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.

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.

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.

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.