Phosphatidylinositol 5-phosphate (PtdIns5P), a novel inositol lipid, has been shown to be the major substrate for the type II PtdInsP kinases (PIPkins) [Rameh et al. (1997) Nature 390, 192^196]. A PtdInsP fraction was prepared from cell extracts by neomycin chromatography, using a protocol devised to eliminate the interaction of acidic solvents with plasticware, since this was found to inhibit the enzyme. The PtdIns5P in this fraction was measured by incubating with [Q Q-32 P]ATP and recombinant PIPkin IIK K, and quantifying the radiolabelled PtdInsP 2 formed. This assay was used on platelets to show that during 10 min stimulation with thrombin, the mass level of PtdIns5P increases, implying the existence of an agoniststimulated synthetic mechanism. ß

Lowe syndrome and Dent-2 disease are caused by mutation of the inositol 5-phosphatase OCRL1. Despite our increased understanding of the cellular functions of OCRL1, the underlying basis for the renal tubulopathy seen in both human disorders, of which a hallmark is low molecular weight proteinuria, is currently unknown. Here, we show that deficiency in OCRL1 causes a defect in endocytosis in the zebrafish pronephric tubule, a model for the mammalian renal tubule. This coincides with a reduction in levels of the scavenger receptor megalin and its accumulation in endocytic compartments, consistent with reduced recycling within the endocytic pathway. We also observe reduced numbers of early endocytic compartments and enlarged vacuolar endosomes in the sub-apical region of pronephric cells. Cell polarity within the pronephric tubule is unaffected in mutant embryos. The OCRL1-deficient embryos exhibit a mild ciliogenesis defect, but this cannot account for the observed impairment of endocytosis. Catalytic activity of OCRL1 is required for renal tubular endocytosis and the endocytic defect can be rescued by suppression of PIP5K. These results indicate for the first time that OCRL1 is required for endocytic trafficking in vivo, and strongly support the hypothesis that endocytic defects are responsible for the renal tubulopathy in Lowe syndrome and Dent-2 disease. Moreover, our results reveal PIP5K as a potential therapeutic target for Lowe syndrome and Dent-2 disease.

Type II phosphatidylinositol phosphate kinases (PIPkins) have recently been found to be primarily phosphatidylinositol 5-phosphate 4-kinases, and their physiological role remains unclear. We have previously shown that a Type II PIPkin [isoform(s) unknown], is localized partly in the nucleus [Divecha, Rhee, Letcher and Irvine (1993) Biochem. J. 289, 617-620], and here we show, by transfection of HeLa cells with green-fluorescent-protein-tagged Type II PIPkins, that this is likely to be the Type IIbeta isoform. Type IIbeta PIPkin has no obvious nuclear localization sequence, and a detailed analysis of the localization of chimaeras and mutants of the alpha (cytosolic) and beta PIPkins shows that the nuclear localization requires the presence of a 17-amino-acid length of alpha-helix (alpha-helix 7) that is specific to the beta isoform, and that this helix must be present in its entirety, with a precise orientation. This resembles the nuclear targeting of the HIV protein Vpr, and Type IIbeta PIPkin is apparently therefore the first example of a eukaryotic protein that uses the same mechanism.

In this study we describe the purification and sequencing of the C isoform of platelet PtdIns4P 5-kinase. Subsequently a cDNA was isolated from a human circulating-leucocyte library, which when sequenced was shown to contain all of the peptides identified in the purified protein. In addition, expression of this cDNA in bacteria led to the production of a protein which was recognized by specific monoclonal antibodies raised to the bovine brain enzyme [Brooksbank, Hutchings, Butcher, Irvine and Divecha (1993) Biochem. J. 291, 77-82] and also led to the appearance of PtdIns4P 5-kinase activity in the bacterial lysates. Interestingly, the cDNA showed no similarity to any of the previously cloned inositide kinases. A search of the DNA databases showed that two proteins from Saccharomyces cerevisiae shared close similarity to this enzyme, one of which, the mss4 gene product, has been implicated in the yeast inositol lipid pathway. These data suggest that the PtdIns4P 5-kinases are a new family of inositide kinases unrelated to the previously cloned phosphoinositide 3/4-kinases.

Thrombin‐stimulated aggregation of human platelets promotes an increase in the phosphatidylinositol 4‐phosphate (PtdIns 4‐P) 5‐kinase (PIPkin) activity in the cytoskeleton. This phenomenon is associated with translocation of PIPkin isoform C to the cytoskeleton and with an increase in the amount of phosphatidylinositol bisphosphate (PtdInsP2) bound to the cytoskeletal pellet. All three of these effects are prevented if the platelets are not stirred or if RGD‐containing peptides are present, demonstrating that they require integrin activation. All three are also abolished by pretreatment with okadaic acid, which also prevents the aggregation‐dependent translocation of pp60(c‐src) to the cytoskeleton. The results point to the existence of a cytoskeletally associated PtdInsP2 pool under the control of integrin‐mediated signals that act via PIPkin C and suggest that a common, okadaic acid‐sensitive mechanism may underlie the aggregation‐dependent translocation of certain signalling molecules to the platelet cytoskeleton.