PMC

Phosphorylation of GAIP by purified CK2. (A) GST-furin cd (1 μg), His6 GAIP, or His6 GAIP (S24A) (5 μg each) were incubated for 1 h at 30°C in the presence (+) or in the absence (−) of purified CK2, [γ-³²P], and 2 mM MgCl₂. Both wild-type GAIP and GAIP (S24A) are phosphorylated by recombinant CK2 (arrow), but phosphorylation of the mutant GAIP is reduced. (B) Equal amounts (5 μg) of GAIP and GAIP (S24A) were incubated with CK2 and processed as in A. Bands corresponding to phosphorylated GAIP were excised, and the amount of [³²P] incorporation was determined by scintillation counting. Phosphorylation of GAIP (S24A) is reduced by 50% compared with wild-type GAIP.

Phosphorylation of GAIP by CCVs. (A) His6 GAIP (lanes 1–12) was mixed with [γ³²-P]ATP, in the absence (lane 1) or presence of increasing concentrations of Mn²⁺ (lanes 2–6 and 13), Mg²⁺ (lanes 7–9), or Zn²⁺ (lanes 10–12). Reactions were started by addition of CCVs and stopped with SDS/BSA. Proteins were acetone precipitated, separated by 12.5% SDS/PAGE, and exposed for autoradiography. Phosphorylation of recombinant GAIP (dots) is favored by Mn²⁺ (lanes 4–6). (B) Migration of recombinant GAIP. His6-tagged proteins were analyzed by SDS/PAGE on a 15% polyacrylamide gel, and stained with Coomassie blue R-250. Migration of GAIP_80–206 (lane 1), wild-type GAIP (lane 2), and GAIP_1–79 (lane 3) are shown. The N terminus of GAIP, GAIP_1–79, migrates more slowly than expected. (C) Phosphorylations were performed as in A in the presence of 3 mM MnCl₂ and with the following His6-tagged proteins: wild-type GAIP (lane 1), GAIP_80–206 (lane 2), GAIP_1–79 (lane 3), GAIP (S24A) (lane 4), or RGS4 (lane 5). Phosphorylated recombinant proteins are marked with a dot. Phosphorylation in the presence of CCVs occurs at the N terminus (1–79) and in the RGS domain (80–206) of GAIP. Mutation of Ser-24 reduced the phosphorylation of GAIP.

Distribution of GAIP in AtT-20#1 cells. (A) AtT-20 cells stably expressing GAIP (clone #1) were homogenized, and the postnuclear supernatant (PNS) was centrifuged at 100,000 × g to yield crude membrane (P) and cytosolic fractions (S). Then, 25 μg of protein/lane were separated by SDS/PAGE, cut in two strips, and immunoblotted with anti-GAIP (N) (lanes 1, 3, and 5) or anti-HA (lanes 2, 4, and 6) and detected by enhanced chemiluminescence. GAIP is found in the PNS (lanes 1 and 2) and in both cytosolic (lanes 3 and 4) and membrane (lanes 5 and 6) fractions. (B) Cells were biosynthetically labeled for 4 h with Easytag, homogenized and fractionated as in A, and solubilized in RIPA buffer. GAIP was immunoprecipitated from the cytosolic (lanes 1 and 3) or crude membrane (lanes 2 and 4) fraction with anti-GAIP (23-217) (lanes 1 and 2) or anti-GAIP (C) (lanes 2 and 3). Immune complexes were separated by SDS/PAGE and detected by autoradiography. Newly synthesized, [³⁵S]GAIP was found mainly in the cytosolic fraction.

The membrane pool of GAIP is phosphorylated. (A) A total of 25 μg (lanes 1 and 2) or 50 μg (lanes 3 and 4) protein from the RM fraction of rat liver were incubated for 3 h with 30 units alkaline phosphatase (AP), separated by SDS/PAGE, followed by immunoblotting with anti-GAIP (N) and enhanced chemiluminescence. After alkaline phosphatase digestion, the slower migrating band (26.5 kDa) of GAIP disappears and only the faster migrating band (28.5 kDa) is seen. (B) AtT-20#1 cells were labeled in vivo with [³²P]orthophosphate for 4 h, and cytosolic (lanes 1, 3, and 5) and membrane fractions (lanes 2, 4, and 6) were prepared and solubilized as in Fig. . GAIP was immunoprecipitated using anti-HA (lanes 1 and 2), anti-GAIP (23–217) (lanes 3 and 4), or anti-GAIP (N) (lanes 5 and 6). Immune complexes were resolved by SDS/PAGE and exposed overnight for autoradiography. Phosphorylated GAIP is detected exclusively in the membrane fraction (P) with all three antibodies.

Immunoprecipitation and phosphoamino acid analysis of ³²P-labeled GAIP in transiently transfected HEK 293T cells. (A) Cells were transiently transfected with a mock pcDNA3 (lanes 1 and 2) or with pcDNA3 GAIP (lanes 3 and 4) and labeled with [³²P]orthophosphate for 2 h. GAIP was immunoprecipitated from cytosolic (S) and membrane (P) fractions with anti-GAIP (C) as in Fig. . ³²P-labeled GAIP (arrows, lanes 3 and 4) is found in both cytosolic (S) and membrane (P) fractions. A faster moving band, 23.1 kDa, is immunoprecipitated from cells labeled with [³²P]orthophosphate after transfection with GAIP (28 kDa), probably resulting from an alternative initiation start site (). (B) In the last 15 min of labeling, cells were incubated with buffer as control (lane 2) or 150 nM PMA (lane 3). The ³²P-labeled GAIP bands were cut out from both the cytosolic and membrane fractions and used for one-dimensional phosphoamino acid analysis on a cellulose-coated plate as described in Experimental Procedures. Lane 1: p-Ser, p-Thr, and p-Tyr standards detected by ninhydrin staining (arrowheads, lane 1). Lanes 2 and 3: Autoradiography p-Ser was the major phosphoamino acid present in both control (lane 2) and PMA-treated cells (lane 3).