Differences between AGAP1, ASAP1 and Arf GAP1 in substrate recognition: interaction with the N-terminus of Arf1

Yoon, Hye‐Young; Jacques, Kerry M.; NEALON, B; Stauffer, Stacey; Premont, Richard T.; Randazzo, Paul A.

doi:10.1016/j.cellsig.2004.02.008

Cited by 21 publications

(17 citation statements)

References 66 publications

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“…These Arf GAPs lack a PH domain N-terminal to the Arf GAP domain. Recent work comparing the structural requirements of Arf for interaction with three Arf GAPs revealed the Nterminus of Arf had a greater role for interacting with ASAP1 and AGAP1 than with Arf GAP1 [44]. Such differences would be anticipated if the catalytic interface differed among the Arf GAPs.…”

Section: Discussionmentioning

confidence: 93%

“…For saturation kinetics, time courses for GTP hydrolysis at the indicated Arf1!GTP concentrations were determined from which initial hydrolysis rates were estimated. An Arf mutant, [L8K]Arf1, was used as a substrate because it loads efficiently with GTP and the k cat /K m is otherwise indistinguishable from wild type Arf1 when using PZA as an enzyme 1 [44]. Tryptophan fluorescence spectra were obtained using a Jobin Yvon Horiba Fluoromax 3 fluorescence spectrophotometer.…”

Section: Miscellaneousmentioning

confidence: 99%

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Regulation of ASAP1 by phospholipids is dependent on the interface between the PH and Arf GAP domains

Magnus¹,

Boja²,

Yoon³

et al. 2005

Cellular Signalling

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ASAP1 is an Arf GAP with a PH domain immediately N-terminal to the catalytic Arf GAP domain. PH domains are thought to regulate enzymes by binding to specific phosphoinositide lipids in membranes, thereby recruiting the enzyme to a site of action. Here, we have examined the functional relationship between the PH and Arf GAP domains. We found that GAP activity requires the cognate PH domain of ASAP1, leading us to hypothesize that the Arf GAP and PH domains directly interact to form the substrate binding site. This hypothesis was supported by the combined results of protection and hydrodynamic studies. We then examined the role of the PH domain in the regulation of Arf GAP activity. The results of saturation kinetics, limited proteolysis, FRET and fluorescence spectrometry support a model in which regulation of the GAP activity of ASAP1 involves a conformational change coincident with recruitment to a membrane surface, and a second conformational change following the specific binding of phosphatidylinositol 4,5-bisphosphate. D

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Section: Discussionmentioning

confidence: 93%

Section: Miscellaneousmentioning

confidence: 99%

Regulation of ASAP1 by phospholipids is dependent on the interface between the PH and Arf GAP domains

Magnus¹,

Boja²,

Yoon³

et al. 2005

Cellular Signalling

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“…As stated earlier, the simplest explanation for the acute decrease in Arf6-GTP is via the activation of a GAP. Studies with the inhibitory myr-Arf6 peptide are consistent because it can inhibit interactions between Arf6 and its GAPs (45), and it does block Arf6-GTP loss in activated platelets (27). However, there are not sufficient data to unequivocally define such a model.…”

Section: Discussionmentioning

confidence: 99%

Primary Platelet Signaling Cascades and Integrin-mediated Signaling Control ADP-ribosylation Factor (Arf) 6-GTP Levels during Platelet Activation and Aggregation

Karim

Choi

Whiteheart

2008

Journal of Biological Chemistry

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Previous studies showed that ADP-ribosylation factor 6 (Arf6) is important for platelet function; however, little is known about which signaling events regulate this small GTP-binding protein. Arf6-GTP was monitored in platelets stimulated with a number of agonists (TRAP, thrombin, convulxin, collagen, PMA, thapsigargin, or A23187) and all led to a time-dependent decrease in Arf6-GTP. ADP and U46619 were without effect. Using inhibitors, it was shown that the decrease of Arf6-GTP is a direct consequence of known signaling cascades. Upon stimulation via PAR receptors, Arf6-GTP loss could be blocked by treatment with U-73122, BAPTA/AM, Ro-31-8220, or Gö6976, indicating requirements for phospholipase C, calcium, and protein kinase C (PKC) ␣/␤, respectively. The Arf6-GTP decrease in convulxin-stimulated platelets showed similar requirements and was also sensitive to piceatannol, wortmannin, and LY294002, indicating additional requirements for Syk and phosphatidylinositol 3-kinase. The convulxin-induced decrease was sensitive to both PKC␣/␤ and ␦ inhibitors. Outside-in signaling, potentially via integrin engagement, caused a second wave of signaling that affected Arf6. Inclusion of RGDS peptides or EGTA, during activation, led to a biphasic response; Arf6-GTP levels partially recovered upon continued incubation. A similar response was seen in ␤3 integrin-null platelets. These data show that Arf6-GTP decreases in response to known signaling pathways associated with PAR and GPVI. They further reveal a second, aggregation-dependent, process that dampens Arf6-GTP recovery. This study demonstrates that the nucleotide state of Arf6 in platelets is regulated during the initial phases of activation and during the later stages of aggregation.

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“…Plasmids-Plasmids for the bacterial expression of [⌬17]Arf1, Arf1, and N-myristoyltransferase have been described previously (27)(28)(29). The plasmid for expression of Arf GAP1-His 6 was obtained from Victor Hsu and was described by Cassel and co-workers (30).…”

Section: Methodsmentioning

confidence: 99%

“…[⌬17]Arf1 was expressed and purified as described (27,33). His 10 [325-724]ASAP1 was expressed and purified as described (31).…”

Section: Methodsmentioning

confidence: 99%

Kinetic Analysis of Arf GAP1 Indicates a Regulatory Role for Coatomer

Luo

Randazzo

2008

Journal of Biological Chemistry

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Arf GAPs are a family of enzymes that catalyze the hydrolysis of GTP bound to Arf. Arf GAP1 is one member of the family that has a critical role in membrane traffic at the Golgi apparatus. Two distinct models for the regulation of Arf GAP1 in membrane traffic have been proposed. In one model, Arf GAP1 functions in a ternary complex with coat proteins and is inhibited by cargo proteins. In another model, Arf GAP1 is recruited to a membrane surface that has defects created by the increased membrane curvature that accompanies transport vesicle formation. Here we have used kinetic and mutational analysis to test predictions of models of regulation of Arf GAP1. We found that Arf GAP1 has a similar affinity for Arf1⅐GTP as another Arf GAP, ASAP1, but the catalytic rate is ≈0.5% that of ASAP1. Coatomer stimulated Arf GAP1 activity; however, different from that predicted from the current model, coatomer affected the K m and not the k cat values. Effects of most mutations in Arf GAP1 paralleled those in ASAP1. Mutation of an arginine that aligned with an arginine presumed to be catalytic in ASAP1 abrogated activity. Peptide from the cytoplasmic tail of cargo proteins inhibited Arf GAP1; however, the unrelated Arf GAP ASAP1 was also inhibited. The curvature of the lipid bilayer had a small effect on activity of Arf GAP1 under the conditions of our experiments. We conclude that coatomer is an allosteric regulator of Arf GAP1. The relevance of the results to the two models of Arf GAP1-mediated regulation of Arf1 is discussed.

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Differences between AGAP1, ASAP1 and Arf GAP1 in substrate recognition: interaction with the N-terminus of Arf1

Cited by 21 publications

References 66 publications

Regulation of ASAP1 by phospholipids is dependent on the interface between the PH and Arf GAP domains

Regulation of ASAP1 by phospholipids is dependent on the interface between the PH and Arf GAP domains

Primary Platelet Signaling Cascades and Integrin-mediated Signaling Control ADP-ribosylation Factor (Arf) 6-GTP Levels during Platelet Activation and Aggregation

Kinetic Analysis of Arf GAP1 Indicates a Regulatory Role for Coatomer

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