Membrane-associated GAIP is a phosphoprotein and can be phosphorylated by clathrin-coated vesicles

Abstract

GAIP (G ␣ interacting protein) is a member of the RGS (regulators of G protein signaling) family and accelerates the turnover of GTP bound to G␣i, G␣q, and G␣13. There are two pools of GAIP-a soluble and a membrane-anchored pool. The membrane-anchored pool is found on clathrin-coated vesicles (CCVs) and pits in rat liver and AtT-20 pituitary cells. By treatment of a GAIP-enriched rat liver fraction with alkaline phosphatase, we found that membranebound GAIP is phosphorylated. By immunoprecipitation carried out on [ 32 P]orthophosphate-labeled AtT-20 pituitary cells stably expressing GAIP, 32 P-labeling was associated exclusively with the membrane pool of GAIP. Phosphoamino acid analysis revealed that phosphorylation of GAIP occurred largely on serine residues. Recombinant GAIP could be phosphorylated at its N terminus with purified casein kinase 2 (CK2). It could also be phosphorylated by isolated CCVs in vitro. Phosphorylation was Mn 2؉ -dependent, using both purified CK2 and CCVs. Ser-24 was identified as one of the phosphorylation sites. Our results establish that GAIP is phosphorylated and that only the membrane pool is phosphorylated, suggesting that GAIP can be regulated by phosphorylation events taking place at the level of clathrin-coated pits and vesicles.G proteins ͉ regulator of G protein signaling ͉ casein kinase 2 S ignal transduction components and G-protein coupled receptor (GPCR) signaling pathways in particular undergo phosphorylation and dephosphorylation events that control their effects on multiple physiological processes (1). For example, GPCR are phosphorylated by G protein-coupled receptor kinases (GRKs) (2, 3), and several G proteins undergo phosphorylation in vivo on their ␣ (4-6) and ␥ (7) subunits, which may regulate their membrane targeting (8) and͞or interaction with partners (9)(10)(11)(12) Recently, a family of more than 20 members, the RGS proteins (for regulators of G protein signaling), have been identified that act as GTPase activating proteins (GAPs) for G␣ subunits (for reviews see refs. 13 and 14). We localized one member of the RGS family, RGS-GAIP (G ␣ interacting protein), by immunocytochemistry and found it to be associated with clathrincoated pits and vesicles (15,16) and further demonstrated that CCVs isolated from rat liver possess GAP activity (16).Clathrin-coated vesicles (CCVs) are known to play a role in G-protein coupled receptor-(2) and receptor-mediated endocytosis at the plasma membrane and in sorting of lysosomal enzymes in the trans-Golgi network (TGN) (17)(18)(19). Formation of CCVs is known to be tightly regulated by phosphorylation and dephosphorylation of adapters and coat proteins (20)(21)(22)(23)(24).GAIP contains nine putative phosphorylation sites-two for protein kinase C (PKC) and seven for casein kinase 2 (CK2) (25). These consensus sites are distributed over all three domains of GAIP: (i) its highly conserved RGS domain (amino acids 86-205) required for GAP activity (26), (ii) its N terminus (amino acids 1-85) most likely responsible for membrane ...

Autoinhibition of the ligand-binding site of GGA1/3 VHS domains by an internal acidic cluster-dileucine motif

“…However, in the presence of 30 mM Mn ϩϩ , the phosphorylation of the GST-GGA1 peptide was stimulated strongly, whereas that of the GST-CI-MPR peptide was reduced, resulting in equivalent phosphorylation. This result is similar to the finding that phosphorylation of the G␣-interacting protein by CK2 is Mn ϩϩ -dependent (16).…”

“…Substitution of an acidic residue at the third position downstream of the serine in GGA2 allowed the molecule to be phosphorylated by CK2. Although the actual kinase that phosphorylates GGA1 and GGA3 in cells has yet to be identified, it should be noted that a CK2-type enzyme has been shown to be associated with clathrin-coated vesicles (16,(21)(22)(23)(24). In preliminary experiments, we found that an activity associated with purified clathrin-coated vesicles phosphorylates the GGA1 peptide.…”

The RGSZ2 Protein Exists in a Complex with μ-Opioid Receptors and Regulates the Desensitizing Capacity of Gz Proteins

“…This is substituted by Ser179 in RGSZ1, Ser150 in RGSZ2, and Ser156 in GAIP (Tesmer et al, 1997;Posner et al, 1999). Moreover, the membrane-bound pool of GAIP is phosphorylated in the N-terminus (Fischer et al, 2000), and at Ser151 that lies inside the RGS domain (Ogier-Denis et al, 2000). The RGS-Rz subfamily binds with the dileucine-rich region of GIPN (GAIP interacting protein N terminus), a putative E3 ubiquitin ligase that links the RGS-Rz proteins with Ga degradation (Fischer et al, 2003).…”

RGSZ1 and GAIP Regulate μ- but Not δ-Opioid Receptors in Mouse CNS: Role in Tachyphylaxis and Acute Tolerance

“…The separated proteins were then transferred to 0.2 mm polyvinylidene difluoride membranes (Bio-Rad). Polyclonal anti-GAIP, -RGSZ1, -GIPC, -Gai2, and -Gaz were diluted typically 1 : 1000 in TBS-0.05% Tween 20 (TTBS) and incubated with the transfer membranes at 61C for 24 h. The anti-GAIP N terminus (1-79) and the 4507 (GAIP 23-217) antibodies, generously provided by T Fischer (De Vries et al, 1998a;Fischer et al, 2000), the RGSZ1 antibody, the Gai2 and the Gaz antibodies Garzón et al, 1997b) were detected with a goat anti-rabbit IgG (H þ L) horseradish peroxidaseconjugate antiserum (BioRad,. While GIPC (Santa Cruz Biotech., Inc, sc-9650) antibody was detected with a donkey anti-goat IgG horseradish peroxidaseconjugate antiserum (sc-2020).…”