A toolkit for expression of Strep-tagged enhanced green fluorescent protein concatemers in mammalian cells.

Green fluorescent protein (GFP) and its variants are widely used tools in life sciences. Recently, we and others have used enhanced green fluorescent protein (EGFP) concatemers for determination of nuclear localization signal strength, as natural fluorescence standards and for mapping mobility in living cell nuclei. In this study, we present a molecular toolbox of Strep-tagged EGFP concatemers ranging from 1 to 12 subunits (Addgene plasmids #122488-122499). EGFP concatemers can be easily fused to targeting motifs of any origin by oligonucleotide ligation. Subsequently, we used liposomal transfection for transient expression of EGFP concatemers in eukaryotic cells. We have tested multiple protocols for further processing of the cells and recommend use of formalin or paraformaldehyde/methanol fixation. After usage of these protocols, we were able to detect concatemers by both GFP fluorescence microscopy and αStrep immunomicroscopy. In addition, we observed a more reliable detection of the StrepTag polypeptide (SA-WSHPQFEK) when using αStrepTag antibody instead of StrepTag binding protein. Summing up, we present a toolbox for expression of a wide range of Strep-tagged EGFP concatemers for multiple applications. By use of EGFP fluorescence and/or StrepTag polypeptide, the expressed concatemers can be easily detected in the cell.

A set of enhanced green fluorescent protein concatemers for quantitative determination of nuclear localization signal strength.

Regulated transport of proteins between nucleus and cytoplasm is an important process in the eukaryotic cell. In most cases, active nucleo-cytoplasmic protein transport is mediated by nuclear localization signal (NLS) and/or nuclear export signal (NES) motifs. In this study, we developed a set of vectors expressing enhanced GFP (EGFP) concatemers ranging from 2 to 12 subunits (2xEGFP to 12xEGFP) for analysis of NLS strength. As shown by in gel GFP fluorescence analysis and αGFP Western blotting, EGFP concatemers are expressed as fluorescent full-length proteins in eukaryotic cells. As expected, nuclear localization of concatemeric EGFPs decreases with increasing molecular weight. By oligonucleotide ligation this set of EGFP concatemers can be easily fused to NLS motifs. After determination of intracellular localization of EGFP concatemers alone and fused to different NLS motifs we calculated the size of a hypothetic EGFP concatemer showing a defined distribution of EGFP fluorescence between nucleus and cytoplasm (n/c ratio = 2). Clear differences of the size of the hypothetic EGFP concatemer depending on the fused NLS motif were observed. Therefore, we propose to use the size of this hypothetic concatemer as quantitative indicator for comparing strength of different NLS motifs.

Dissection of cell cycle-dependent dynamics of Dnmt1 by FRAP and diffusion-coupled modeling.

DNA methyltransferase 1 (Dnmt1) reestablishes methylation of hemimethylated CpG sites generated during DNA replication in mammalian cells. Two subdomains, the proliferating cell nuclear antigen (PCNA)-binding domain (PBD) and the targeting sequence (TS) domain, target Dnmt1 to the replication sites in S phase. We aimed to dissect the details of the cell cycle-dependent coordinated activity of both domains. To that end, we combined super-resolution 3D-structured illumination microscopy and fluorescence recovery after photobleaching (FRAP) experiments of GFP-Dnmt1 wild type and mutant constructs in somatic mouse cells. To interpret the differences in FRAP kinetics, we refined existing data analysis and modeling approaches to (i) account for the heterogeneous and variable distribution of Dnmt1-binding sites in different cell cycle stages; (ii) allow diffusion-coupled dynamics; (iii) accommodate multiple binding classes. We find that transient PBD-dependent interaction directly at replication sites is the predominant specific interaction in early S phase (residence time Tres ≤ 10 s). In late S phase, this binding class is taken over by a substantially stronger (Tres ∼22 s) TS domain-dependent interaction at PCNA-enriched replication sites and at nearby pericentromeric heterochromatin subregions. We propose a two-loading-platform-model of additional PCNA-independent loading at postreplicative, heterochromatic Dnmt1 target sites to ensure faithful maintenance of densely methylated genomic regions.

Human inositol 1,4,5-trisphosphate 3-kinase isoform B (IP3KB) is a nucleocytoplasmic shuttling protein specifically enriched at cortical actin filaments and at invaginations of the nuclear envelope.

Recent studies have shown that inositol 1,4,5-trisphosphate 3-kinase isoform B (IP3KB) possesses important roles in the development of immune cells. IP3KB can be targeted to multiple cellular compartments, among them nuclear localization and binding in close proximity to the plasma membrane. The B isoform is the only IP3K that is almost ubiquitously expressed in mammalian cells. Detailed mechanisms of its targeting regulation will be important in understanding the role of Ins(1,4,5)P(3) phosphorylation on subcellular calcium signaling and compartment-specific initiation of pathways leading to regulatory active higher phosphorylated inositol phosphates. Here, we identified an exportin 1-dependent nuclear export signal ((134)LQRELQNVQV) and characterized the amino acids responsible for nuclear localization of IP3KB ((129)RKLR). These two targeting domains regulate the amount of nuclear IP3KB in cells. We also demonstrated that the localization of IP3KB at the plasma membrane is due to its binding to cortical actin structures. Intriguingly, all three of these targeting activities reside in one small polypeptide segment (amino acids 104-165), which acts as a multitargeting domain (MTD). Finally, a hitherto unknown subnuclear localization of IP3KB could be demonstrated in rapidly growing H1299 cells. IP3KB is specifically enriched at nuclear invaginations extending perpendicular between the apical and basal surface of the nucleus of these flat cells. Such nuclear invaginations are known to be involved in Ins(1,4,5)P(3)-mediated Ca(2+) signaling of the nucleus. Our findings indicate that IP3KB not only regulates cytoplasmic Ca(2+) signals by phosphorylation of subplasmalemmal and cytoplasmic Ins(1,4,5)P(3) but may also be involved in modulating nuclear Ca(2+) signals generated from these nuclear envelope invaginations.

Nucleocytoplasmic shuttling of human inositol phosphate multikinase is influenced by CK2 phosphorylation.

Human inositol phosphate multikinase (IPMK) is a multifunctional protein in cellular signal transduction, namely, a multispecific inositol phosphate kinase, phosphatidylinositol 3-kinase, and a scaffold within the mTOR-raptor complex. To fulfill these nuclear and cytoplasmic functions, intracellular targeting of IPMK needs to be regulated. We show here that IPMK, which has been considered to be a preferentially nuclear protein, is a nucleocytoplasmic shuttling protein, whose nuclear export is mediated by classical nuclear export receptor CRM1. We identified a functional nuclear export signal (NES) additionally to its previously described nuclear import signal (NLS). Furthermore, we describe a mechanism by which the activity of the IPMK-NLS is controlled. Protein kinase CK2 binds endogenous IPMK and phosphorylates it at serine 284. Interestingly, this phosphorylation can decrease nuclear localization of IPMK cell type specifically. A controlled nuclear import of IPMK may direct its actions either toward nuclear inositol phosphate (InsPx) metabolism or cytoplasmic actions on InsPx, phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P2], as well as mTOR-raptor.

A toolkit for graded expression of green fluorescent protein fusion proteins in mammalian cells.

Green fluorescent protein (GFP) and GFP-like proteins of different colors are important tools in cell biology. In many studies, the intracellular targeting of proteins has been determined by transiently expressing GFP fusion proteins and analyzing their intracellular localization by fluorescence microscopy. In most vectors, expression of GFP is driven by the enhancer/promoter cassette of the immediate early gene of human cytomegalovirus (hCMV). This cassette generates high levels of protein expression in most mammalian cell lines. Unfortunately, these nonphysiologically high protein levels have been repeatedly reported to artificially alter the intracellular targeting of proteins fused to GFP. To cope with this problem, we generated a multitude of attenuated GFP expression vectors by modifying the hCMV enhancer/promoter cassette. These modified vectors were transiently expressed, and the expression levels of enhanced green fluorescent protein (EGFP) alone and enhanced yellow fluorescent protein (EYFP) fused to another protein were determined by fluorescence microscopy and/or Western blotting. As shown in this study, we were able to (i) clearly reduce the expression of EGFP alone and (ii) reduce expression of an EYFP fusion protein down to the level of the endogenous protein, both in a graded manner.

A novel 6-pyrophosphorylating IP6 kinase (IP6-6K) discovered in the protozoon Trichomonas vaginalis.

The parasitic protozoon Trichomonas vaginalis is the pathogen of trichomoniasis, the most common non-viral, sexually transmitted disease in humans. Inositol phosphates function in the pathomechanisms of a number of human pathogenic protozoa. Recent findings point to a role of inositol phosphates in T. vaginalis' adaption to oxygen exposure during change of host. Six inositol phosphate kinase genes (tvip6k1-4, tvipk1-2) were identified in the T. vaginalis genome by us all coding for proteins containing canonical sequence motifs of the major group of animal inositol phosphate kinases (PDKG, SSLL, DFG/A). When characterizing the purified protein product of tvip6k1, we discovered that the major activity of the highly active enzyme (˜2 µmol/min/mg) is a conversion of InsP6 to 6PP-InsP5 and not 5PP-InsP5 as by animal isoforms. Thus TvIP6K1 is a novel IP6-6K. The enzyme also converts Ins(1,3,4,5,6)P5 to products pyrophosphorylated both at 6- and 4-phosphate still having a free 5-hydroxyl. In addition, the enzyme has a minor selectivity to phosphorylate the 3-OH in Ins(1,2,4,5)P4 and Ins(1,2,4,5,6)P5. To present knowledge this novel enzyme is restricted to protozoa. Since its structure is predicted to be distinctly different from animal IP6K (IP6-5K) forms, TvIP6-6K may become a promising target to search for novel trichomoniasis specific drugs.

Intracellular localization of human Ins(1,3,4,5,6)P5 2-kinase.

InsP6 is an intracellular signal with several proposed functions that is synthesized by IP5K [Ins(1,3,4,5,6)P5 2-kinase]. In the present study, we overexpressed EGFP (enhanced green fluorescent protein)-IP5K fusion proteins in NRK (normal rat kidney), COS7 and H1299 cells. The results indicate that there is spatial microheterogeneity in the intracellular localization of IP5K that could also be confirmed for the endogenous enzyme. This may facilitate changes in InsP6 levels at its sites of action. For example, overexpressed IP5K showed a structured organization within the nucleus. The kinase was preferentially localized in euchromatin and nucleoli, and co-localized with mRNA. In the cytoplasm, the overexpressed IP5K showed locally high concentrations in discrete foci. The latter were attributed to stress granules by using mRNA, PABP [poly(A)-binding protein] and TIAR (TIA-1-related protein) as markers. The incidence of stress granules, in which IP5K remained highly concentrated, was further increased by puromycin treatment. Using FRAP (fluorescence recovery after photobleaching) we established that IP5K was actively transported into the nucleus. By site-directed mutagenesis we identified a nuclear import signal and a peptide segment mediating the nuclear export of IP5K.

Nuclear accumulation of SHIP1 mutants derived from AML patients leads to increased proliferation of leukemic cells.

The inositol 5-phosphatase SHIP1 acts as negative regulator of intracellular signaling in myeloid cells and is a tumor suppressor in myeloid leukemogenesis. After relocalization from the cytoplasm to the plasma membrane SHIP1 terminates PI3-kinase mediated signaling processes. Furthermore, SHIP1 is also found in distinct puncta in the cell nucleus and nuclear SHIP1 has a pro-proliferative function. Here we report the identification of five nuclear export signals (NESs) which regulate together with the two known nuclear localization signals (NLSs) the nucleocytoplasmic shuttling of SHIP1. Mutation of NLSs reduced the nuclear import and mutation of NESs decreased the nuclear export of SHIP1 in the acute myeloid leukemia (AML) cell line UKE-1. Interestingly, four SHIP1 mutants (K210R, N508D, V684E, Q1153L) derived from AML patients showed a nuclear accumulation after expression in UKE-1 cells. In addition, overexpression of the AML patient-derived mutation N508D caused an increased proliferation rate of UKE-1 cells in comparison to wild type SHIP1. Furthermore, we identified serine and tyrosine phosphorylation as a molecular mechanism for the regulation of nucleocytoplasmic shuttling of SHIP1 where tyrosine phosphorylation of distinct residues i.e. Y864, Y914, Y1021 reduces nuclear localization, whereas serine phosphorylation at S933 enhances nuclear localization of SHIP1. In summary, our data further implicate nuclear SHIP1 in cellular signaling and suggest that enhanced accumulation of SHIP1 mutants in the nucleus may be a contributory factor of abnormally high proliferation of AML cells.

The families of kinases removing the Ca2+ releasing second messenger Ins(1,4,5)P3.

The formation and degradation of the second messenger D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] are of great metabolic importance, because of its role in the mediation of calcium release from intracellular stores. The concentration of Ins(1,4,5)P3 in the cell is regulated by three signaling enzymes: phospholipase C isoforms release Ins(1,4,5)P3 from the plasma membrane by hydrolysis of phosphatidyl inositol 4,5-bisphosphate, whereas inositol phosphate 5-phosphatases remove it by dephosphorylation and a group of inositol phosphate kinases eliminate it by further phosphorylation at its 3- or 6-hydroxy group. The latter group is formed by the three isoforms of Ins(1,4,5)P3 3-kinase (IP3K) and inositol phosphate multikinase. In this article the tissue specific gene expression, molecular structure, role in calcium oscillations, regulation by calcium calmodulin, by phosphorylation and by intracellular localization of the IP3K isoforms are discussed. Another important aspect is the evolution of diverse inositol phosphate metabolizing enzymes from a eukaryotic founder by different mechanisms of gene diversification. Finally the role of IPMK in calcium signaling will be elucidated in more detail.

The tumor suppressor SHIP1 colocalizes in nucleolar cavities with p53 and components of PML nuclear bodies.

The inositol 5-phosphatase SHIP1 is a negative regulator of signaling processes in haematopoietic cells. By converting PI(3,4,5)P3 to PtdIns(3,4)P2 at the plasma membrane, SHIP1 modifies PI3-kinase mediated signaling. We have recently demonstrated that SHIP1 is a nucleo-cytoplasmic shuttling protein and SHIP1 nuclear puncta partially colocalize with FLASH, a component of nuclear bodies. In this study, we demonstrate that endogenous SHIP1 localizes to intranucleolar regions of both normal and leukemic haematopoietic cells. In addition, we report that ectopically expressed SHIP1 accumulates in nucleolar cavities and colocalizes with the tumor suppressor protein p53 and components of PML nuclear bodies (e.g. SP100, SUMO-1 and CK2). Moreover, SHIP1 also colocalizes in nucleolar cavities with components of the ubiquitin-proteasome pathway. By using confocal microscopy data, we generated 3D-models revealing the enormous extent of the SHIP1 aggresomes in the nucleolus. Furthermore, treatment of cells with the proteasome inhibitor MG132 causes an enlargement of nucleolar SHIP1 containing structures. Unexpectedly, this accumulation can be partially prevented by treatment with the inhibitor of nuclear protein export Leptomycin B. In recent years, several proteins aggregating in nucleolar cavities were shown to be key factors of neurodegenerative diseases and cancerogenesis. Our findings support current relevance of nuclear localized SHIP1.

Efficacious inhibition of Importin α/ß-mediated nuclear import of human inositol phosphate multikinase.

Human inositol phosphate multikinase (IPMK) is a nucleocytoplasmic shuttling protein involved in multiple signal transduction pathways located both in the nucleus and in the cytoplasm. To efficaciously inhibit the conventional nuclear import of IPMK, we first examined the effect of different inhibitors and cellular stressors on nuclear import of enhanced green fluorescent protein monomer and octamer, both fused with a monopartite nuclear localization signal (NLS), in HeLa and H1299 cells. Most efficacious inhibition of conventional nuclear protein import was observed when using Importazole and hydrogen peroxide. Therefore, these substances were then applied to examine nuclear import mechanisms of IPMK. Thereby, we demonstrated that nuclear accumulation of IPMK is significantly lessened, but not abrogated by inhibition of conventional protein import. This indicates that IPMK is imported into the nucleus by both conventional and non-conventional pathways. Furthermore, intracellular distribution of an IPMK mutant with inactivated NLS is unaffected by inhibition of conventional protein import. Obviously, the conventional import of IPMK is entirely mediated by interaction of the Importin α/ß heterodimer with IPMK's sole NLS motif (R(320)HRKIYTKKHH). Future research should focus on the hitherto unknown non-conventional import of IPMK and the potential impact of its dysregulation on IPMK signaling pathways regulating cellular growth and proliferation.

A novel Entamoeba histolytica inositol phosphate kinase catalyzes the formation of 5PP-Ins(1,2,3,4,6)P(5).

The parasitic protozoan Entamoeba histolytica is able to invade human tissues by secreting proteolytic enzymes. This secretion is regulated by inositol phosphate-mediated Ca(2+) release from internal stores. To further investigate the inositol phosphate metabolism of Entamoeba histolytica four putative inositol phosphate kinase genes (ehipk1-4) were identified and their expression analyzed by real-time quantitative PCR using RNA of trophozoites. Furthermore inositol phosphate kinase EhIPK1 was recombinantly expressed, purified and enzymatically characterized. Its main activity is the conversion of InsP(6) to 5PP-Ins(1,2,3,4,6)P(5), one of the main inositol phosphates found in Entamoeba histolytica. Remarkably, EhIPK1 possesses several additional enzymatic activities, e.g. the phosphorylation of the Ca(2+)-releasing second messenger Ins(1,4,5)P(3).We were able to identify several compounds with inhibitory potential against EhIPK1. Because of the important role of inositol phosphates in the invasion of human tissues by Entamoeba histolytica, inositol phosphate metabolizing enzymes are interesting targets for novel therapeutic approaches.

A Plasmodium multi-domain protein possesses multiple inositol phosphate kinase activities.

The synchronization of intraerythrocytic maturation of Plasmodium parasites is an important factor in the malaria infection process. Synchronization is mediated by inositol phosphate (InsP(x))-induced Ca(2+)-release from internal stores. To further investigate the InsP(x) metabolism in these parasites a Plasmodium protein possessing inositol phosphate kinase (IPK) activity was recombinantly expressed, purified and enzymatically characterized for the first time. Its main activity is the conversion of the Ca(2+)-releasing second messenger Ins(1,4,5)P(3) to Ins(1,3,4,5)P(4), an important factor in chromatin remodeling and also in Ca(2+)-release. This protein possesses several additional IPK activities pointing to a potential role as inositol phosphate multikinase. Interestingly, we have also identified three putative subdomains of histone deacetylase in this protein possibly linking InsP(x)- and acetylation-mediated transcription regulation. Furthermore, we examined the inhibitory potential of >40 polyphenolic substances against its kinase activity. Because of the important role of InsP(x)-induced Ca(2+)-release in the development of Plasmodium parasites, IPKs are interesting targets for novel antimalarial approaches.

The inositol 5-phosphatase SHIP1 is a nucleo-cytoplasmic shuttling protein and enzymatically active in cell nuclei.

The inositol 5-phosphatase SHIP1 is a negative regulator of signaling processes in hematopoietic cells. SHIP1 mediates its regulatory function after relocalization from the cytoplasm to the plasma membrane where it converts its substrate PI(3,4,5)P(3) to PI(3,4)P(2) thereby terminating PI3-kinase mediated signaling. In addition, SHIP1 converts Ins(1,3,4,5)P(4) to Ins(1,3,4)P(3) thereby regulating inositol phosphate metabolism. Here we report, that SHIP1 can be detected in nuclear puncta of Jurkat cells by confocal microscopy after expression of SHIP1 from a tetracycline inducible vector. SHIP1-containing nuclear puncta partially co-localize with FLASH, a multifunctional nuclear protein that has been linked to apoptotic signaling and transcriptional control. Nuclear localization was confirmed for endogenously expressed SHIP1 in the myeloid leukemia cell line TF1. In addition, enzymatically active SHIP1 was found in nuclear fractions of Jurkat cells with a similar specific activity as cytoplasmic SHIP1. Further analysis revealed that SHIP1 is a nucleocytoplasmic shuttling protein which is actively imported into and exported out of the nucleus. Nuclear import is mediated by two canonical nuclear localization signals (NLS) i.e. K(327)KSK and K(547)KLR. Mutational inactivation of each NLS motif inhibited nuclear import and reduced the proliferation of cells indicating a functional role of nuclear SHIP1 for cell growth. Our data indicate that SHIP1 is partly localized in the nucleus and suggest that SHIP1 plays a role for nuclear phosphoinositide and/or nuclear inositol phosphate signaling.

Subcellular localisation of human inositol 1,4,5-trisphosphate 3-kinase C: species-specific use of alternative export sites for nucleo-cytoplasmic shuttling indicates divergent roles of the catalytic and N-terminal domains.

The three isoforms of human Ins(1,4,5)P3 3-kinase (IP3K) show remarkable differences in their intracellular targeting. Whereas predominant targeting to the cytoskeleton and endoplasmic reticulum has been shown for IP3K-A and IP3K-B, rat IP3K-C shuttles actively between the nucleus and cytoplasm. In the present study we examined the expression and intracellular localisation of endogenous IP3K-C in different mammalian cell lines using an isoform-specific antibody. In addition, human IP3K-C, showing remarkable differences to its rat homologue in the N-terminal targeting domain, was tagged with EGFP and used to examine active transport mechanisms into and out of the nucleus. We found both a nuclear import activity residing in its N-terminal domain and a nuclear export activity sensitive to treatment with leptomycin B. Different from the rat isoform, an exportin 1-dependent nuclear export site of the human enzyme resides outside the N-terminal targeting domain in the catalytic enzyme domain. A phylogenetic survey of vertebrate IP3K sequences indicates that in each of the three isoforms a nuclear export signal has evolved in the catalytic domain either de novo (IP3K-A) or as a substitute for an earlier evolved corresponding N-terminal signal (IP3K-B and IP3K-C). In higher vertebrates, and in particular in primates, re-export of nuclear IP3K activity may be guaranteed by the mechanism discovered.

The human homologue of yeast ArgRIII protein is an inositol phosphate multikinase with predominantly nuclear localization.

The function of the transcription regulator ArgRIII in the expression of several genes involved in the metabolism of arginine in yeast has been well studied. It was previously reported that it is also an inositol phosphate multikinase and an important factor of the mRNA export pathway [reviewed by Shears (2000) Bioessays 22, 786-789]. In the present study we report the cloning of a full-length 1248-bp cDNA encoding a human inositol phosphate multikinase (IPMK). This protein has a calculated molecular mass of 47.219 kDa. Functionally important motifs [inositol phosphate-binding site, ATP-binding site, catalytically important SSLL (Ser-Ser-Leu-Leu) domain] are conserved between the human IPMK and yeast ArgRIII. Bacterially expressed protein demonstrated an inositol phosphate multikinase activity similar to that of yeast ArgRIII. Ins(1,4,5)P3 is phosphorylated at positions 3 and 6 up to Ins(1,3,4,5,6)P5. The human IPMK fused with a fluorescent protein tag is localized predominantly in the nucleus when transiently expressed in mammalian cells. A basic cluster in the protein's C-terminus is positively involved in nuclear targeting. These findings are consistent with the concept of a nuclear inositol phosphate signalling and phosphorylation pathway in mammalian cells.

Rat inositol 1,4,5-trisphosphate 3-kinase C is enzymatically specialized for basal cellular inositol trisphosphate phosphorylation and shuttles actively between nucleus and cytoplasm.

The calcium-liberating second messenger inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) is converted to inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4) by Ins(1,4,5)P3 3-kinases (IP3Ks) that add a fourth phosphate group to the 3-position of the inositol ring. Two isoforms of IP3Ks (named A and B) from different vertebrate species have been well studied. Recently the cloning and examination of a human full-length cDNA encoding a novel isoform, termed human IP3K-C (HsIP3K-C), has been reported. In the present study we report the cloning of a full-length cDNA encoding a rat homologue of HsIP3K-C with a unique mRNA expression pattern, which differs remarkably from the tissue distribution of HsIP3K-C. Of the rat tissues examined, rat IP3K-C (RnIP3K-C) is mainly present in heart, brain, and testis and shows the strongest expression in an epidermal tissue, namely tongue epithelium. RnIP3K-C has a calculated molecular mass of approximately 74.5 kDa and shows an overall identity of approximately 75% with HsIP3K-C. A bacterially expressed, enzymatically active and Ca2+-calmodulin-regulated fragment of this isoform displays remarkable enzymatic properties like a very low Km for Ins(1,4,5)P3 ( approximately 0.2 microm), substrate inhibition by high concentrations of Ins(1,4,5)P3, allosteric product activation by Ins(1,3,4,5)P4 in absence of Ca2+-calmodulin (Ka(app) 0.52 microm), and the ability to efficiently phosphorylate a second InsP3 substrate, inositol 2,4,5-trisphosphate, to inositol 2,4,5,6-tetrakisphosphate in the presence of Ins(1,3,4,5)P4. Furthermore, the RnIP3K-C fused with a fluorescent protein tag is actively transported into and out of the nucleus when transiently expressed in mammalian cells. A leucine-rich nuclear export signal and an uncharacterized nuclear import activity are localized in the N-terminal domain of the protein and determine its nucleocytoplasmic shuttling. These findings point to a particular role of RnIP3K-C in nuclear inositol trisphosphate phosphorylation and cellular growth.