAP) that negatively regulates the p21ras (Ras) family of signaling proteins by accelerating GTP hydrolysis. Germline loss of function mutations of NF1 lead to Neurofibromatosis type I. Carriers of the mutations develop benign neurofibromas and are predisposed to neuronal tumors, but also to juvenile myelomonocytic leukemia (JMML), MDS, and AML. In addition, in children suffering from CMML without germline NF1 mutations, acquired loss of function mutations of NF1 have been reported. In contrast, the role of NF1 in adult myeloid malignancies has not been studied in detail. Therefore, we first evaluated NF1 gene expression in 272 AML cases which were analyzed by Affymetrix HG-U133 Plus 2.0 microarrays. These included cases with t(15;17)(q22;q12) (n=15), t(8;21)(q22;q22) (n=16), inv(16)(p13q22) (n=7), t(11q23)/MLL-rearrangement (n=10), complex aberrant karyotype (n=47), normal karyotype (n=97), and with various other genetic abnormalities (n=80). The median NF1 expression intensity was 131.6 (range 35.2 - 457.5). 68 cases showed an expression intensity of NF1 below 98.6 (first quartile). In this cohort cases with t(8;21) (n=10) or complex karyotype (n=18) were over-represented (Chi-square: p<0.0001 and p=0.021, respectively), while cases with normal karyotype (n=16) were under-represented (Chi-square: p=0.016). In 54/68 of the latter cases with low expression material was availabel for FISH analysis with a probe spanning the NF1 locus. Remarkably, in 11/54 of these cases (20.4%) a NF1 deletion was observed by interphase FISH (% of cells with NF1 deletion median 90% (range: 60-99%). The mean±SD NF1 expression intensity in cases with NF1 deletion was 60.4±17.7 as compared to 75.5±18.0 in cases with 2 NF1 copies (p=0.023). Chromosome banding analysis in these 11 cases revealed a complex karyotype (n=7), a normal karyotype (n=2), an inv(3)(q21q26), and a 5q-deletion accompanied by trisomy 21, respectively. To further investigate the incidence of NF1 deletion in myeloid malignancies 425 additional patients were analyzed by FISH for NF1 deletion using a 420 kb probe spanning the NF1 gene. A heterozygous NF1 deletion was observed in 30/425 (7.1%) patients: de novo AML: 13/142 (9.2%), s-AML: 5/39 (12.8%), t-AML: 4/13 (31%), CMML: 5/99 (5.6%), MDS: 0/122 (0%), MPN: 3/10 (30%). Chromosome banding analysis in the NF1-deleted cases revealed a normal karyotype (n=4), an inv(16)(p13q22) (n=6), an inv(3)(q21q26) (n=6), a complex aberrant karyotype (n=5) or other abnormalities (n=9). In conclusion, NF1 deletions occur in 11% of AML, 5% of CMML and 3/10 MPN and therefore are a frequent and important alternative genetic mechanism for activating the RAS pathway in adult myeloid malignancies. Disclosures:Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Schindela:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership.

Genome-wide Scan of 500 000 Single-Nucleotide Polymorphisms Among Responders and Nonresponders to Interferon Beta Therapy in Multiple Sclerosis

Interferon beta is 1 of 2 first-line treatments for relapsing-remitting multiple sclerosis (MS). However, not all patients respond to interferon beta therapy, and to date there is a lack of surrogate markers that reliably correlate with responsiveness to interferon beta therapy in MS. To identify allelic variants that influence response to interferon beta therapy in patients with MS. Genome-wide scan. Academic research. Patients Two hundred patients having relapsing-remitting MS treated with interferon beta and having a follow-up period of at least 2 years were classified as responders or nonresponders to treatment based on stringent clinical criteria. In the first phase of the study, a pooling-based genome-wide association study of 428 867 single-nucleotide polymorphisms (SNPs) was performed in 53 responders and 53 nonresponders to interferon beta therapy. After applying several selection criteria, 383 SNPs were individually genotyped in an independent validation cohort of 49 responders and 45 nonresponders to interferon beta therapy using a different genotyping platform. Eighteen SNPs had uncorrected P < .05 associated with interferon beta responder status in the validation cohort. Of these, 7 SNPs were located in genes that code for alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid-type glutamate receptor GRIA3, type 1 interferon-related proteins ADAR and IFNAR2, cell cycle-dependent protein CIT, zinc finger proteins ZFAT and ZFHX4, and guanosine triphosphatase-activating protein STARD13. This study supports an underlying polygenic response to interferon beta treatment in MS and highlights the importance of the glutamatergic system in patient response to interferon beta therapy.

NMR Structural Studies on Human p190-A RhoGAPFF1 Revealed that Domain Phosphorylation by the PDGF-Receptor α Requires Its Previous Unfolding

p190-A and -B Rho GAPs (guanosine triphosphatase activating proteins) are the only cytoplasmatic proteins containing FF domains. In p190-A Rho GAP, the region containing the FF domains has been implicated in binding to the transcription factor TFII-I. Moreover, phosphorylation of Tyr308 within the first FF domain inhibits this interaction. Because the structural determinants governing this mechanism remain unknown, we sought to solve the structure of the first FF domain of p190-A Rho GAP (RhoGAPFF1) and to study the potential impact of phosphorylation on the structure.We found that RhoGAPFF1 does not fold with the typical (α1–α2–310–α3) arrangement of other FF domains. Instead, the NMR data obtained at 285 K show an α1–α2–α3–α4 topology. In addition, we observed that specific contacts between residues in the first loop and the fourth helix are indispensable for the correct folding and stability of this domain.The structure also revealed that Tyr308 contributes to the domain hydrophobic core. Furthermore, the residues that compose the target motif of the platelet-derived growth factor receptor α kinase form part of the α3 helix. We observed that the phosphorylation reaction requires a previous step including domain unfolding, a process that occurs at 310 K. In the absence of phosphorylation, the temperature-dependent RhoGAPFF1 folding/unfolding process is reversible. However, phosphorylation causes an irreversible destabilization of the RhoGAPFF1 structure, which probably accounts for the inhibitory effect that it exerts on the TFII-I interaction.Our results link the ability of a protein domain to be phosphorylated with conformational changes in its three-dimensional structure.

Neurofibromatosis type 1 and associated malignancies

Neurofibromatosis type 1 (NF1) is a common autosomal dominant neurocutaneous disorder with a predisposition to the development of benign and malignant tumors. Mutations in the NF1 gene result in loss of function of neurofibromin, a guanosine triphosphatase-activating protein that helps maintain the proto-oncogene Ras in its inactive form. Loss of neurofibromin results in increased proliferation and tumorigenesis. As a result, people with NF1 are at increased risk for the development of nervous and non-nervous system malignancies. Malignancy is a major source of morbidity and mortality in NF1. The natural history of NF1-associated malignancies is often different than that of their sporadic counterparts and, as such, management strategies need to be adjusted accordingly.

ARAP1 Regulates Endocytosis of EGFR

Signaling through the EGF receptor is regulated by endocytosis. ARAP1 is a protein with Arf guanosine triphosphatase-activating protein (GAP) and Rho GAP domains. We investigated the role of ARAP1 in EGF receptor endocytic trafficking. Following EGF treatment of cells, ARAP1 rapidly and transiently associated with the edge of the cell and punctate structures containing Rab5, rabaptin 5 and EGFR but not early embryonic antigen 1 (EEA1). EGF associated with the ARAP1-positive punctate structures prior to EEA1-positive early endosomes. Recruitment of ARAP1 to the punctate structures required active Rab5 and an additional signal from EGFR. Decreasing ARAP1 levels with small interfering RNA accelerated association of EGF with EEA1 endosomes and degradation of EGFR. Phosphorylation of extracellular-signal-regulated kinase (ERK) and c-Jun-amino-terminal kinase (JNK) was diminished and more transient in cells with reduced levels of ARAP1 than in controls. Based on these findings, we propose that ARAP1 regulates the endocytic traffic of EGFR and, consequently, the rate of EGFR signal attenuation.

New member of the guanosine triphosphatase activating protein family in the human epididymis

The effect of the guanosine triphosphatase activating proteins (GAPs) on spermatogenesis has been studied for years, though no GAPs have been explored in epididymis, an essential organ for sperm maturation. In this study, a new GAP member, designated as MacGAP, was cloned in human epididymis. The MacGAP gene encodes a protein of 618 amino acids with a putative size of 70 kDa and harbors the conserved RhoGAP domain. The N-terminal and C-terminal peptides of MacGAP were expressed and their corresponding antisera were prepared. The antisera against N-terminal peptide could detect antigen as low as 0.3 ng, and its specificity was also confirmed. However, the antisera against C-terminal peptide failed to detect its antigen because of its low sensitivity. Immunohistochemistry showed that the MacGAP protein was dependent on epididymis and had a region-specific expression pattern, with high expression in the epithelial cells'basal section in the caput region. The results have created a foundation for further interpretation of the biological effects of GAPs in sperm maturation.

Regulation of Glucose Transporter 4 Translocation by the Rab Guanosine Triphosphatase-Activating Protein AS160/TBC1D4: Role of Phosphorylation and Membrane Association

Insulin-stimulated translocation of the glucose transporter GLUT4 to the plasma membrane in muscle and fat cells depends on the phosphatidylinositide 3-kinase/Akt pathway. The downstream target AS160/TBC1D4 is phosphorylated upon insulin stimulation and contains a TBC domain (Tre-2/Bub2/Cdc16) that is present in most Rab guanosine triphosphatase-activating proteins. TBC1D4 associates with GLUT4-containing membranes under basal conditions and dissociates from membranes with insulin. Here we show that the association of TBC1D4 with membranes is required for its inhibitory action on GLUT4 translocation under basal conditions. Whereas the insulin-dependent dissociation of TBC1D4 from membranes was not required for GLUT4 translocation, its phosphorylation was essential. Many agonists that stimulate GLUT4 translocation failed to trigger TBC1D4 translocation to the cytosol, but in most cases these agonists stimulated TBC1D4 phosphorylation at T642, and their effects on GLUT4 translocation were inhibited by overexpression of the TBC1D4 phosphorylation mutant (TBC1D4-4P). We postulate that TBC1D4 acts to impede GLUT4 translocation by disarming a Rab protein found on GLUT4-containing-membranes and that phosphorylation of TBC1D4 per se is sufficient to overcome this effect, allowing GLUT4 translocation to the cell surface to proceed.

Human CHN1 mutations hyperactivate alpha2-chimaerin and cause Duane's retraction syndrome.

Duane's retraction syndrome (DRS) is a complex congenital eye movement disorder caused by aberrant innervation of the extraocular muscles by axons of brainstem motor neurons. Studying families with a variant form of the disorder (DURS2-DRS), we have identified causative heterozygous missense mutations in CHN1, a gene on chromosome 2q31 that encodes alpha2-chimaerin, a Rac guanosine triphosphatase-activating protein (RacGAP) signaling protein previously implicated in the pathfinding of corticospinal axons in mice. We found that these are gain-of-function mutations that increase alpha2-chimaerin RacGAP activity in vitro. Several of the mutations appeared to enhance alpha2-chimaerin translocation to the cell membrane or enhance its ability to self-associate. Expression of mutant alpha2-chimaerin constructs in chick embryos resulted in failure of oculomotor axons to innervate their target extraocular muscles. We conclude that alpha2-chimaerin has a critical developmental function in ocular motor axon pathfinding.