Dynamin-mediated Nephrin phosphorylation regulates glucose-stimulated insulin release in pancreatic beta cells

J Biol Chem. 2012 Aug 17;287(34):28932-42. doi: 10.1074/jbc.M112.389452. Epub 2012 Jun 20.


We have previously demonstrated a role for Nephrin in glucose stimulated insulin release (GSIR). We now hypothesize that Nephrin phosphorylation is required for GSIR and that Dynamin influences Nephrin phosphorylation and function. MIN6-C3 Nephrin-deficient pancreatic beta cells and human islets were transfected with WT-Nephrin or with a mutant Nephrin in which the tyrosine residues responsible for SH2 domain binding were substituted with phenylalanine (3YF-Nephrin). GSIR and live images of Nephrin and vesicle trafficking were studied. Immunoprecipitation experiments and overexpression of WT-Dynamin or dominant negative Dynamin mutant (K44A-Dynamin) in WT-Nephrin, 3YF-Nephrin, or Nephrin siRNA-transfected cells were utilized to study Nephrin-Dynamin interaction. In contrast to WT-Nephrin or to single tyrosine mutants, 3YF-Nephrin did not positively affect GSIR and led to impaired cell-cell contacts and vesicle trafficking. K44A-Dynamin prevented the effect of Nephrin on GSIR in the absence of protein-protein interaction between Nephrin and Dynamin. Nephrin gene silencing abolished the positive effects of WT-Dynamin on GSIR. The effects of protamine sulfate and vanadate on Nephrin phosphorylation and GSIR were studied in MIN6 cells and human islets. WT-Nephrin phosphorylation after glucose occurred at Tyr-1176/1193 and resulted in improved GSIR. On the contrary, protamine sulfate-induced phosphorylation at Tyr-1176/1193/1217 was associated with Nephrin degradation and impaired GSIR. Vanadate, which prevented Nephrin dephosphorylation after glucose stimulation, improved GSIR in human islets and MIN6 cells. In conclusion, Dynamin-dependent Nephrin phosphorylation occurs in response to glucose and is necessary for Nephrin-mediated augmentation of GSIR. Pharmacological modulation of Nephrin phosphorylation may thus facilitate pancreatic beta cell function.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Substitution
  • Dynamins / genetics
  • Dynamins / metabolism*
  • Gene Silencing
  • Glucose / metabolism*
  • Glucose / pharmacology
  • HEK293 Cells
  • Humans
  • Insulin / metabolism*
  • Insulin Secretion
  • Insulin-Secreting Cells / cytology
  • Insulin-Secreting Cells / metabolism*
  • Membrane Proteins / genetics
  • Membrane Proteins / metabolism*
  • Mutation, Missense
  • Phosphorylation / drug effects
  • Phosphorylation / physiology
  • Proteolysis*
  • Sweetening Agents / metabolism
  • Sweetening Agents / pharmacology
  • Vanadates / pharmacology


  • Insulin
  • Membrane Proteins
  • Sweetening Agents
  • nephrin
  • Vanadates
  • Dynamins
  • Glucose