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.
The formation of distant metastases is a complex process involving escape of cancer cells from the primary tumor, dissemination to distant organs, and finally re-colonization and expansion (1). For metastatic dissemination, the cancer cell must acquire the ability to migrate, which is associated with cytoskeletal re-arrangements. Migrating cells extend actinbased filopodia and lamellipodia at the leading edge. To initiate this process, a local formation of F-actin is required, which can be mediated by actin nucleating (e.g. Arp2/3 and formins (2)), F-actin bundling (e.g. fascin (3)), and by F-actin cross-linking proteins (e.g. filamins (4)). The actin-severing proteins ADF/ cofilin and gelsolin depolymerize F-actin and thus increase actin turnover (5). The balance between stimulation of actinpolymerizing proteins and actin-depolymerizing proteins is tightly regulated by distinct signaling pathways (e.g. phospholipase C and phosphoinositide 3-kinase (6 -8)). As activation of these pathways can also result in induction of proliferation, "fine-tuning" of continuous signal inputs determines the cellular response. This fine-tuning is mediated by various small molecules, including calcium, cyclic AMP, phosphatidylinositol phosphates, and inositol phosphates. Among the inositides, membranous phosphatidylinositol 4,5-bisphosphate plays a central role in the control of migration as it regulates the activity of cofilin, gelsolin, and profilin (9, 10) and serves as a substrate for the production of the calcium-mobilizing second messenger inositol 1,4,5-trisphosphate (Ins(1,4,5)P 3 ).2 Phospholipase C-mediated hydrolysis of phosphatidylinositol 4,5-bisphosphate increases Ins(1,4,5)P 3 levels and subsequent calcium release from the endoplasmic reticulum. Calcium plays an important role in cell migration because calcium transients activate gelsolin and indirectly ADF/cofilin (9, 10).Inositol 1,4,5-trisphosphate 3-kinase isoenzymes (ITPKA, ITPKB, and ITPKC) metabolize Ins(1,4,5)P 3 to inositol-1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P 4 ), and thus regulate Ins(1,4,5)P 3 -induced calcium signals (11). The ITPK isoenzymes are highly conserved in their catalytically active C-terminal domains but show large differences in their N-terminal regulatory domains mediating mainly cellular targeting. The isoenzymes differ in subcellular localization and tissue expression patterns. ITPKB and ITPKC mRNAs are ubiquitously expressed, whereas mRNA of ITPKA was only identified in neurons and testis (12). In neurons, ITPKA was shown to be targeted to F-actin via an N-terminal actin binding domain (amino acids 1-66 (13)) and was suggested to be relevant for long term potentiation and spatial learning (14,15
InsP(6) [Ins(1,2,3,4,5,6)P6; phytate] is the most abundant inositol phosphate in mammalian cells with cytosolic/nuclear concentrations of up to 50 μM. We noticed that InsP6 in culture medium at a concentration of ≤50 μM significantly stimulates H1299 tumour cell growth, whereas larger concentrations of InsP6 inhibit growth. A detailed study of the fate of 30 μM InsP6 added to H199 cells revealed a major fraction of InsP6 initially precipitates as cell-surface metal complexes, but becomes slowly re-solubilized by extracellular dephosphorylation first to InsP3 isomers and subsequently to free myo-inositol. The precipitated metal-InsP6 complex is endocytosed in a receptor-independent but intact-glycocalyx-dependent manner and appears in lysosomes, where it is immediately dephosphorylated to Ins(1,2,4,5,6)P5 and very slowly to free inositol. By RNA knockdown, we identified secreted and lysosome targeted MINPP1 (multiple inositol-polyphosphate phosphatase 1), the mammalian 3-phytase, to be essentially involved both in extracellular and in lysosomal InsP6 dephosphorylation. The results of the present study indicate that tumour cells employ this enzyme to utilize the micronutrients myo-inositol and metal-phosphate when encountering extracellular InsP6 and thus to enhance their growth potential.
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