From approximately 5 × 10 6 yeast transformants we recovered a total of 43 different positive clones expressing putative VDR interactors. To identify novel interactors of human VDR we carried out a Y2H assay by screening full length human VDR against a bone marrow cDNA library contained in the pAD-Gate1 to 3 vector system, as described by Maier et al. Genome-wide screening of a human cDNA library for VDR interacting proteins delivered PIM-1 kinase as candidate interactor These findings suggest involvement of the VDR-PIM-1 interaction in calcitriol-mediated growth and especially differentiation of keratinocytes. Here we identify PIM-1 kinase as a novel VDR interacting protein whose overexpression significantly enhances and knock-down suppresses both, endogenous calcitriol response gene transcription (osteopontin) and an extrachromosomal DR3 (direct repeat of 2 hexamers spaced by 3 nucleotides) reporter response. PIM-1 (proviral insertion of Moloney murine leukemia virus), which is a serine/threonine kinase of the calcium/calmodulin-regulated kinase (CAMK) family, phosphorylates different downstream effectors and has accordingly been proposed to play a key role in cell-cycle progression, cell survival, and differentiation of a variety of cell types. ĭespite an already substantial quantity of literature on VDR biology - for example, 1α,25- dihydroxyvitamin D3 signaling classification of agonistic/antagonistic VDR ligands synthesized with the goal to improve the biological profile of the natural hormone for therapeutic application descriptions of associations between VDR and a variety of interactors as listed in the Primos ( ) and Biogrid ( ) database as well as descriptions of structural motifs and conformational changes responsible for transcriptional control - there remain important regulatory aspects and mechanisms that have not been comprehensively elucidated.īy performing a genome-wide yeast two-hybrid (Y2H) screen using human VDR as bait in order to detect new protein-protein interactions (PPIs) to obtain a more complete understanding of the mode of action of VDR, we were able to identify a previously unknown binding partner, PIM-1 (NCBI accession no. These effects occur within minutes after hormone administration, are too rapid to involve changes in gene expression but instead direct an increase in numerous second messengers that can, though, ultimately affect transcription through secondary cross-talk with other (kinase) signal transduction cascades. Secondly, calcitriol can also mediate ‘rapid responses’ that are elicited by still debatable receptor(s) located near or associated with the plasma membrane and its caveolae compartments. The binding of calcitriol or one of its agonists triggers the release of corepressors, accompanied by subsequent recruitment of coactivators of the p160 family and finally of more general cofactors such as CBP/p300 to mediate local chromatin remodeling and stable assembly of the basal transcriptional machinery. Corepressor proteins such as hairless, Alien, NCoR (nuclear receptor corepressor) or SMRT (silencing mediator for retinoic acid receptor and thyroid hormone receptor) link non-liganded DNA-bound VDR or antagonist-destabilized nuclear hormone receptor (NHR)-coactivator complexes to enzymes with histone deacetylase (HDAC) activity and as a result promote condensation of chromatin to repress basal transcription. Genomic control of primary calcitriol-responsive genes is primarily achieved by binding of (phosphorylated) vitamin D receptor (VDR)-retinoid X receptor (RXR) heterodimers in a non-symmetrical head to tail arrangement to vitamin D response elements (VDRE) located within the promotor regions of target genes. ![]() These results, taken together with previous reports of involvement of kinase pathways in VDR transactivation, underscore the biological relevance of this novel protein-protein interaction. We further demonstrate that PIM-1 modulates calcitriol signaling in HaCaT keratinocytes by enhancing both endogenous calcitriol response gene transcription (osteopontin) and an extrachromosomal DR3 reporter response. Resultsīased on data obtained from genome-wide yeast two-hybrid screenings, domain mapping studies, intracellular co-localization approaches as well as reporter transcription assay measurements, we show here that the C-terminus of human PIM-1 kinase isoform2 (amino acid residues 135–313), a serine/threonine kinase of the calcium/calmodulin-regulated kinase family, directly interacts with VDR through the receptor’s DNA-binding domain. The vitamin D3 receptor (VDR) is responsible for mediating the pleiotropic and, in part, cell-type-specific effects of 1,25-dihydroxyvitamin D3 (calcitriol) on the cardiovascular and the muscle system, on the bone development and maintenance, mineral homeostasis, cell proliferation, cell differentiation, vitamin D metabolism, and immune response modulation.
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