The present study reports a previously unknown protein-protein interaction between human VDR and PIM-1 kinase and an involvement of a futher kinase in calcitriol signaling. VDR is an evolutionarily conserved member of the hormone-responsive NHR superfamily, expressed in malignant specimens (e.g. carcinomas and melanomas) and numerous mammalian tissues and, most crucially for our study, in bone and skin where it can be detected in a variety of cell types, including keratinocytes, fibroblasts, Langerhans cells, melanocytes, endothelial cells, and B- and T-lymphocytes [20, 21].
PIM-1, originally identified as a locus frequently activated by proviral insertion of the Moloney murine leukemia virus , encodes a highly conserved 313 AA serine/threonine kinase of the calcium/calmodulin-regulated kinase (CAMK) family whose expression is stimulated by a variety of cytokines, hormones and mitogens. PIM-1 appears to be involved in the control of cell growth, differentiation, apoptosis and malignant transformation [9, 15, 17, 23]. Putative PIM-1 kinase substrates and interactors include the c-Myb transcription factor nuclear adapter protein p100 , the G1-specific cell-cycle regulator Cdc25A , HP1 , the cdk inhibitor p21cip1/waf1, the nuclear mitotic apparatus protein NuMA  and the proapoptotic protein Bad , which are to a large extent involved in regulating cell-cycle progression and apoptosis. Nevertheless, the altogether small number of reported substrates would seem unlikely to be able to account for the broad spectrum of PIM-1 kinase functions, implying that it most probably interacts with additional, to date unidentified cellular substrates in specific physiological environments.
Expression of PIM-1 occurs in a wide range of tissues: hematopoietic and lymphoid tissue, prostate, testis, ovary, small intestine, colon, hippocampus and oral epithelia. It is significantly upregulated in B-cell lymphomas and erythroleukemias and has, via multi-tissue Northern blotting, been detected in a number of squamous cell carcinoma-derived keratinocyte lines, and in early and late passages of both spontaneously immortalized keratinocytes and normal human epidermal cells where its expression was substantially higher after confluence than during the logarithmic growth phase and correlated with increased keratinocyte differentiation [30–34]. There is an obvious overlap in the expression of VDR and PIM-1 kinase in a variety of calcitriol-responsive tissues.
Support for the biological significance of our discovery of a physical interaction between human VDR and PIM-1 kinase is the fact that most nuclear hormone receptors are phosphoproteins [extensively studied examples are ERα , PR , and mouse RARα ], whereby phosphorylation status can apparently govern receptor activity under the prevailing cellular conditions. Human VDR has been shown to be phosphorylated by protein kinase C (PKC) at serine 51 in the α-helical region that forms the two zinc fingers  and by protein kinase A (PKA) at serine 182 , which together attenuate the receptor’s VDRE-binding, RXR heterodimerization and transactivation functions. Conversely, calcitriol-stimulated, casein kinase II-catalyzed phosphorylation of human VDR at serine 208 [39–41] does not affect the receptor’s kinetics for hormone binding, its partitioning into the nucleus or its ability to associate with DNA but instead modulates its affinity for the 220 MW DRIP205/TRAP220 subunit of the large DRIP (VDR interacting proteins) mediator complex that directly anchors liganded VDR to components of the RNA polymerase II holoenzyme and hence amplifies its ability to transactivate target promoters. In addition, activated calcium/calmodulin-dependent kinase CaMKIV, which exhibits 30% identity and 45% similarity to PIM-1 (http://www.biobase.international.com), dramatically stimulates calcitriol-activated reporter gene expression by increasing VDR phosphorylation levels and results in enhanced interaction between VDR and SRC coactivator proteins in mammalian two-hybrid studies . Calcitriol also activates mitogen-activated protein (MAPK) kinases in normal human keratinocytes  and other cell types , thereby providing a rapid mechanism for the hormone to promote its own receptor-mediated action and suggesting a possible connection between the well-established gene regulatory mechanisms mediated through the VDR and the rapid response cascades. Functional studies on other steroid hormone and retinoid receptors have similarly implicated phosphorylation in the regulation of DNA association , hormone binding , nuclear localization  and transcriptional activation [48, 49]. Similar modifications have also been reported for a variety of NHR coregulators, including SRC-1, TIF2, RAC3, p300, CBP, NcoR and SMRT [50, 51], which may either enhance their interaction with NHRs as well as their abilitiy to recruit histone acetyltransferase (HAT) complexes (in the case of coactivators) or induce the redistribution of corepressor from the nucleus to the cytoplasm, thereby lessening the potential association between corepressors and NHRs [52–54].
Our domain mapping approaches have nevertheless indicated that the interaction with PIM-1 kinase does not involve the ligand-binding domain of VDR in contrary to the majority of co-activators. The interaction between PIM-1 and VDR is instead restricted to the receptor’s DNA binding domain, a type of interaction that, to our knowledge, has been described only 3 times previously: (i) between VDR and the bone-specific transcription factor Runx2 as a prerequisite to upregulate rat osteocalcin expression in osteoblasts ; (ii) between VDR and acute promyelocytic leukemia zinc finger protein (PLZF) necessary for the regulation of calcitriol-induced monocytic differentiation in hematopoietic cells ; and (iii), in a different experimental context, between VDR and the oncogenic nucleoporin CAN/Nup214, a component of the nuclear pore complex .
Because yeast two-hybrid experiments do not allow any conclusions about protein-protein interactions in a native cellular environment, we also studied PIM-1 and VDR in the spontaneously immortalized human HaCaT cell line which is known to behave phenotypically like normal basal epidermal keratinocytes in terms of growth and differentiation and to develop in the absence of serum and supplementary exogenous growth factors, features which make it a widely used cell culture system for such studies and an important model for the highly proliferative epidermis . As visualized by overexpressing YFP-VDR and tomato-tagged PIM-1, we could observe significant co-localization of the two proteins in the nucleus of HaCaT keratinocytes, which confirms that both proteins could indeed have the opportunity to interact with each other at the physiological level. We were subsequently able to increase transcription of CYP24A1 and osteopontin mRNA, the latter a classic VDR response gene [59, 60], by approximately 50% upon extrachromosomal overexpression of PIM-1 kinase. Furthermore, we similarly observed a clear enhancement of extrachromosomal DR3 reporter response upon PIM-1 overespression and respectively a clear reduction in DR3 reporter response by PIM-1 shRNA mediated knock-down, suggesting that PIM-1 kinase indeed can modulate calcitriol signaling. In an earlier study, Thompson et al. analyzed whether PIM-1 was capable of modulating androgen signaling because androgens are known to play a key role in male physiology as well as in the promotion or progression of prostate cancer and PIM-1 kinase was reported to be markedly overexpressed under such conditions . Ruling out simple explanations like altered receptor levels or DNA binding characteristics, they observed that, under transient transfection conditions, PIM-1 attenuated the transcriptional activity of AR, another NHR superfamily member (and in parallel experiments also of a LBD-deleted AR, GR and ERα), in a dose-dependent fashion in several cell types, but found no evidence for direct phosphorylation of AR by PIM-1 or, in contrast to our results, for a direct protein-protein interaction . They therefore suggested that PIM-1 likely influences AR activity via an indirect mechanism that possibly involves phosphorylation of a coregulator and/or a component of the transcriptional machinery. We could show that PIM-1 binds to the DNA-binding domain of VDR and modulates calcitriol signaling, however, we did not analyse the phosphorylation of VDR by PIM-1. Therefore it remains unclear whether VDR or a different component of the pathway is modulated by PIM-1. Interestingly, and in this context it was reported that PIM-1 enhances the transcriptional activity of c-myb, another transcription factor, by direct phosphorylating the DNA-binding domain as well as the phosphorylation of the transcriptional co-activator p100 [24, 62].
In a final pull-down of VDR-interacting protein complexes we investigated the protein complex of VDR by using the facility of a LXXLL containing peptide (DRIP-2) to capture the VDR-interacting co-activator protein complex in cell lysates. We detected the PIM-1 isoform 2 in the eluted fraction containing also VDR, but there is no detection of endogene PIM-1 in the fraction without DRIP-2 capture, so PIM-1 indeed seems to be included into the VDR-complex captured by DRIP-2 peptide, and potentially a member of the DRIP205-VDR co-activator complex .