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Chromatin-Associated Protein Kinase C-&Theta Molecular Cell Article Chromatin-Associated Protein Kinase C-q Regulates an Inducible Gene Expression Program and MicroRNAs in Human T Lymphocytes Elissa L. Sutcliffe,1 Karen L. Bunting,3,8 Yi Qing He,1,8 Jasmine Li,1 Chansavath Phetsouphanh,6 Nabila Seddiki,6 Anjum Zafar,1 Elizabeth J. Hindmarsh,1 Christopher R. Parish,1 Anthony D. Kelleher,6 Russell L. McInnes,4 Toshiki Taya,5 Peter J. Milburn,2 and Sudha Rao1,7,* 1Department of Immunology 2Biomolecular Resource Facility John Curtin School of Medical Research, Australian National University, Canberra, ACT 2601, Australia 3Division of Hematology and Medical Oncology, Department of Medicine, Weill Cornell Medical College, Cornell University, New York, NY 10065, USA 4Agilent Technologies, 347 Burwood Highway, Forest Hill VIC 3131, Australia 5Agilent Technologies Japan, Hachioji Site, 9-1 Takakura-Cho, Hachioji-Shi, Tokyo 192-8510, Japan 6National Centre in HIV Epidemiology and Clinical Research, St. Vincent’s Centre for Applied Medical Research, University of New South Wales, Sydney NSW 2010, Australia 7Discipline of Biomedical Sciences, Faculty of Applied Science, The University of Canberra, Canberra ACT 2601, Australia 8These authors contributed equally to this work *Correspondence: [email protected] DOI 10.1016/j.molcel.2011.02.030 SUMMARY provides the exquisite control necessary for the initiation of specific transcription programs. Traditionally, the action of Studies in yeast demonstrate that signaling kinases signaling kinases was thought to occur predominantly in the have a surprisingly active role in the nucleus, where cytoplasm. Pioneering studies in yeast, however, have shown they tether to chromatin and modulate gene expres- that signal transduction kinases translocate to the nucleus and sion programs. Despite these seminal studies, the stably associate with the promoter and transcribed regions of nuclear mechanism of how signaling kinases control genes to regulate expression (Pascual-Ahuir et al., 2006; Pokho- transcription of mammalian genes is in its infancy. lok et al., 2006). These chromatin-tethered kinases have been shown to have both a structural role as part of transcription Here, we provide evidence for a hitherto unknown q complexes, as well as an enzymatic role by phosphorylating function of protein kinase C-theta (PKC- ), which their target proteins (de Nadal and Posas, 2010; Edmunds and physically associates with the regulatory regions of Mahadevan, 2006). inducible immune response genes in human T cells. There is growing evidence in higher eukaryotes that signal Chromatin-anchored PKC-q forms an active nuclear transduction kinases can exhibit a distinct function in both the complex by interacting with RNA polymerase II, the cytoplasm and the nucleus. For example, upstream kinases of histone kinase MSK-1, and the adaptor molecule the NF-kB pathway, IKK1 and NIK, shuttle between the cyto- 14-3-3z. ChIP-on-chip reveals that PKC-q binds to plasm and nucleus in resting cells to facilitate basal NF-kB tran- promoters and transcribed regions of genes, as scriptional activity (Birbach et al., 2002). IKK-a was shown to well as to microRNA promoters that are crucial for have an alternative role as a histone kinase that directly phos- cytokine regulation. Our results provide a molecular phorylates H3S10 at the promoters of NF-kB-responsive genes (Anest et al., 2003; Yamamoto et al., 2003). The p38 mitogen- explanation for the role of PKC-q not only in normal activated protein kinase (MAPK) is another signaling kinase T cell function, but also in circumstances of its that is recruited to the chromatin of muscle-specific loci where ectopic expression in cancer. it targets the SWI-SNF chromatin remodeling complex (Simone et al., 2004). It has also been demonstrated that inflammatory stimuli signal via p38 to chromatin (Saccani et al., 2002). INTRODUCTION Translocation of signaling kinases to the nucleus could provide an efficient mechanism whereby cells communicate Intracellular signal transduction often involves a complex extracellular signals generated at the plasma membrane to the cascade of phosphorylation events that enable cells to respond nucleus. Indeed, PKC isozymes have been shown to be capable appropriately to extracellular stimuli. Kinases play a crucial role of residency within the nucleus (Martelli et al., 1999), and it has in these signaling pathways as they transiently associate with been known for some time that these kinases can phosphorylate binding partners and transfer phosphate groups onto target histones in vitro (Inoue et al., 1977; Yu et al., 1998). Some of the substrates. The activation and concerted action of such proteins nuclear substrates identified for PKCs in vivo include DNA 704 Molecular Cell 41, 704–719, March 18, 2011 ª2011 Elsevier Inc. Molecular Cell PKC-q Binds Target Genes and MicroRNAs in T Cells topoisomerase I, lamin B, myogenin, nucleolin, p53, TdT and the ciation between endogenous PKC-q and histones H3 and H4 in vitamin D3 receptor (Martelli et al., 1999). Importantly, phosphoi- T cells (Figure 1E). Additionally, PKC-q associates with two indi- nositide signaling, which is central to the PKC activation cators of active chromatin, Pol II and histone H3 acetylated at pathway, appears to be intact at both the plasma membrane lysine-9 (H3K9ac) (Figure 1F). The anti-phospho-Pol II antibody and in the nucleus (Visnjic and Banfic, 2007). Despite increasing recognizes this protein when it is phosphorylated at serine-5 (de- evidence that PKC signaling kinases localize to the nucleus, their noted anti-Pol IIp herein). PKC-q failed to coimmunoprecipite the nuclear-specific functions and substrate specificities have re- repressive marks H2A.Z and HP1-a (data not shown). Taken mained unclear. The Ca2+-independent PKC isoform, PKC-q, together, nuclear PKC-q exists in the proximity of nucleosomes has previously been shown to associate with centrosomes and within active chromatin. kinetochore structures of the mitotic spindle within the nucleus of murine erythroleukemia cells (Passalacqua et al., 1999). This PKC-q Is Preferentially Recruited to the Regulatory kinase is best known for its critical role in efficient T cell activation Regions of Activated Genes and Forms a Nuclear (Isakov and Altman, 2002) and for its importance in proliferation, Complex with Pol II and MSK-1 in Human T Cells differentiation, and survival of mature T cells (Hayashi and Alt- Since PKC-q colocalized in the vicinity of nucleosomes in both man, 2007). Dysregulation of the PKC family has also been impli- resting and activated T cells, we investigated whether PKC-q cated in pathologies such as cancer and tumor metastasis functions as a chromatin-tethered kinase, similar to those (Griner and Kazanietz, 2007; Martiny-Baron and Fabbro, 2007). recently reported (Pokholok et al., 2006; Proft et al., 2006). Here, we demonstrate a nuclear role for PKC-q as a compo- Sequential ChIP showed increased co-occupancy between nent of an activating transcriptional complex in human T cells. histone H3 not only with PKC-q and catalytically active PKC-q We have shown that PKC-q physically associates with the prox- (denoted as PKC-qp herein), but also with the histone kinase imal promoter and coding regions of immune response genes MSK-1 on the CD69 proximal promoter in activated T cells (ST) after T cell activation. Chromatin-tethered PKC-q is intimately (Figure 2A). PKC-q and MSK-1 were also found to coexist on associated with the presence of RNA polymerase II, the histone this stretch of DNA in activated T cells (Figure 2B). A stimulus- kinase MSK-1, and the adaptor protein 14-3-3z. This event dependent increase in PKC-q recruitment was observed across appears to be coupled with PKC-q catalytic activity. ChIP-on- the CD69 gene, with maximal occupancy detected at +0.5 Kb in chip showed that PKC-q also localizes to the regulatory regions the 50-transcribed region following 24 hr of P/I activation (Fig- of a distinct cluster of microRNAs and negatively regulates their ure 2C). Similarly, a transient increase in MSK-1 occupancy transcription. These findings exemplify a more general role for was detected across the CD69 gene, with levels peaking signaling kinases as regulators of gene transcription in mamma- at +0.5 Kb in the transcribed region following 4 hr of P/I (Fig- lian cells that act by two distinct mechanisms: (1) cytoplasmic ure 2D). PKC-q and MSK-1 occupancy were also detected in signaling to the nucleus and (2) direct association with chro- resting T cells in the 50-transcribed region (Figures 2C and 2D), matin-bound transcription complexes at activated target genes which may reflect basal transcription of this gene (Sutcliffe in the nucleus. et al., 2009). PKC-q catalytic activity is substantially influenced by phos- RESULTS phorylation of serine-695 in its hydrophobic motif (Liu et al., 2002). A transient increase was observed in active PKC-qp PKC-q Localizes in the Nucleus of T Cells and Coresides (phosphorylated serine-695) at the CD69 promoter after 4 hr with Active Chromatin P/I (Figure 2E). Recruitment of PKC-qp was closely coupled to To determine whether PKC-q resides in the nucleus of human the early stages of transcriptional activation at the CD69 T cells, we performed immunoblot of nuclear extracts from Ju- promoter, while being sustained in the 50-transcribed region (Fig- rkat T cells that were either untreated or stimulated with phorbol ure 2E). Recruitment of Pol II to gene promoters and coding 12-myristate 13-acetate/calcium ionomycin (P/I). PKC-q was regions is a well-characterized mark of active gene transcription. present in the nucleus of both nonstimulated (NS) and P/I-stim- Sequential ChIP revealed increased co-occupancy of both ulated (ST) T cells (Figure 1A). To confirm the purity of nuclear PKC-q and MSK-1 with Pol II on the CD69 gene promoter in acti- preparations, we carried out immunofluorescence staining for vated T cells (Figure 2F).
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