Resistance to inhibitors of cholinesterase 8A catalyzes release of Galphai-GTP and nuclear mitotic apparatus protein (NuMA) from NuMA/LGN/Galphai-GDP complexes

Abstract

Resistance to inhibitors of cholinesterase (Ric) 8A is a guanine nucleotide exchange factor that activates certain G protein alpha-subunits. Genetic studies in Caenorhabditis elegans and Drosophila melanogaster have placed RIC-8 in a previously uncharacterized G protein signaling pathway that regulates centrosome movements during cell division. Components of this pathway include G protein subunits of the Galphai class, GPR or GoLoco domain-containing proteins, RGS (regulator of G protein signaling) proteins, and accessory factors. These proteins interact to regulate microtubule pulling forces during mitotic movement of chromosomes. It is unclear how the GTP-binding and hydrolysis cycle of Galphai functions in the context of this pathway. In mammals, the GoLoco domain-containing protein LGN (GPSM2), the LGN- and microtubule-binding nuclear mitotic apparatus protein (NuMA), and Galphai regulate a similar process. We find that mammalian Ric-8A dissociates Galphai-GDP/LGN/NuMA complexes catalytically, releasing activated Galphai-GTP in vitro. Ric-8A-stimulated activation of Galphai caused concomitant liberation of NuMA from LGN. We conclude that Ric-8A efficiently utilizes GoLoco/Galphai-GDP complexes as substrates in vitro and suggest that Ric-8A-stimulated release of Galphai-GTP and/or NuMA regulates the microtubule pulling forces on centrosomes during cell division.

Fig. 1.

Purification of the LGN/Gαi-1/NuMA (LGN BD) complex. (A) A soluble Sf9 cell lysate containing rat His-6-LGN was supplemented with purified myristoylated Gαi-1 from E. coli and rapidly adsorbed to Ni-NTA agarose. The resin was eluted with imidazole, and a portion of the eluate was resolved by SDS/PAGE; the gel was stained with Coomassie brilliant blue. (B) The eluate containing LGN/Gαi-1 was immediately supplemented with a 5-fold molar excess (to LGN) of myristoylated Gαi-1, loaded onto a Hi-trap Q column (GE Healthcare), and resolved with a linear NaCl gradient. Fractions of the eluate were resolved by SDS/PAGE. The fractions that were pooled are noted (Q pool). (C) The Q pool of LGN/Gαi-1 complex was incubated with a 4-fold molar excess of NuMA (LGN BD) and a molar equivalent of myristoylated-Gαi-1, ultracentrifuged, and gel-filtered over tandem Superdex 75/200 columns. Fractions of the gel filtration eluate were resolved by SDS/PAGE and stained. Fractions containing the LGN/Gαi-1/NuMA complex were pooled (Complex Pool) and used for subsequent assays.

Fig. 2.

GTP[γS] binding to GoLoco protein/Gαi-1 complexes. (A) Full-length LGN/Gαi-1 (triangles), LGN/Gαi-1/NuMA (squares) complexes (50 nM), and free Gαi-1 (circles) (200 nM) were used to examine intrinsic (filled symbols) and 200 nM Ric-8A-stimulated (open symbols) rates of GTP[γS] binding. (B) LGN_short/Gαi-1 (triangles), AGS3_short/Gαi-1 (squares) complexes (50 nM), and free Gαi-1 (circles) (200 nM) were used to examine intrinsic (filled symbols) and 200 nM Ric-8A-stimulated (open symbols) rates of GTP[γS] binding. Each reaction contained 10 mM MgCl₂ and 10 μM[³⁵S]GTP[γS] (10,000 cpm/pmol). Results are shown as the mean of triplicate experiments ± standard deviation.

Fig. 3.

Release of GDP from complexes containing myristoylated and unmodified Gαi-1 and LGN_short. Myristoylated Gαi-1 and unmodified Gαi-1 (100 nM) were bound to [α-³²P]GDP and incubated with LGN_short (0, ▪; 5 nM, □;10 nM, •; 50 nM, ○; 100 nM, ▴; 200 nM, ▵; 500 nM, ▾ ;1 μM, ▿;5 μM, ♦) at 4°C for 5 min. Release of GDP from unmodified Gαi-1 was initiated at 30°C by adding 0 (A) or 200 nM (B) Ric-8A. Release of GDP from myristoylated Gαi-1 was initiated at 25°C by adding 0 (C) or 200 nM (D) Ric-8A. Duplicate aliquots of each reaction mixture were removed and quenched at the indicated times with AlF^–₄ quench buffer. Results are shown as the mean of duplicate experiments (± standard deviation). The intrinsic (E) and Ric-8A-stimulated (F) rates of GDP release from myristoylated Gαi(•) and unmodified Gαi (○) are plotted logarithmically against LGN_short concentration by using origin 6.0 (Microcal Software).

Fig. 4.

Ric-8A activates the LGN_short/myristoylated Gαi-1 complex catalytically. (A) Myristoylated Gαi-1/LGN_short complex (≈5 μM) was incubated with 50 μM GTP[γS], ≈1 mM free Mg⁺², and 0 (red), 100 nM (black), 500 nM (green), 1 μM (blue), or 5 μM (orange) Ric-8A for 15 min at 25°C. (B) Unmodified Gαi-1/LGN_short complex (≈5 μM) was incubated with 50 μM GTP[γS], ≈1 mM free Mg⁺², and 0 (red), 1 μM (black), 5 μM (green), or 25 μM (blue) Ric-8A for 15 min at 25°C or ≈10 mM free Mg⁺² and 25 μM Ric-8A (orange) for 15 min at 30°C. Reaction mixtures were loaded onto tandem Superdex 75/200 columns and resolved. The UV absorbance traces of the column eluates are shown.

Fig. 5.

Ric-8A dissociates the LGN/Gαi-1/NuMA complex, activating myristoylated Gαi-1 and liberating NuMA. LGN/Gαi-1/NuMA complex (10 μM, ≈40 μM Gαi-1) was incubated for 10 (red) or 30 (blue) min at 30°C with 100 μM GDP (A), 100 μM GTP[γS] (B), 100 μM GDP and 1 μM Ric-8A (C), or 100 μM GTP[γS] and 1 μM Ric-8A (D). The reaction mixtures were loaded onto tandem Superdex 75/200 columns and resolved. Fractions from each experiment were analyzed by SDS/PAGE. Each silver-stained gel is shown below the UV absorbance of the gel filtration eluate. The positions of gel filtration mass standards and the fractions where NuMA dimer appeared are noted.