Regulators and Effectors of Arf GTPases in Neutrophils

Figure 4

AGAP2 efficiently stimulates GTP hydrolysis on Arf1 and GAP activity is stimulated by products of PI3K, PtdIns(3)P, and PtdIns(3,5)P₂. Recombinant myristoylated Arf1 was purified from E. coli as previously described [20]. AGAP2 cDNA was inserted into the pACHLT-A baculovirus shuttle vector and cotransfected with linearized BaculoGold viral DNA into sf9 cells. Culture supernatants were used to infect sf9 cells with an MOI of 10. Insect cells were collected 48 h after infection and His6-AGAP2 was purified from sf9 lysates by chromatography on Ni-trap columns. (a) GTPα³²P was loaded onto Arf1 in the presence of 1 mg/mL of liposomes composed of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine (molar ratio 40.55 : 31 : 28.45) for 30 min at 30°C. AGAP2 at the indicated concentrations was mixed with 0.3 μM GTPα³²P-loaded Arf1 and incubated for 30 min at 30°C in GAP buffer (20 mM Tris pH 8.0, 2 mM DTT, 100 mM NaCl, 1 mM MgCl₂, and 100 μg/mL liposomes). (b) GTPα³²P was loaded onto Arf1 in the presence of 1 mg/mL of liposomes composed of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine (molar ratio 40.55 : 31 : 28.45), and liposome-supplemented PtdIns(3)P or PtdIns(3,5)P₂ (molar ratio 37.4 : 28.5 : 26.2 : 7.9) for 30 min at 30°C. AGAP2 (10 nM) was mixed with 0.3 μM GTPα³²P-loaded Arf1 and incubated at 30°C in GAP buffer for indicated time points. Reactions were stopped by dilution in ice-cold stop buffer (20 mM Tris pH 8.0, 1 mM DTT, and 10 mM MgCl₂). Samples were filtered on Gelman GN-6 membranes and bound nucleotides were eluted with 2 M LiCl. GTP was separated from GDP by chromatography using polyethylenimine cellulose TLC plates developed in 1 M LiCl/1 M formic acid. The GTPα³²P/GDPα³²P ratios were calculated after exposure of TLC plates to a phosphorimager.

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(b)