14-3-3 proteins are essential signalling hubs for beta cell survival.
OBJECTIVE
Diabetes is characterised by pancreatic beta cell death and dysfunction, resulting from unbalanced pro-survival and pro-death signalling. The 14-3-3 proteins are molecular adaptors that integrate numerous signalling pathways, including the v-raf-leukaemia viral oncogene 1 (RAF1)/B cell leukaemia/lymphoma 2 (BCL-2)-associated agonist of cell death (BAD) pathway, which we have previously implicated in insulin-dependent beta cell survival. Nevertheless, the roles of 14-3-3 proteins in beta cell fate and function have not been investigated.


METHODS
We examined the abundance, localisation, modulation and roles of 14-3-3 proteins using quantitative RT-PCR, immunoblot or imaging. MIN6 cells or mouse islets cells were manipulated with inhibitors, short interfering RNA (siRNA) or plasmids overexpressing 14-3-3.


RESULTS
We first characterised the abundance and subcellular location of all seven 14-3-3 isoforms in mouse and human beta cells. Most isoforms were cytoplasmic, except 14-3-3σ, which appeared to be nuclear. Analysis of 14-3-3 abundance under stress conditions revealed distinct modulation in mouse islets and MIN6 cells. Generalised 14-3-3 inhibition promoted apoptosis and dysfunction, and siRNA-mediated knockdown revealed isoform-specific roles in caspase-3-dependent beta cell apoptosis, with a clear role for 14-3-3ζ. Overabundance of 14-3-3ζ sequestered BAD-BCL2-associated X protein (BAX) from mitochondria, attenuated Dp5 (also known as Hrk) and Puma (also known as Bbc3) induction, and increased survival in response to pro-inflammatory cytokines or thapsigargin. Anti-apoptotic insulin treatment increased the sequestration of BAD/BAX by 14-3-3ζ. BAD mutants that were unable to bind 14-3-3ζ localised to mitochondria and induced apoptosis.


CONCLUSIONS
This first study of the 14-3-3 family in beta cells revealed specific regulation, localisation and anti-apoptotic roles among the isoforms. Focus on 14-3-3ζ revealed its importance in preventing BAD-BAX mitochondrial localisation and protecting beta cells from multiple stresses. Thus, some 14-3-3 proteins are pro-survival signalling hubs.
Diabetes Research Group, Department of Cellular and Physiological Sciences, University of British Columbia, 5358 Life Sciences Building, 2350 Health Sciences Mall, Vancouver, BC, Canada V6T 1Z3.
G E
GE
Lim
M
M
Piske
J D
JD
Johnson
D048948:14-3-3 Proteins / Q000378:metabolism*; D000328:Adult; D000818:Animals; D017209:Apoptosis; D001786:Blood Glucose / Q000378:metabolism; D002460:Cell Line; D002470:Cell Survival; D003593:Cytoplasm / Q000378:metabolism; D006801:Humans; D007328:Insulin / Q000378:metabolism; D050417:Insulin-Secreting Cells / Q000166:cytology*; D051379:Mice; D008810:Mice, Inbred C57BL; D008875:Middle Aged; D020033:Protein Isoforms; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D015398:Signal Transduction
D048948:14-3-3 Proteins; D001786:Blood Glucose; D007328:Insulin; D020033:Protein Isoforms; D019253:Proto-Oncogene Proteins c-bcl-2