Three phosphatidylinositol 4-kinase isoforms, PI4K 230, 92 and 55 have been cloned and sequenced allowing a much wider characterization than the previously employed enzymological typing into type II and III enzymes. PI4K 230 and 92 contain a highly conserved catalytic core, PI4K55 one with a much lower degree of similarity. Candidate kinase motifs, deduced from the protein kinase super family, are absolutely conserved in all isoforms. Kinase activities are described based on their sensitivity and reactivity towards wortmannin, phenylarsine oxide (PAO) and 5'-p-fluorosulfonylbenzoyladenosine (FSBA). Localization of all isoforms in the cell is reported. All enzymes contain nuclear localization and export sequence motifs (NLS and NES) leading to the expectation that they can be transferred to the nucleus. PI4K230 has been found in the nucleolus, PI4K92 in the nucleus, additionally further broadening the function of these enzymes. In the cytoplasm of neuronal cells, PI4K230 is distributed evenly on membranes that are ultra structurally cisterns of the rough endoplasmatic reticulum, outer membranes of mitochondria, multivesicular bodies, and are in close vicinity of synaptic contacts. PI4K92 is functionally characterized as a key enzyme regulating Golgi disintegration/reorganization during mitosis probably via phosphorylation by cyclin-dependent kinases on well-defined sites. PI4K55 is involved in the production of second messengers, diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (InsP3) at the plasma membrane, moreover, in the endocytotic pathway in the cytoplasm.

(2001) Eur. J. Biochem. 268, 2099 -2106). Characterization proved three of them (anti-pSer-294, anti-pSer-496, and anti-pThr-504 antibody) to be highly specific, recognizing solely PI4K92 phosphorylated at these sites, respectively. Indirect immunofluorescence reveals that PI4K92 phosphorylated on Ser-294 localizes exclusively at the Golgi. The enzyme phosphorylated on Ser-496 and Thr-504 is detected in nuclear speckles. Phosphorylation of Ser-294 on PI4K92 increases the lipid kinase activity and thus serves better in maintaining Golgi function and morphology (compare Hausser, A., Storz, P., Martens, S., Link, G., Toker, A., and Pfizenmaier, K. (2005) Nat. Cell Biol. 7, 880 -886). Microinjection of anti-pSer-496, but not of antipSer-294 or anti-pThr-504 antibody, into the cytoplasm or into the nucleus of HS68 cells leads to development of hotspots, probably representing aggregated PI4K92, and in later stages, cells become apoptotic and finally die. The association of phosphorylated PI4K92 with nuclear speckles is dynamic and follows the morphological alteration of speckles upon inhibition of mRNA transcription with ␣-amanitin. Overexpressed PI4K92 phosphorylated on Ser-294 is not transported to the nucleus, and that phosphorylated on Ser-496 is found in the nucleus and mislocalized at the Golgi complex. We conclude that nuclear phosphatidylinositol 4-phosphate, and consequently, synthesis of polyphosphoinositides are required for a correct nuclear function.In the past 20 years, evidence has been accumulated for the presence of intranuclear polyphosphoinositides that form the components of a phosphoinositide-phospholipase C cycle (for review, see Ref. 1), which is independent from that at the plasma and other cytoplasmic membranes (2, 3). In a recent study, the pleckstrin homology domain of phospholipase C ␦1 has been used as a probe to show that phosphatidylinositol (4,5)-bisphosphate (PtdIns(4,5)P 2 ) 2 is present intranuclear and is not detectable in the nuclear membrane (4). Alternatively, a monoclonal antibody used for indirect immunofluorescence staining procedures reveals PtdIns(4,5)P 2 in distinct subnuclear domains, identified as "nuclear speckles" (Refs. 5 and 6, and see also Refs. 7 and 8). Most probably, these latter polyphosphoinositides are not present in membranes but are associated with proteins (2, 3, 9). In principal, it explains why they are not extractable with Triton X-100, which, however, completely removes the nuclear double membrane (9).Several distinct functions are well known for polyphosphoinositides in the cytoplasm in addition to their role as precursor for the generation of second messengers, inositol (1,4,5)-trisphosphate and diacylglycerol. In the middle of the 1990s, it was revealed that PtdIns(4,5)P 2 itself may serve as a regulator or effector molecule on its own right (for review, see ref. 10). In many cell types, stimulation with growth factors or hormones results in marked changes in cellular morphology. This alteration is thought to be due to a rearrangement of actin via inf...

Three phosphatidylinositol 4-kinase isoforms, PI4K 230, 92 and 55 have been cloned and sequenced allowing a much wider characterization than the previously employed enzymological typing into type II and III enzymes. PI4K 230 and 92 contain a highly conserved catalytic core, PI4K55 one with a much lower degree of similarity. Candidate kinase motifs, deduced from the protein kinase super family, are absolutely conserved in all isoforms. Kinase activities are described based on their sensitivity and reactivity towards wortmannin, phenylarsine oxide (PAO) and 5'-p-fluorosulfonylbenzoyladenosine (FSBA). Localization of all isoforms in the cell is reported. All enzymes contain nuclear localization and export sequence motifs (NLS and NES) leading to the expectation that they can be transferred to the nucleus. PI4K230 has been found in the nucleolus, PI4K92 in the nucleus, additionally further broadening the function of these enzymes. In the cytoplasm of neuronal cells, PI4K230 is distributed evenly on membranes that are ultra structurally cisterns of the rough endoplasmatic reticulum, outer membranes of mitochondria, multivesicular bodies, and are in close vicinity of synaptic contacts. PI4K92 is functionally characterized as a key enzyme regulating Golgi disintegration/reorganization during mitosis probably via phosphorylation by cyclin-dependent kinases on well-defined sites. PI4K55 is involved in the production of second messengers, diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (InsP3) at the plasma membrane, moreover, in the endocytotic pathway in the cytoplasm.