Identification of the universal cofactor (auxilin 2) in clathrin coat dissociation

Eur J Cell Biol. 2000 May;79(5):336-42. doi: 10.1078/S0171-9335(04)70037-0.

Abstract

Uncoating of clathrin-coated vesicles in neuronal cells requires hsc70 in concert with the cofactor auxilin which contains a J-domain as well as a domain with homology to dual specific phosphatases and tensin, known as PTEN. The question of whether an analogous factor operates in other cell types has until now remained unanswered. Here we show that it is the recently discovered and widely expressed cyclin G-associated protein kinase which fulfils the function of neuronal auxilin in hsc70-mediated clathrin coat dissociation. GAK possesses a J-domain, which stimulates the hsc70 ATPase, it competes with auxilin for clathrin binding and at sufficiently high concentrations acts as a clathrin assembly protein. Moreover, GAK binds to the gamma- and alpha-appendage domains of the adaptor proteins AP-1 and AP-2 in vitro and phosphorylates their medium chains. Cells that transiently overexpress GAK are impaired in respect of receptor-mediated endocytosis. In transfected cells clathrin is dislodged from coated pits/vesicles and co-localizes with GFP-GAK in the form of large aggregates. The cellular distribution of membrane-associated adaptors was unaffected by overexpression of GAK. Our results point to a hsc70/auxilin-based uncoating system as a ubiquitous feature of eukaryotic cells.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Carrier Proteins / metabolism
  • Cell Fractionation
  • Clathrin / metabolism*
  • Clathrin-Coated Vesicles / immunology
  • Clathrin-Coated Vesicles / metabolism*
  • Cyclin G
  • Cyclin G1
  • Cyclins / chemistry
  • Cyclins / genetics
  • Cyclins / immunology
  • Cyclins / metabolism*
  • Endocytosis
  • Genes, Reporter
  • HSC70 Heat-Shock Proteins
  • HSP70 Heat-Shock Proteins*
  • HeLa Cells
  • Humans
  • Immunohistochemistry
  • Intracellular Signaling Peptides and Proteins
  • Molecular Chaperones / metabolism
  • Phosphorylation
  • Protein Binding
  • Protein Structure, Tertiary
  • Protein-Serine-Threonine Kinases / chemistry
  • Protein-Serine-Threonine Kinases / genetics
  • Protein-Serine-Threonine Kinases / immunology
  • Protein-Serine-Threonine Kinases / metabolism*
  • Radioligand Assay
  • Rats
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / immunology
  • Recombinant Fusion Proteins / metabolism
  • Transfection

Substances

  • CCNG1 protein, human
  • Carrier Proteins
  • Ccng1 protein, rat
  • Clathrin
  • Cyclin G
  • Cyclin G1
  • Cyclins
  • Gak protein, rat
  • HSC70 Heat-Shock Proteins
  • HSP70 Heat-Shock Proteins
  • HSPA8 protein, human
  • Hspa8 protein, rat
  • Intracellular Signaling Peptides and Proteins
  • Molecular Chaperones
  • Recombinant Fusion Proteins
  • GAK protein, human
  • Protein-Serine-Threonine Kinases