Phosphorylation-dependent Regulation of Connecdenn/DENND1 Guanine Nucleotide Exchange Factors

J Biol Chem. 2015 Jul 17;290(29):17999-18008. doi: 10.1074/jbc.M115.636712. Epub 2015 Jun 8.


Connecdenn 1/2 are DENN (differentially expressed in normal and neoplastic cells) domain-bearing proteins that function as GEFs (guanine nucleotide exchange factors) for the small GTPase Rab35. Disruption of connecdenn/Rab35 function leads to defects in the recycling of multiple cargo proteins from endosomes with altered cell function, yet the regulation of connecdenn GEF activity is unexplored. We now demonstrate that connecdenn 1/2 are autoinhibited such that the purified, full-length proteins have significantly less Rab35 binding and GEF activity than the isolated DENN domain. Both proteins are phosphorylated with prominent phosphorylation sites between residues 500 and 600 of connecdenn 1. A large scale proteomics screen revealed that connecdenn 1 is phosphorylated at residues Ser-536 and Ser-538 in an Akt-dependent manner in response to insulin stimulation of adipocytes. Interestingly, we find that an Akt inhibitor reduces connecdenn 1 interaction with Rab35 after insulin treatment of adipocytes. Remarkably, a peptide flanking Ser-536/Ser-538 binds the DENN domain of connecdenn 1, whereas a phosphomimetic peptide does not. Moreover, connecdenn 1 interacts with 14-3-3 proteins, and this interaction is also disrupted by Akt inhibition and by mutation of Ser-536/Ser-538. We propose that Akt phosphorylation of connecdenn 1 downstream of insulin activation regulates connecdenn 1 function through an intramolecular interaction.

Keywords: DENN domain; Rab; Rab35; cell biology; connecdenn; endocytosis; endosome; guanine nucleotide exchange factor (GEF); intracellular trafficking; membrane trafficking.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 14-3-3 Proteins / metabolism
  • 3T3-L1 Cells
  • Animals
  • Death Domain Receptor Signaling Adaptor Proteins / chemistry
  • Death Domain Receptor Signaling Adaptor Proteins / metabolism*
  • Guanine Nucleotide Exchange Factors / chemistry
  • Guanine Nucleotide Exchange Factors / metabolism*
  • HEK293 Cells
  • Humans
  • Insulin / metabolism
  • Mice
  • Phosphorylation
  • Protein Interaction Maps
  • Protein Structure, Tertiary
  • Proto-Oncogene Proteins c-akt / metabolism
  • Signal Transduction
  • rab GTP-Binding Proteins / metabolism*


  • 14-3-3 Proteins
  • DENND1A protein, human
  • DENND1B protein, human
  • Death Domain Receptor Signaling Adaptor Proteins
  • Guanine Nucleotide Exchange Factors
  • Insulin
  • Proto-Oncogene Proteins c-akt
  • RAB35 protein, human
  • rab GTP-Binding Proteins