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

Gopinath Kulasekaran1, Nadya Nossova1, Andrea L Marat1

  • 1Department of Neurology and Neurosurgery, Montreal Neurological Institute, McGill University, Montreal, Quebec H3A 2B4, Canada.

The Journal of Biological Chemistry
|June 10, 2015
PubMed

Insights

Connecdenn proteins regulate Rab35, crucial for cell function. Akt-dependent phosphorylation of Connecdenn 1 by insulin controls its guanine nucleotide exchange factor (GEF) activity and cargo recycling.

Area of Science:

  • Cell Biology
  • Molecular Signaling
  • Protein Regulation

Background:

  • Connecdenn proteins (differentially expressed in normal and neoplastic cells) are guanine nucleotide exchange factors (GEFs) for Rab35, essential for endosomal recycling and cell function.
  • The regulation of Connecdenn GEF activity, particularly in response to extracellular signals, remains largely uncharacterized.

Purpose of the Study:

  • To investigate the regulatory mechanisms governing Connecdenn 1 and Connecdenn 2 GEF activity.
  • To determine the role of phosphorylation in modulating Connecdenn function, specifically in response to insulin signaling in adipocytes.

Main Methods:

  • Purification of full-length Connecdenn proteins and isolated DENN domains.
  • Assays to measure Rab35 binding and GEF activity.
  • Proteomics screening to identify phosphorylation sites.
  • Insulin stimulation experiments in adipocytes with and without Akt inhibitors.
  • Analysis of Connecdenn 1 interaction with Rab35 and 14-3-3 proteins using peptide-based assays and mutational analysis.

Main Results:

  • Connecdenn 1/2 exhibit autoinhibition, with reduced GEF activity compared to isolated DENN domains.
  • Connecdenn 1 is phosphorylated at Ser-536 and Ser-538 in an Akt-dependent manner upon insulin stimulation.
  • Akt inhibition reduces Connecdenn 1 interaction with Rab35 and 14-3-3 proteins, suggesting phosphorylation regulates these interactions.

Conclusions:

  • Akt-mediated phosphorylation of Connecdenn 1 at Ser-536/Ser-538, downstream of insulin signaling, is a key regulatory mechanism.
  • This phosphorylation event likely relieves autoinhibition, modulating Connecdenn 1's interaction with Rab35 and 14-3-3 proteins to control cargo recycling.
  • The findings reveal an intramolecular regulatory mechanism for Connecdenn GEF activity in response to metabolic cues.
Keywords:
DENN domainRabRab35cell biologyconnecdennendocytosisendosomeguanine nucleotide exchange factor (GEF)intracellular traffickingmembrane trafficking

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