develop and age normally under chow diet and maintain normal glucose tolerance (unpublished observations). As a matter of fact, although PKCd can serve as a pro-apoptotic signal, depending on the cellular context, it can also elicit anti-apoptotic and survival signals in a variety of cell systems [16–18]. These proliferative effects might involve a direct interference of PKCd with cell cycle regulation [19,20]. Intriguingly, proliferation of differentiated b- cells is a rare event although proteins which are important for cell cycle progression are expressed [21]. In adult mice less than 0.4% of b-cells stain positive for BrdU, in cultured human islet preparations only 0.3% of the cells proliferate [21–23]. Proliferation is tightly controlled by the sequential expression and activation of cell cycle regulators, such as cyclins and cyclin-dependent kinases (CDKs). The mitogenic activity of cyclin-CDK complexes is limited through binding of transiently expressed cell cycle inhibitors [24]. Inhibitors of the Cip/Kip family, p21Cip1/WAF1, p27kip1 and p57Kip2 are ubiquitously expressed proteins that slow down proliferation and cell cycle progression at G1/S or G2/M phase transitions [25]. While p57Kip2 regulates cell cycling mainly during development, p21Cip1/WAF1 and p27kip1 accumulate in mitogen-starved cells and mediate cell cycle arrest upon DNA damage [26–28]. In accordance with a minor role of p21Cip1/WAF1 during development, mice deficient of p21Cip1/WAF1 show normal growth and differentiation of the endocrine pancreas [22]. In contrast, mice that specifically overexpress p21Cip1/WAF1 in b-cells have impaired b-cell replication and develop age-related hyperglycemia due to increased apoptosis [29]. The activity of p21Cip1/WAF1 is regulated further by its subcellular distribution which is controlled by phosphorylation of p21Cip1/WAF1 at residues located in the C-terminal domain in proximity to the nuclear localization sequence [30]. PKB/Akt- mediated phosphorylations at Ser146 and at Thr145 sequester p21Cip1/WAF1 into the cytosol [31]. In vitro phosphorylation assays have further shown that PKCd can phosphorylate directly p21Cip1/WAF1 at Ser146, which triggers its cytosolic accumulation and influences the stabilization of p21Cip1/WAF1 [20]. In the present study, we examined the role PKCd plays in proliferation and survival of insulin-secreting cells. Our results suggest that PKCd phosphorylates the cell cycle inhibitor p21Cip1/WAF1 at Ser146, which favors its nuclear extrusion and supports proliferation under stress-free conditions. However, under stress conditions such as free fatty acids PKCd turns into a pro-apoptotic kinase. Results PKCd affects proliferation and apoptosis of insulin-secreting cells The first observation that PKCd may influence cell growth was made with INS-1E cells which were transfected with either an active PKCd (PKCdWT) or an inactive, kinase dead PKCd (PKCdKN, Fig. 1A). Surprisingly, PKCdWT transfected cells displayed 2.4 times more nuclei stained positive for the proliferation marker Ki67 when compared to untransfected control cells (Fig. 1B). The phosphorylation of PKCd at Ser643 and Thr505 was increased proportionally to the protein amount in PKCdWT INS-1E cells under standard culture conditions, which is indicative for an active PKCd (Fig. 1A). In PKCdKN INS-1E cells phosphorylation of PKCd at Thr505, a phosphorylation site of PDK1, is also increased proportionally to the protein amount, while phosphorylation at the autophosphorylation site Ser643 is reduced (Fig. 1A) [32–34]. It is noteworthy that the PKCdKN mutant remains inactive regardless of the degree of phosphory- lation. These observations suggest that PKCd supports prolifera- tion of INS-1E cells. PKCdelta in Insulin Secreting Cells Figure 1. Under non-stress condition PKCd promotes prolifer- ation of insulin secreting INS-1E cells. (A) Representative Western blots demonstrating expression and phosphorylation at Thr505 and at Ser643 of PKCd of control INS-1E cells and cells overexpressing PKCdWT or PKCdKN. Tubulin was used as loading control. Molecular weight markers are shown on the right. (B) Percentage of control, PKCdWT and PKCdKN INS-1E cells staining positive for Ki67 after 2 d culture. Data are expressed as means 6 SEM of n = 3–4 independent experiments. * (p,0.05) represents significance to control cells. doi:10.1371/journal.pone.0028828.g001 PKCd controls cytosolic-nuclear trafficking of p21Cip1/WAF1 in INS-1E cells and primary mouse islet cells The analysis of proteins which regulate proliferation revealed that PKCdWT cells expressed a significantly lower amount of the cell cycle inhibitor p21Cip1/WAF1 than control or PKCdKN INS-1E cells, while no change in expression of p27kip1 was apparent (Fig. 2A, B). Notably, due to the shorter length of rodent p21Cip1/WAF1 compared to the human orthologue, the protein band of p21Cip1/WAF1 displayed an apparent molecular weight lower than 21 kDa. This band was specific for p21Cip1/WAF1, as a protein of the same size was detected in bleomycin-treated wild type mouse embryonic fibroblasts (MEFs) but not in p21Cip1/WAF1-deficient MEFs (Fig. S1A). Moreover, the specificity of two antibodies used in this study was confirmed by immunocytochemistry, which revealed a p21Cip1/WAF1-specific staining in bleomycin-treated MEFs that was completely absent in untreated cells or in p21Cip1/WAF1-deficient cells exposed to the DNA damaging agent (Fig. S1B). The reduced expression of p21Cip1/WAF1 in PKCdWT INS-1E cells may be responsible for accelerated proliferation. Even more interesting is that the analysis of the subcellular distribution of p21Cip1/WAF1 using confocal laser scanning microsco- py showed that PKCdWT cells expressed p21Cip1/WAF1 almost exclusively in cytoplasm. In contrast, in PKCdKN cells and to a lesser extent in control INS-1E cells p21Cip1/WAF1 nuclear accumulation of PLoS ONE | www.plosone.org 2 December 2011 | Volume 6 | Issue 12 | e28828