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

The GGAs (Golgi-localizing, γ-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 (γ-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.

[1]  S. Kornfeld,et al.  Interaction of the Cation-dependent Mannose 6-Phosphate Receptor with GGA Proteins* , 2002, The Journal of Biological Chemistry.

[2]  J. Bonifacino,et al.  Structural basis for acidic-cluster-dileucine sorting-signal recognition by VHS domains , 2002, Nature.

[3]  Thomas Earnest,et al.  Structural basis for recognition of acidic-cluster dileucine sequence by GGA1 , 2002, Nature.

[4]  Raymond J. Deshaies,et al.  Multisite Phosphorylation and the Countdown to S Phase , 2001, Cell.

[5]  M. Lindsay,et al.  GGAs: roles of the different domains and comparison with AP-1 and clathrin. , 2001, Molecular biology of the cell.

[6]  K. Nakayama,et al.  Golgi-localizing, γ-Adaptin Ear Homology Domain, ADP-ribosylation Factor-binding (GGA) Proteins Interact with Acidic Dileucine Sequences within the Cytoplasmic Domains of Sorting Receptors through Their Vps27p/Hrs/STAM (VHS) Domains* , 2001, The Journal of Biological Chemistry.

[7]  S. Kornfeld,et al.  Gamma subunit of the AP-1 adaptor complex binds clathrin: implications for cooperative binding in coated vesicle assembly. , 2001, Molecular biology of the cell.

[8]  J. Bonifacino,et al.  Sorting of Mannose 6-Phosphate Receptors Mediated by the GGAs , 2001, Science.

[9]  V. Lehto,et al.  Binding of GGA2 to the Lysosomal Enzyme Sorting Motif of the Mannose 6-Phosphate Receptor , 2001, Science.

[10]  S. Emr,et al.  Yeast Gga coat proteins function with clathrin in Golgi to endosome transport. , 2001, Molecular biology of the cell.

[11]  P. Madsen,et al.  The sortilin cytoplasmic tail conveys Golgi–endosome transport and binds the VHS domain of the GGA2 sorting protein , 2001, The EMBO journal.

[12]  J. Bonifacino,et al.  The GGAs Promote ARF-Dependent Recruitment of Clathrin to the TGN , 2001, Cell.

[13]  O. Zhdankina,et al.  Yeast GGA proteins interact with GTP‐bound Arf and facilitate transport through the Golgi , 2001, Yeast.

[14]  H. Pelham,et al.  A Selective Transport Route from Golgi to Late Endosomes That Requires the Yeast Gga Proteins , 2000, The Journal of cell biology.

[15]  K. Nakayama,et al.  Adaptor γ Ear Homology Domain Conserved in γ-Adaptin and GGA Proteins That Interact with γ-Synergin , 2000 .

[16]  L. Wan,et al.  Membrane-associated GAIP is a phosphoprotein and can be phosphorylated by clathrin-coated vesicles. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[17]  M. Robinson,et al.  A Family of Proteins with γ-Adaptin and Vhs Domains That Facilitate Trafficking between the Trans-Golgi Network and the Vacuole/Lysosome , 2000, The Journal of cell biology.

[18]  J. Bonifacino,et al.  GGAs: a family of ADP ribosylation factor-binding proteins related to adaptors and associated with the Golgi complex. , 2000, The Journal of cell biology.

[19]  R. Kahn,et al.  A family of ADP-ribosylation factor effectors that can alter membrane transport through the trans-Golgi. , 2000, Molecular biology of the cell.

[20]  O. Lohi,et al.  Vear, a Novel Golgi-associated Protein with VHS and γ-Adaptin “Ear” Domains* , 2000, The Journal of Biological Chemistry.

[21]  M. A. Downs,et al.  Epsin Binds to Clathrin by Associating Directly with the Clathrin-terminal Domain , 2000, The Journal of Biological Chemistry.

[22]  L. Wan,et al.  PACS-1 Defines a Novel Gene Family of Cytosolic Sorting Proteins Required for trans-Golgi Network Localization , 1998, Cell.

[23]  G. Thomas,et al.  Intracellular trafficking of furin is modulated by the phosphorylation state of a casein kinase II site in its cytoplasmic tail. , 1995, The EMBO journal.

[24]  C. Allende,et al.  Protein kinase CK2: an enzyme with multiple substrates and a puzzling regulation , 1995, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[25]  R. Frank,et al.  Phosphorylation of the cytoplasmic domain of the bovine cation-independent mannose 6-phosphate receptor. Serines 2421 and 2492 are the targets of a casein kinase II associated to the Golgi-derived HAI adaptor complex. , 1990, The Journal of biological chemistry.

[26]  S. Morris,et al.  Analysis of 100-180-kDa phosphoproteins in clathrin-coated vesicles from bovine brain. , 1990, The Journal of biological chemistry.

[27]  D. Branton,et al.  Assembled clathrin in erythrocytes. , 1987, The Journal of biological chemistry.

[28]  S. Puszkin,et al.  Brain clathrin light chain 2 can be phosphorylated by a coated vesicle kinase. , 1985, Proceedings of the National Academy of Sciences of the United States of America.