Autoinhibition of the ligand-binding site of GGA1/3 VHS domains by an internal acidic cluster-dileucine motif.
The GGAs (Golgi-localizing, gamma-adaptin ear homology domain, ARF-binding proteins) are a family of proteins implicated in protein trafficking from the Golgi to endosomes/lysosomes. These proteins have modular structures with an N-terminal VHS (VPS-27, Hrs, and STAM) domain followed by a GAT (GGA and TOM1) domain, a connecting hinge segment, and a C-terminal GAE (gamma-adaptin ear) domain. Isolated VHS domains have been shown to bind specifically to acidic cluster (AC)-dileucine motifs present in the cytoplasmic tails of the mannose 6-phosphate receptors. Here we report that full-length cytoplasmic GGA1 and GGA3 but not GGA2 bind the cation-independent mannose 6-phosphate receptor very poorly because of autoinhibition. This inhibition is caused by the binding of an AC-LL sequence present in the hinge segment to the ligand-binding site in the VHS domain. The inhibition depends on the phosphorylation of a serine located three residues upstream of the AC-LL motif. The serine is phosphorylated by casein kinase 2 in in vitro assays. Substitution of the GGA1 inhibitory sequence into the analogous location in GGA2, which lacks the AC-LL motif, results in autoinhibition of the latter protein. These data indicate that the activity of GGA1 and GGA3 is regulated by cycles of phosphorylation/dephosphorylation.
Doray
Balraj
B
Division of Hematology, Washington University School of Medicine, St. Louis, MO 63110, USA.
Bruns
Kerry
K
Ghosh
Pradipta
P
Kornfeld
Stuart A
SA
D020727:ADP-Ribosylation Factors / Q000737:chemistry / Q000378:metabolism*; D033942:Adaptor Proteins, Vesicular Transport; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D002417:Cattle; D002460:Cell Line; D002522:Chlorocebus aethiops; D004151:Dipeptides / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008024:Ligands; D008565:Membrane Proteins / Q000378:metabolism; D010766:Phosphorylation; D020033:Protein Isoforms / Q000737:chemistry / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014162:Transfection
D033942:Adaptor Proteins, Vesicular Transport; D002352:Carrier Proteins; D004151:Dipeptides; C407306:GGA adaptor proteins; D008024:Ligands; D008565:Membrane Proteins; D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins; C038361:leucylleucine; D020727:ADP-Ribosylation Factors