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PMID: 10760275 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S.

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

Proceedings of the National Academy of Sciences of the United States of America ·Vol. 97 ·No. 8 ·2000-04-11 ·Pages 4040-5

Fischer T, Elenko E, Wan L, Thomas G, Farquhar MG

Abstract

GAIP (G alpha interacting protein) is a member of the RGS (regulators of G protein signaling) family and accelerates the turnover of GTP bound to Galphai, Galphaq, and Galpha13. 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 membrane-bound 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.

MeSH Terms
Animals Base Sequence Cell Line DNA Primers Manganese/metabolism Mice Phosphoproteins/metabolism Phosphorylation Protein Kinases/metabolism RGS Proteins Rats
Chemicals
DNA Primers Phosphoproteins RGS Proteins regulator of G-protein signalling 19 Manganese Protein Kinases
Authors & Affiliations
5 authors, click to expand affiliations / ORCID
Fischer T
Department of Cellular and Molecular Medicine, University of California San Diego, La Jolla, CA 92093-0651, USA.
Elenko E
Wan L
Thomas G
Farquhar M G
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Article Info
Journal
Proceedings of the National Academy of Sciences of the United States of America
Abbr.
Proc Natl Acad Sci U S A
ISSN
0027-8424
DOI
10.1073/pnas.97.8.4040
Published
2000-04-11
Pages
4040-5
Language
English
Region
United States
NLM ID
7505876
PMCID
PMC18141
Subset
IM
Grants
NCI NIH HHS · CA67754 · United States
NIDDK NIH HHS · R01 DK017780 · United States
NIDDK NIH HHS · DK17780 · United States
NCI NIH HHS · CA58689 · United States
NCI NIH HHS · T32 CA067754 · United States
External Links
PubMed source DOI: 10.1073/pnas.97.8.4040 PMC Full Text View journal details
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