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1 (CK1) d and e as kinases downstream of the CCK2R that phosphorylate PKD2 at Ser244. Our data show that PKD2 interacts with and phosphorylates HDAC7, leading to its nuclear exclusion and concomitant derepression of HDAC7- regulated genes such as nur77. Only active PKD2 that also accumulates in the nucleus induces ef?cient translocation of HDAC7 from the cytoplasm to the nucleus and maximum Nur77 reporter activity. Gastrin-induced nuclear exclusion of HDAC7 and Nur77 reporter activity is critically dependent on PKD2. In conclusion, we establish a novel pathway that includes the coordinate regulation of PKD2 by CK1d/e and PKCZ downstream of the CCK2R, leading to nuclear accumu- lation of active PKD2, which results in nuclear exclusion of HDAC7 and transcriptional activation of nur77. Thus, com- Received:

20 April 2007;

accepted:

18 September 2007;

published online:

25 October

2007 *Corresponding author. Department of Internal Medicine I, University of Ulm, Robert Koch Strasse 8, Ulm 89081, Germany. Tel.: ?

49 731 50024308;

Fax: ?

49 731 50024302;

E-mail: [email protected]

6 Present address: Division of Biological Sciences, University of California, San Diego, La Jolla, CA, USA The EMBO Journal (2007) 26, 4619C4633 | &

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2007 European Molecular Biology Organization The EMBO Journal VOL

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4619 partment-speci?c signaling accomplished by phosphorylation at three critical sites determines the precise function of PKD2 as a mediator of CCK2R-induced transcriptional activation in gastric cancer cells. Results Role of the catalytic activity for nuclear accumulation of PKD2 Work from our laboratory established that PKD2 shuttles continuously between the cytoplasm and the nucleus in human epithelial tumor cells (Auer et al, 2005). In AGS-B cells, endogenous PKD2 as well as ectopically expressed enhanced green ?uorescence (eGFP)-PKD2 is localized pre- dominantly in the cytoplasm and relocalizes to the nucleus upon treatment of cells with gastrin (Figure 1A and B). Nuclear accumulation of PKD2 in response to gastrin was time dependent, reaching a maximum between

20 and

30 min of incubation (Supplementary Figure S1A). Treatment with leptomycin B (LMB) that blocks Crm-1-dependent nuclear export (Kudo et al, 1999) led to nuclear accumulation of both endogenous and ectopically expressed PKD2 (Figure 1A and B). Next, we determined whether gastrin-induced catalytic activity of PKD2 (Sturany et al, 2002) was required and suf?cient for its nuclear accumulation. Similar to wild-type Figure

1 Role of catalytic activity for nuclear accumulation of PKD2. (A) Subcellular localization of endogenous PKD2. AGS-B cells incubated with solvent (?) or

100 nM gastrin (G) for

40 min or

10 ng/ml LMB for

1 h were ?xed followed by anti-PKD2/Alexa

488 immunostaining and confocal laser scanning microscopy (LSCM). Fin was calculated. Size bar indicates

5 mm. (B) Subcellular localization of eGFP-PKD2 and its mutants in the presence and absence of gastrin. AGS-B cells were transfected with eGFP-PKD2 (wt-PKD2), eGFP-PKD2-S706/710 E or eGFP-PKD2- D695 A and treated as described in panel A. Images were captured by in vivo LSCM and Fin was calculated. (C) Lysates of AGS-B cells transfected with eGFP-PKD2 together with empty vector (?) or constitutively active PKCZ for

48 h were analyzed by anti-pPKD2-Ser706/710 (pPKD2), anti- PKD2 or anti-PKCZ western analysis (WB). (D) Effect of active PKCZ on PKD2 nuclear ........