Human phosphatidylinositol 4-kinase isoform PI4K92. Expression of the recombinant enzyme and determination of multiple phosphorylation sites.

Human phosphatidylinositol 4-kinase, isoform PI4K92, was expressed as His6 tagged protein in Sf9 cells reaching a level of approximately 5% of cellular protein. The enzyme can be purified nearly to homogeneity in a single step by absorption/desorption on Ni/nitriloacetic acid agarose magnetic beads. High Km values in the millimolar range for ATP and PtdIns as well as only a moderate inhibition by adenosine and a sensitivity to Wortmannin (IC50 approximately 300 nM) characterize the enzyme as a type 3 PI4K. The enzyme produces PtdIns4P as product. The isolated enzyme is a phosphoprotein, additionally phosphate is incorporated by incubation with ATP/Mg or ATP/Mn. Phosphorylation sites were mapped by MALDI-MS and LC-MS/MS at the following positions: S258, T263, S266, S277, S294, T423, S496, T504. Accordingly, a stretch of 81 amino acids between the common and the C-terminal catalytic domain was designated phosphorylation domain.     

Mammalian phosphatidylinositol 4-kinases

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.     

Inhibition of phosphatidylinositol 4-kinase results in a significant reduced respiratory burst in formyl-methionyl-leucyl-phenylalanine-stimulated human neutrophils

The effects of phenylarsine oxide and a monoclonal antibody directed against type II phosphatidylinositol 4-kinase (PI4K) on the N-formyl-methionyl-leucyl-phenylalanine (fMLP)-stimulated respiratory burst and the PI4K activity in neutrophils were investigated. Fluorescence microscopic imaging showed that the antibody labeled with IANBD amide (N,N'-dimethyl-N-(iodoacetyl)-N'-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)ethylenediamine) could enter into the cytosol possibly by endocytosis. It was found that the antibody inhibited the fMLP-stimulated respiratory burst but had little effect on the phorbol myristate acetate-activated respiratory burst in neutrophils, whereas phenylarsine oxide inhibited both. It was found that even at higher concentration, the antibody could not completely inhibit the cell response. Using cells preincubated with human immunoglobulin G of the same concentration as the control, the maximal inhibition of the fMLP-stimulated respiratory burst by the antibody against type II PI4K was found to be about 70%, whereas the PI4K activity was inhibited by only about 40%. The discrepancy in depressing the cell response and the enzyme activity may be the result of depletion of the phosphatidylinositol 4,5-bisphosphate or phosphatidylinositol 3,4,5-trisphosphate pools during the incubation of cells with the antibody. Both the 40% inhibition of PI4K activity and 70% depression of the respiratory burst by the type II PI4K antibody may imply that at least 40% of the phosphatidylinositol 4,5-biphosphate was synthesized promptly by all forms of PI4K and phosphatidylinositol-4-phosphate 5-kinase in the fMLP-activated cells. The results suggest that PI4K plays a central role in either phospholipase C or PI3K signaling and that PI3K, PI4K, and phosphatidylinositol 4-phosphate 5-kinase must be considered as an integrated family for the phosphatidylinositol 3,4,5-trisphosphate initiated signaling.     

Nucleolar localization of phosphatidylinositol 4-kinase PI4K230 in various mammalian cells.

BACKGROUND: Previous immunohistochemical investigations could not detect PI4K230, an isoform of mammalian phosphatidylinositol 4-kinases (also called type III alpha), in the nucleus and nucleolus of cells in spite of its predicted nuclear localization signals. METHODS: Immunofluorescent detection of PI4K230 and other PI4K isoforms was performed on formaldehyde (PFA) or ethanol fixed cells and rat brain cryosections. Costaining with nucleolin and the effect of siRNA, Triton X-100, DNase, and RNase treatments were also tested to determine the localization of PI4K230. RESULTS: PI4K230 gives a prominent signal in the nucleolus of ethanol fixed rat brain cryosections and of several cell types in addition to its presence in the nucleus and cytoplasm. The PI4K230 immunoreactivity of the nucleolus is masked in PFA fixed cells, but it can be restored by treatment of PFA fixed cells with hot wet citrate buffer or by washing the cryosections with PBS prior to PFA fixation. Nucleolar PI4K230 occurs in a Triton X-100 resistant complex. Treatment of COS-7 cells with siRNA targeting PI4K230 and permeabilized B50 cells with DNase or RNase results in the loss of PI4K230 signal from the nucleolus. CONCLUSION: These experiments suggest the participation of PI4K230 in a DNase and RNase sensitive complex with a unique localization and function in the nucleolus.     

Mimicking phosphorylation of the small heat-shock protein alphaB-crystallin recruits the F-box protein FBX4 to nuclear SC35 speckles.

The mammalian small heat shock protein alphaB-crystallin can be phosphorylated at three different sites, Ser19, Ser45 and Ser59. We compared the intracellular distribution of wild-type, nonphosphorylatable and all possible pseudophosphorylation mutants of alphaB-crystallin by immunoblot and immunocytochemical analyses of stable and transiently transfected cells. We observed that pseudophosphorylation at two (especially S19D/S45D) or all three (S19D/S45D/S59D) sites induced the partial translocation of alphaB-crystallin from the detergent-soluble to the detergent-insoluble fraction. Double immunofluorescence studies showed that the pseudophosphorylation mutants localized in nuclear speckles containing the splicing factor SC35. The alphaB-crystallin mutants in these speckles were resistant to mild detergent treatment, and also to DNase I or RNase A digestion, indicating a stable interaction with one or more speckle proteins, not dependent on intact DNA or RNA. We further found that FBX4, an adaptor protein of the ubiquitin-protein isopeptide ligase SKP1/CUL1/F-box known to interact with pseudophosphorylated alphaB-crystallin, was also recruited to SC35 speckles when cotransfected with the pseudophosphorylation mutants. Because SC35 speckles also react with an antibody against alphaB-crystallin endogenously phosphorylated at Ser45, our findings suggest that alphaB-crystallin has a phosphorylation-dependent role in the ubiquitination of a component of SC35 speckles.     

Distinct Golgi populations of phosphatidylinositol 4-phosphate regulated by phosphatidylinositol 4-kinases

Phosphatidylinositol 4-phosphate (PI4P) regulates biosynthetic membrane traffic at multiple steps and differentially affects the surface delivery of apically and basolaterally destined proteins in polarized cells. Two phosphatidylinositol 4-kinases (PI4Ks) have been localized to the Golgi complex in mammalian cells, type III PI4Kbeta (PI4KIIIbeta) and type II PI4Kalpha (PI4KIIalpha). Here we report that PI4KIIIbeta and PI4KIIalpha localize to discrete subcompartments of the Golgi complex in Madin-Darby canine kidney (MDCK) cells. PI4KIIIbeta was enriched in early Golgi compartments, whereas PI4KIIalpha colocalized with markers of the trans-Golgi network (TGN). To understand the temporal and spatial control of PI4P generation across the Golgi complex, we quantitated the steady state distribution of a fluorescent PI4P-binding domain relative to cis/medial Golgi and TGN markers in transiently transfected MDCK cells. The density of the signal from this PI4P reporter was roughly 2-fold greater in the early Golgi compartments compared with that of the TGN. Furthermore, this ratio could be modulated in vivo by overexpression of catalytically inactive PI4KIIIbeta and PI4KIIalpha or in vitro by the PI4KIIIbeta inhibitor wortmannin. Our data suggest that both PI4KIIIbeta and PI4KIIalpha contribute to the compartmental regulation of PI4P synthesis within the Golgi complex. We discuss our results with respect to the kinetic effects of modulating PI4K activity on polarized biosynthetic traffic in MDCK cells.     

Multiple roles for phosphatidylinositol 4-kinase in biosynthetic transport in polarized Madin-Darby canine kidney cells.

Phosphatidylinositols (PI) play important roles in regulating numerous cellular processes including cytoskeletal organization and membrane trafficking. The control of PI metabolism by phosphatidylinositol kinases has been the subject of extensive investigation; however, little is known about how phosphatidylinositol kinases regulate traffic in polarized epithelial cells. Because phosphatidylinositol 4-kinase (PI4K)-mediated phosphatidylinositol 4-phosphate (PI(4)P) production has been suggested to regulate biosynthetic traffic in yeast and mammalian cells, we have examined the role of PI4Kbeta in protein delivery in polarized MDCK cells, at different levels of the biosynthetic pathway. Expression of wild type PI4Kbeta had no effect on the rate of transport of influenza hemagglutinin (HA) through the Golgi complex, but inhibited the rate of trans-Golgi network (TGN)-to-cell surface delivery of this protein. By contrast, expression of dominant-negative, kinase-dead PI4Kbeta (PI4Kbeta(D656A)) inhibited intra-Golgi transport but stimulated TGN-to-cell surface delivery of HA. Moreover, expression of PI4Kbeta(D656A) significantly increased the solubility in cold Triton X-100 of HA staged in the TGN, suggesting that altered association of HA with lipid rafts may be responsible for the enhanced transport rate. Both wild type and kinase-dead PI4Kbeta inhibited basolateral delivery of vesicular stomatitis virus G protein, suggesting an effector function for PI4Kbeta in the regulation of basolateral traffic. Thus, by contrast with the observed requirement for PI4Kbeta activity and PI(4)P for efficient transport in yeast, our data suggest that changes in PI(4)P levels can stimulate and inhibit Golgi to cell surface delivery in mammalian cells.     

The protein kinase C-dependent phosphorylation of serine 166 is controlled by the phospholipid species bound to the phosphatidylinositol transfer protein alpha

The charge isomers of bovine brain PI-TPalpha (i.e. PI-TPalphaI containing a phosphatidylinositol (PI) molecule and PI-TPalphaII containing a phosphatidylcholine (PC) molecule) were phosphorylated in vitro by rat brain protein kinase C (PKC) at different rates. From the double-reciprocal plot, it was estimated that the V(max) values for PI-TPalphaI and II were 2.0 and 6.0 nmol/min, respectively; the K(m) values for both charge isomers were about equal, i.e. 0.7 micrometer. Phosphorylation of charge isomers of recombinant mouse PI-TPalpha confirmed that the PC-containing isomer was the better substrate. Phosphoamino acid analysis of in vitro and in vivo (32)P-labeled PI-TPalphas showed that serine was the major site of phosphorylation. Degradation of (32)P-labeled PI-TPalpha by cyanogen bromide followed by high pressure liquid chromatography and sequence analysis yielded one (32)P-labeled peptide (amino acids 104-190). This peptide contained Ser-148, Ser-152, and the consensus PKC phosphorylation site Ser-166. Replacement of Ser-166 with an alanine residue confirmed that indeed this residue was the site of phosphorylation. This mutation completely abolished PI and PC transfer activity. This was also observed when Ser-166 was replaced with Asp, implying that this is a key amino acid residue in regulating the function of PI-TPalpha. Stimulation of NIH3T3 fibroblasts by phorbol ester or platelet-derived growth factor induced the rapid relocalization of PI-TPalpha to perinuclear Golgi structures concomitant with a 2-3-fold increase in lysophosphatidylinositol levels. This relocalization was also observed for Myc-tagged wtPI-TPalpha expressed in NIH3T3 cells. In contrast, the distribution of Myc-tagged PI-TPalpha(S166A) and Myc-tagged PI-TPalpha(S166D) were not affected by phorbol ester, suggesting that phosphorylation of Ser-166 was a prerequisite for the relocalization to the Golgi. A model is proposed in which the PKC-dependent phosphorylation of PI-TPalpha is linked to the degradation of PI.     

Courier service for phosphatidylinositol: PITPs deliver on demand

Phosphatidylinositol is the parent lipid for the synthesis of seven phosphorylated inositol lipids and each of them play specific roles in numerous processes including receptor-mediated signalling, actin cytoskeleton dynamics and membrane trafficking. PI synthesis is localised to the endoplasmic reticulum (ER) whilst its phosphorylated derivatives are found in other organelles where the lipid kinases also reside. Phosphorylation of PI to phosphatidylinositol (4,5) bisphosphate (PI(4,5)P₂) at the plasma membrane and to phosphatidylinositol 4-phosphate (PI4P) at the Golgi are key events in lipid signalling and Golgi function respectively. Here we review a family of proteins, phosphatidylinositol transfer proteins (PITPs), that can mobilise PI from the ER to provide the substrate to the resident kinases for phosphorylation. Recent studies identify specific and overlapping functions for the three soluble PITPs (PITPα, PITPβ and PITPNC1) in phospholipase C signalling, neuronal function, membrane trafficking, viral replication and in cancer metastases.     

Phosphoinositide-dependent kinase-2 is a distinct protein kinase enriched in a novel cytoskeletal fraction associated with adipocyte plasma membranes

By recombining subcellular components of 3T3-L1 adipocytes in a test tube, early insulin signaling events dependent on phosphatidylinositol 3-kinase (PI 3-kinase) were successfully reconstituted, up to and including the phosphorylation of glycogen synthase kinase-3 by the serine/threonine kinase, Akt (Murata, H., Hresko, R.C., and Mueckler, M. (2003) J. Biol. Chem. 278, 21607-21614). Utilizing the advantages provided by a cell-free methodology, we characterized phosphoinositide-dependent kinase 2 (PDK2), the putative kinase responsible for phosphorylating Akt on Ser-473. Immunodepleting cytosolic PDK1 from an in vitro reaction containing plasma membrane and cytosol markedly inhibited insulin-stimulated phosphorylation of Akt at the PDK1 site (Thr-308) but had no effect on phosphorylation at the PDK2 site (Ser-473). In contrast, PDK2 activity was found to be highly enriched in a novel cytoskeletal subcellular fraction associated with plasma membranes. Akt isoforms 1-3 and a kinase-dead Akt1 (K179A) mutant were phosphorylated in a phosphatidylinositol 3,4,5-trisphosphate-dependent manner at Ser-473 in an in vitro reaction containing this novel adipocyte subcellular fraction. Our data indicate that this PDK2 activity is the result of a kinase distinct from PDK1 and is not due to autophosphorylation or transphosphorylation of Akt.     

Cyclin-dependent kinase-5 is involved in neuregulin-dependent activation of phosphatidylinositol 3-kinase and Akt activity mediating neuronal survival

The phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway plays an important role in mediating survival signals in wide variety of neurons and cells. Recent studies show that Akt also regulates metabolic pathways to regulate cell survival. In this study, we reported that cyclin-dependent kinase-5 (Cdk5) regulates Akt activity and cell survival through the neuregulin-mediated PI 3-kinase signaling pathway. We found that brain extracts of Cdk5-/-mice display a lower PI 3-kinase activity and phosphorylation of Akt compared with that in wild type mice. Moreover, we demonstrated that Cdk5 phosphorylated Ser-1176 in the neuregulin receptor ErbB2 and phosphorylated Thr-871 and Ser-1120 in the ErbB3 receptor. We identified the Ser-1120 sequence RSRSPR in ErbB3 as a novel phosphorylation consensus sequence of Cdk5. Finally, we found that Cdk5 activity is involved in neuregulin-induced Akt activity and neuregulin-mediated neuronal survival. These findings suggest that Cdk5 may exert a key role in promoting neuronal survival by regulating Akt activity through the neuregulin/PI 3-kinase signaling pathway.     

Protein kinase C phosphorylation of desmin at four serine residues within the non-alpha-helical head domain

We reported that phosphorylation by either cAMP-dependent protein kinase or protein kinase C (Ca2+/phospholipid-dependent enzyme) in vitro induces disassembly of the desmin filaments (Inagaki, M., Gonda, Y., Matsuyama, M., Nishizawa, K., Nishi, Y., and Sato, C. (1988) J. Biol. Chem. 263, 5970-5978). For this subunit protein, Ser-29, Ser-35, and Ser-50 within the non-alpha-helical head domain were shown to be the sites of phosphorylation for cAMP-dependent protein kinase (Geisler, N., and Weber, K. (1988) EMBO J. 7, 15-20). In the present work, we identified the sites of desmin phosphorylated in vitro by other protein kinase which affects the filament structure. The protein kinase C-phosphorylated desmin was hydrolyzed with trypsin, and the phosphorylated peptides were isolated by reverse-phase chromatography. Sequential analysis of the purified phosphopeptides, together with the known primary sequence, revealed that Ser-12, Ser-29, Ser-38, and Ser-56 were phosphorylated by protein kinase C. All four sites are located within the non-alpha-helical head domain of desmin. Ser-12, Ser-38, and Ser-56, specifically phosphorylated by protein kinase C, have arginine residues at the carboxyl-terminal side (Arg-14, Arg-42, and Arg-59, respectively). Ser-29 phosphorylated by both protein kinase C and cAMP-dependent protein kinase has arginine residues at the amino and carboxyl termini (Arg-27 and Arg-33). These findings support the view that the head domain-specific phosphorylation strongly influences desmin filament structure; however, each protein kinase differed with regard to site recognition on this domain.     

Coordination of Golgi functions by phosphatidylinositol 4-kinases

Phosphatidylinositol 4-kinases (PI4Ks) regulate vesicle-mediated export from the Golgi apparatus via phosphatidylinositol 4-phosphate (PtdIns4P) binding effector proteins that control vesicle budding reactions and regulate membrane dynamics. Evidence has emerged from the characterization of Golgi PI4K effectors that vesicle budding and lipid dynamics are tightly coupled via a regulatory network that ensures that the appropriate membrane composition is established before a transport vesicle buds from the Golgi. An important hub of this network is protein kinase D, which regulates the activity of PI4K and several PtdIns4P effectors that control sphingolipid and sterol content of Golgi membranes. Other newly identified PtdIns4P effectors include Vps74/GOLPH3, a phospholipid flippase called Drs2 and Sec2, a Rab guanine nucleotide exchange factor (GEF). These effectors orchestrate membrane transformation events facilitating vesicle formation and targeting. In this review, we discuss how PtdIns4P signaling is integrated with membrane biosynthetic and vesicle budding machineries to potentially coordinate these crucial functions of the Golgi apparatus.     

Phosphatidylinositol 4-phosphate is required for translation initiation in Saccharomyces cerevisiae

The small natural product wortmannin inhibits protein synthesis by modulating several phosphatidylinositol (PI) metabolic pathways. A primary target of wortmannin in yeast is the plasma membrane-associated PI 4-kinase (PI4K) Stt4p, which is required for actin cytoskeleton organization. Here we show that wortmannin treatment or inactivation of Stt4p, but not disorganization of the actin cytoskeleton per se, leads to a rapid attenuation of translation initiation. Interestingly, inactivation of Pik1p, a wortmannin-insensitive, functionally distinct PI4K, implicated in the regulation of Golgi functions and secretion, also results in severe translation initiation defects with a marked increase of the phosphorylation of the translation initiation factor eIF2alpha. Because wortmannin largely phenocopies the effects of rapamycin (e.g. it triggers nuclear accumulation of Gln3p), it likely also inhibits the PI kinase-related, target of rapamycin (TOR) kinases. Importantly, however, neither inactivation of Stt4p nor Pik1p significantly affects TOR-controlled readouts other than translation initiation, indicating that these PI4Ks do not simply function upstream of TOR. Together, our results reveal the existence of a novel translation initiation control mechanism in yeast that is tightly coupled to the synthesis of distinct PI4P pools.     

Protein kinase D regulates vesicular transport by phosphorylating and activating phosphatidylinositol-4 kinase IIIbeta at the Golgi complex

Protein kinase D (PKD) regulates the fission of vesicles originating from the trans-Golgi network. We show that phosphatidylinositol 4-kinase IIIbeta (PI4KIIIbeta) - a key player in the structure and function of the Golgi complex - is a physiological substrate of PKD. Of the three PKD isoforms, only PKD1 and PKD2 phosphorylated PI4KIIIbeta at a motif that is highly conserved from yeast to humans. PKD-mediated phosphorylation stimulated lipid kinase activity of PI4KIIIbeta and enhanced vesicular stomatitis virus G-protein transport to the plasma membrane. The identification of PI4KIIIbeta as one of the PKD substrates should help to reveal the molecular events that enable transport-carrier formation.