PKCdelta in Insulin Secreting Cells Figure 4. Phosphorylation of p21Cip1/WAF1 depends on PKCd activity. Western blot analysis of cell lysates from control, PKCdWT and PKCdKN INS-1E cells for p21Cip1/WAF1 phosphorylation at (A) Ser146 and (B) Thr145. The densitometric analysis of phospho-Ser146 staining of p21Cip1/WAF1 to GAPDH as loading control is given as means 6 SEM of n = 3 independent experiments. * (p,0.05) and ** (p,0.01) indicate significance to the respective band of INS-1E cells set to 100%. (C) Western blots for Ser643 P-PKCd, Ser146 P-p21Cip1/WAF1, p21Cip1/WAF1 and tubulin as loading control of cell lysates from INS-1E cells after 2 d control culture (Con), after serum starvation for 16 h (Ø FCS), after 90 min treatment with the PKCd inhibitor rottlerin (10 mM) and after stimulation with PMA (1 mM for 2 min) in the presence or absence of rottlerin (10 mM for 90 min). doi:10.1371/journal.pone.0028828.g004 This study deciphers a direct link between PKCd and the cell cycle inhibitor p21Cip1/WAF1 which may influence b-cell prolifer- ation. The mechanism which drives PKCd from a proliferative into a pro-apoptotic role under stress conditions remains to be elucidated. Discussion The present study discloses the cell cycle inhibitor p21Cip1/WAF1 as a target of PKCd in insulin-secreting cells. Phosphorylation of p21Cip1/WAF1 at Ser146 by PKCd leads to its nuclear extrusion, thereby favoring cell proliferation and survival. The fact that p21Cip1/WAF1 is a substrate of PKCd is consistent with a previous report [20] and supported by our observation that both RNA interference as well as a pharmacological inhibitor of PKCd suppressed phosphorylation of p21Cip1/WAF1, whereas the PKC activator PMA increased p21Cip1/WAF1 phosphorylation. Further- more, inhibition of PKCd activity by expression of a kinase-inactive PKCd mutant reduced phosphorylation and increased nuclear accumulation of p21Cip1/WAF1. In contrast, in PKCdWT-expressing cells p21Cip1/WAF1 was phosphorylated at Ser146 and largely confined to the cytoplasm. Interestingly, inhibition of PKB and ERK1/2 did not diminish phosphorylation of the cell cycle inhibitor, indicating that PKCd is the major regulator of p21Cip1/WAF1 in insulin-secreting cells. Thus, our data suggest that in proliferating insulin-secreting cells PKCd supports proliferation, at least in part, by reducing nuclear accumulation and stability of p21Cip1/WAF1 (Fig. 7). In pancreatic slices of PKCdWT mice, proliferation was not detectable. Similarly, in p21Cip1/WAF1 KO mice, proliferation of pancreatic islet cells was also not significantly increased (0.4% in WT PLoS ONE | www.plosone.org 5 December 2011 | Volume 6 | Issue 12 | e28828