A PH-like domain of the Rab12 guanine nucleotide exchange factor DENND3 binds actin and is required for autophagy

J Biol Chem. 2018 Mar 23;293(12):4566-4574. doi: 10.1074/jbc.RA117.001446. Epub 2018 Jan 19.

Abstract

Rab GTPases are key regulators of membrane trafficking, and many are activated by guanine nucleotide exchange factors bearing a differentially expressed in normal and neoplastic cells (DENN) domain. By activating the small GTPase Rab12, DENN domain-containing protein 3 (DENND3) functions in autophagy. Here, we identified a structural domain (which we name PHenn) containing a pleckstrin homology subdomain that binds actin and is required for DENND3 function in autophagy. We found that a hydrophobic patch on an extended β-turn of the PHenn domain mediates an intramolecular interaction with the DENN domain of DENND3. We also show that DENND3 binds actin through a surface of positively charged residues on the PHenn domain. Substitutions that blocked either DENN or actin binding compromised the role of DENND3 in autophagy. These results provide new mechanistic insight into the structural determinants regulating DENND3 in autophagy and lay the foundation for future investigations of the DENN protein family.

Keywords: DENN domain; DENND3; GTPase; PH domain; PHenn domain; Rab; Rab12; actin; autophagy; guanine-nucleotide exchange factor (GEF); intramolecular interaction; structural biology; structure-function.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Actins / chemistry
  • Actins / genetics
  • Actins / metabolism*
  • Autophagy*
  • Blood Proteins / chemistry
  • Blood Proteins / metabolism*
  • Crystallography, X-Ray
  • Guanine Nucleotide Exchange Factors / chemistry
  • Guanine Nucleotide Exchange Factors / genetics
  • Guanine Nucleotide Exchange Factors / metabolism*
  • HEK293 Cells
  • Humans
  • Phosphoproteins / chemistry
  • Phosphoproteins / metabolism*
  • Phosphorylation
  • Protein Binding
  • Protein Conformation
  • Protein Domains
  • rab GTP-Binding Proteins / chemistry
  • rab GTP-Binding Proteins / genetics
  • rab GTP-Binding Proteins / metabolism*

Substances

  • Actins
  • Blood Proteins
  • DENND3 protein, human
  • Guanine Nucleotide Exchange Factors
  • Phosphoproteins
  • platelet protein P47
  • Rab12 protein, human
  • rab GTP-Binding Proteins

Associated data

  • PDB/6B3Y