AbstractIt is widely recognized that Hsp27 is a downstream substrate of the p38 MAPK cascade whereas the role of PKD family members in mediating receptor‐stimulated Hsp27 Ser‐82 phosphorylation has not been evaluated. Here, we show that neurotensin induced a rapid and striking increase in Hsp27 Ser‐82 phosphorylation in PANC‐1 cells, which was closely correlated with stimulation of activation loop phosphorylation of PKDs and p38 MAPK Thr180/Tyr182 phosphorylation. Treatment of PANC‐1 cells with either the selective PKC inhibitor GF‐I or the p38 MAPK inhibitor SB202190 partially reduced neurotensin‐induced Hsp27 Ser‐82 phosphorylation. However, treatment of the cells with a combination of GF‐I and SB202190 virtually abolished neurotensin‐induced Hsp27 Ser‐82 phosphorylation. Overexpression of PKD in stably transfected PANC‐1 cells increased the magnitude and prolonged the duration of Hsp27 Ser‐82 phosphorylation in response to neurotensin. Either PKD or PKD2 gene silencing utilizing siRNAs targeting distinct PKD or PKD2 sequences reduced neurotensin‐stimulated Hsp27 Ser‐82 phosphorylation, but cotransfection of siRNAs targeting both, PKD and PKD2, markedly decreased neurotensin‐induced Hsp27 Ser‐82 phosphorylation. Knockdown of PKD and PKD2 abolished Hsp27 phosphorylation in cells treated with SB202190. Thus, neurotensin induces Hsp27 Ser‐82 phosphorylation through p38 MAPK‐ and PKC/PKD‐dependent pathways in PANC‐1 cells. Our results demonstrate, for the first time, that neurotensin induces a striking increase in Hsp27 phosphorylation on Ser‐82 in PANC‐1 cells through convergent p38 MAPK, PKD, and PKD2 signaling. J. Cell. Biochem. 103: 648–662, 2008. © 2007 Wiley‐Liss, Inc.