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Phosphorylation at Ser244 by CK1 determines nuclear localization and substrate targeting of PKD2 J von Blume1,6 , Uwe Knippschild2 , Franck Dequiedt3 , Georgios Giamas2 , Alexander Beck4 , Alexandra Auer1 , Johan Van Lint5 , Guido Adler1 and Thomas Seufferlein1, *

1 Department of Internal Medicine I, University of Ulm, Ulm, Germany,

2 Department of Surgery 1, University of Ulm, Ulm, Germany,

3 Laboratory of Molecular and Cellular Biology, FUSAGx, Gembloux, Belgium,

4 PANATecs, Tu ¨bingen, Germany and

5 Department of Molecular Cell Biology, Molecular Medicine of Protein Kinases, KU Leuven, Belgium Protein kinase D2 (PKD2), a member of the PKD family of serine/threonine kinases, is localized in various subcellular compartments including the nucleus where the kinase accumulates upon activation of G-protein-coupled recep- tors.

We de?ne three critical post-translational modi?ca- tions required for nuclear accumulation of PKD2 in response to activation of the CCK2 receptor (CCK2R): phos- phorylation at Ser706 and Ser710 within the activation loop by PKCg leading to catalytic activity and phosphorylation at Ser244 within the zinc-?nger domain, which is crucial for blocking nuclear export of active PKD2 by preventing its interaction with the Crm-1 export machinery. We identify CK1d and e as upstream activated kinases by CCK2R that phosphorylate PKD2 at Ser244. Moreover, nuclear accumu- lation of active PKD2 is a prerequisite for ef?cient phos- phorylation of its nuclear substrate, HDAC7. Only nuclear, active PKD2 mediates CCK2R-induced HDAC7 phosphoryla- tion and Nur77 expression. Thus, we de?ne a novel, com- partment-speci?c signal transduction pathway downstream of CCK2R that phosphorylates PKD2 at three speci?c sites, results in nuclear accumulation of the active kinase and culminates in ef?cient phosphorylation of nuclear PKD2 substrates in human gastric cancer cells. The EMBO Journal (2007) 26, 4619C4633. doi:10.1038/ sj.emboj.7601891;

Published online

25 October

2007 Subject Categories: signal transduction Keywords: CK1;

compartment-speci?c signalling;

gastric cancer;

HDACs;

PKD2 Introduction Protein kinase D2 (PKD2) is a member of the PKD family of serine/threonine kinases, which belongs to the CAMK super- family (Manning et al, 2002) and comprises PKD1/PKCm, PKD2 (Sturany et al, 2001) and PKD3/PKCn (Hayashi et al, 1999). PKDs exhibit a homologous catalytic domain, but vary with respect to their subcellular localization and expression (Rykx et al, 2003;

Rozengurt et al, 2005). They are major targets for tumor-promoting phorbol esters and activated by G-protein-coupled receptors (GPCRs) via PKCs (Rozengurt et al, 2005). The best-characterized isoform is PKD1, which has been implicated in the regulation of Golgi function, cell proliferation, apoptosis and migration (Van Lint et al, 2002). Recently, we demonstrated that gastrin, an important acid secretagogue and a trophic factor for the gastrointestinal mucosa, activates PKD2 in human gastric cancer cells ex- pressing the CCK2 receptor (CCK2R) (Sturany et al, 2001). The subcellular localization plays an important role in de?ning the biological effect of a given kinase signal. The biological function of PKD1 is regulated, for example, by its localization at the plasma membrane (Oancea et al, 2003). PKD1 has also been demonstrated to mediate nuclear export of class IIa histone deacetylases in thymocytes and lympho- cytes (Vega et al, 2004;

Dequiedt et al, 2005;

Matthews et al, 2006), but the compartment(s) in which this process occurs are not de?ned. In resting human epithelial cells, PKD2 is largely detectable in the cytoplasm. It is not actively retained there, but shuttles continuously between cytoplasm and nucleus most likely due to the combined activity of a nuclear localization signal in the linker region between the C1a and C1b domains and a nuclear export signal in the C1a domain. The activity of the nuclear export signal is dependent on the nuclear transport receptor, Crm-1. Upon activation of the CCK2R, PKD2 accumulates in the nucleus by an as yet unknown mechanism (Auer et al, 2005). The potential role of PKD2 nuclear accumulation for ef?cient substrate phos- phorylation is currently elusive. Here we demonstrate that nuclear accumulation of PKD2 mediated by CCK2R requires three phosphorylation events, phosphorylation at Ser706 and Ser710, that is catalytic activ- ity, and phosphorylation at a novel site in the zinc-?nger linker domain of PKD2, Ser244. We identify casein kinase