Phosphatidylinositol-4-phosphate 5-kinases act downstream of surface receptors

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 signaling molecules to the platelet cytoskeleton (Hinchliffe, 1996).

Substitution of phenylalanine for tyrosine at codon 809 (Y809F) of the human colony-stimulating factor 1 (CSF-1) receptor (CSF-1R) impairs ligand-stimulated tyrosine kinase activity, prevents induction of c-MYC and cyclin D1 genes, and blocks CSF-1-dependent progression through the G1 phase of the cell cycle. An unbiased genetic screen was designed to isolate genes that restore the ability of CSF-1 to stimulate growth in cells that express mutant CSF-1R (Y809F). This screen led to identification of a truncated form of the murine type Ibeta phosphatidylinositol 4-phosphate 5-kinase (mPIP5K-Ibeta). This truncated protein lacks residues 1 to 238 of mPIP5K-Ibeta and is catalytically inactive. When cells expressing CSF-1R (Y809F) are transfected with mPIP5K-Ibeta (delta1-238), CSF-1-dependent induction of c-MYC and cyclin D1 is restored and ligand-dependent cell proliferation is sustained. CSF-1 normally triggers the rapid disappearance of CSF-1R (Y809F) from the cell surface; however, transfection of cells with mPIP5K-Ibeta (delta1-238) stabilizes CSF-1R (Y809F) expression on the cell surface, resulting in elevated levels of ligand-activated CSF-1R (Y809F). These results suggest a role for PIP5K-Ibeta in receptor endocytosis and that the truncated enzyme compensates for a mitogenically defective CSF-1R by interfering with this process (Davis, 1997).

Tumor necrosis factor-alpha (TNF-alpha) binding to its receptors leads to a diversity of biological responses. The actions of TNF are the result of the interaction of cytoplasmic proteins that bind directly to the intracellular domains of the two TNF receptors, p55 and p75. A novel interaction is reported between the juxtamembrane region of the p55 TNF receptor and a newly discovered 47-kDa isoform of phosphatidylinositol-4-phosphate 5-kinase (PIP5K), a member of the enzyme family that generates the key signaling messenger, phosphatidylinositol 4,5-bisphosphate. The interaction is found to be specific for the p55 TNF receptor and is not observed with the p75 TNF receptor, the Fas antigen, or the p75 neurotrophin receptor, which are other members of the TNF receptor superfamily. In vitro experiments using recombinant fusion proteins verify the authenticity of the interaction between the p55 receptor and PIP5KIIbeta, a new isoform of PIP5K, but not the previously identified 53-kDa PIP5KIIalpha. Treatment of HeLa cells with TNF-alpha results in an increased PIP5K activity. These results indicate that phosphatidylinositol turnover may be linked to stimulation of the p55 TNF receptor and suggest that a subset of TNF responses may result from the direct association of PIP5KIIbeta with the p55 TNF receptor (Castellino, 1997).

Phosphatidylinositol-5-phosphate 4-kinase (PIP4K) is required for the production of phosphoinositol-4,5-hisphosphate (PIP2), which has been closely associated with growth factor signalling. The possibility that phosphoinositide kinases may be take part in signal transduction through interactions with the epidermal growth factor (EGF) receptor and the ErbB family of tyrosine kinase receptors was tested. Interactions of the Type IIbeta isoform of PIP4K were observed with the EGF receptor family members in a number of diverse cell lines, including A431, PC12 and MCF7 cells but not with the N6F TrkA receptor. Co-immunoprecipitation experiments indicate that PIP4K interacts with not only the EGF receptor, but also selectively with members of the ErbB tyrosine kinase family. These results demonstrate another enzyme substrate for EGF receptors that facilitates the production of phosphoinositides at the cell membrane (Castellino, 1999).

Subcellular localization of phosphatidylinositol-4-phosphate 5-kinases

Phosphoinositide signal transduction pathways in nuclei use enzymes that are indistinguishable from their cytosolic analogues. Distinct phosphatidylinositol phosphate kinases (PIPKs), the type I and type II isoforms, are concentrated in nuclei of mammalian cells. The cytosolic and nuclear PIPKs display comparable activities toward the substrates phosphatidylinositol 4-phosphate and phosphatidylinositol 3-phosphate. Indirect immunofluorescence revealed that these kinases are associated with distinct subnuclear domains, identified as 'nuclear speckles', which also contained pre-mRNA processing factors. A pool of nuclear phosphatidylinositol bisphosphate (PIP2), the product of these kinases, was also detected at these same sites by monoclonal antibody staining. The localization of PIPKs and PIP2 to speckles is dynamic in that both PIPKs and PIP2 reorganize along with other speckle components upon inhibition of mRNA transcription. Because PIPKs have roles in the production of most phosphatidylinositol second messengers, these findings demonstrate that phosphatidylinositol signaling pathways are localized at nuclear speckles. Surprisingly, the PIPKs and PIP2 are not associated with invaginations of the nuclear envelope or any nuclear membrane structure. The putative absence of membranes at these sites suggests novel mechanisms for the generation of phosphoinositides within these structures (Boronenkov, 1998).

In many different cell types, including smooth muscle cells, phosphatidylinositol (4)-phosphate 5-kinase plays a critical role in the regulation of membrane concentrations of phosphatidylinositol (4,5)-bisphosphate and formation of inositol (1,4,5)-trisphosphate. In unstimulated porcine trachealis smooth muscle, 70% of total cellular phosphatidylinositol (4)-phosphate 5-kinase activity is associated with cytoskeletal proteins and only trace activity is detectable in isolated sarcolemma. Using two different preparations, cytoskeleton-associated phosphatidyl inositol (4)-phosphate 5-kinase was studied under conditions that attempted to mimic the ionic and thermal cytoplasmic environment of living cells. The cytoskeleton-associated enzyme, studied using phosphatidylinositol (4)-phosphate substrate concentrations that produce phosphatidylinositol 4,5-bisphosphate at about 10% of the maximal rate, is sensitive to free [Mg2+], has an absolute requirement for phosphatidylserine, phosphatidic acid, or phosphatidylinositol, and includes type I isoforms. At 0.5 mM free [Mg2+], physiological spermine concentrations, 0.2-0.4 mM, increases phosphatidylinositol (4)-phosphate 5-kinase activity two to four times compared to controls run without spermine. The EC50 for spermine-evoked increases in activity is 0.17 +/- 0.02 mM. Spermine-evoked enzyme activity is a function of both free [Mg2+] and substrate concentration. Cytoskeleton-associated phosphatidylinositol (4)-phosphate 5-kinase is inhibited by free [Ca2+] over a physiological range for cytoplasm--10(-8) to 10(-5) M, an effect independent of the presence of calmodulin. Na+ over the range 20 to 50 mM also inhibits this enzyme activated by 5 mM Mg2+ but has no effect on spermine-activated enzyme. Na+, Ca2+, and spermine appear to be physiological modulators of smooth muscle cytoskeleton-bound phosphatidylinositol (4)-phosphate 5-kinase (Chen, 1998).

Membrane trafficking downstream of phosphatidylinositol-4-phosphate 5-kinases

The FAB1 gene of budding yeast is predicted to encode a protein of 257 kDa that exhibits significant sequence homology to a human type II PI(4)P 5-kinase (PIP5K-II). The recently cloned human PIP5K-II specifically converts PI(4)P to PI(4,5)P2. The region of highest similarity between Fab1p and PIP5K-II includes a predicted nucleotide binding motif, which is likely to correspond to the catalytic domain of the protein. Interestingly, neither PIP5K-II nor Fab1p exhibit significant homology with cloned PI 3-kinases or PI 4-kinases. fab1 mutations result in the formation of aploid and binucleate cells (hence the name FAB). In addition, loss of Fab1p function causes defects in vacuole function and morphology, cell surface integrity, and cell growth. Experiments with a temperature conditional fab1 mutant reveal that their vacuoles rapidly (within 30 min) enlarge to more than double the size upon shifting cells to the nonpermissive temperature. Additional experiments with the fab1 ts mutant together with results obtained with fab1 vps (vacuolar protein sorting defective) double mutants indicate that the nuclear division and cell surface integrity defects observed in fab1 mutants are secondary to the vacuole morphology defects. Based on these data, it is proposed that Fab1p is a PI(4)P 5-kinase and that the product of the Fab1p reaction, PIP2, functions as an important regulator of vacuole homeostasis perhaps by controlling membrane flux to and/or from the vacuole. Furthermore, a comparison of the phenotypes of fab1 mutants and other yeast mutants affecting PI metabolism suggests that phosphoinositides may serve as general regulators of several different membrane trafficking pathways (Yamamoto, 1995).

Regulated fusion of secretory granules with the plasma membrane in secretory cells requires ATP, Ca2+ and cytosolic as well as membrane proteins. ATP-dependent steps in Ca(2+)-activated secretion from PC12 cells require three cytosolic PEP proteins (priming in exocytosis proteins, PEP1-3), the identity of which will provide insights into the required ATP-using reactions. PEP3 was recently identified as phosphatidylinositol transfer protein (PtdInsTP), and PEP1 consists of the type I phosphatidylinositol-4-phosphate 5-kinase (PtdInsP5K). The roles of PEP3/PtdInsTP and PEP1/PtdInsP5K in sequential phosphoinositide recruitment and phosphorylation explains their synergistic activity in ATP-dependent priming. Moreover, inhibition of Ca(2+)-activated secretion by PtdIns(4,5)P2-specific antibodies and phospholipase C implies that 5-phosphorylated inositides play a novel, necessary role in the regulated secretory pathway. The results indicate that lipid kinase-mediated phosphorylation is an important basis for ATP use in the exocytotic pathway (Hay, 1995).

Clathrin-coated pits are sites of concentration of ligand-bound signaling receptors. Several such receptors are known to recruit, bind, and activate the heterodimeric phosphatidylinositol-3-kinase, resulting in the generation of phosphatidylinositol 3,4, 5-trisphosphate. Dioctanoyl-phosphatidylinositol-3,4,5-P3 binds specifically and saturably to soluble AP-2 and with greater affinity to AP-2 within assembled coat structures. Soluble -myo-inositol hexakisphosphate shows converse behavior. Binding to bovine brain clathrin-coated vesicles is evident only after detergent extraction. These observations and evidence for recognition of the diacylglyceryl backbone as well as the inositol phosphate headgroup are consistent with AP-2 interaction with membrane phosphoinositides in coated vesicles and with soluble inositol phosphates in cytoplasm. A discrete binding domain is identified near the N terminus of the AP-2 alpha subunit, and an expressed fusion protein containing this sequence exhibits specific, high affinity binding that is virtually identical to the parent protein. This region of the AP-2 alpha sequence also shows the greatest conservation between a Caenorhabditis elegans homolog and mammalian alpha, consistent with a function in recognition of an evolutionarily unchanging low molecular weight ligand. Binding of phosphatidylinositol 3,4, 5-trisphosphate to AP-2 inhibits the protein's clathrin binding and assembly activities. These findings are discussed in the context of the potential roles of phosphoinositides and AP-2 in the internalization and trafficking of cell surface receptors (Gaidarov, 1996).

Pleckstrin homology (PH) domains may act as membrane localization modules through specific interactions with phosphoinositide phospholipids. These interactions could represent responses to second messengers, with scope for regulation by soluble inositol polyphosphates. A biosensor-based assay was used here to probe interactions between PH domains and unilamellar liposomes containing different phospholipids and to demonstrate specificity for distinct phosphoinositides. The dynamin PH domain specifically interact with liposomes containing phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2] and, more weakly, with liposomes containing phosphatidylinositol-4-phosphate [PI(4)P]. This correlates with phosphoinositide activation of the dynamin GTPase. The functional GTPase of a dynamin mutant lacking the PH domain, however, cannot be activated by PI(4,5)P2. The phosphoinositide-PH domain interaction can be abolished selectively by point mutations in the putative binding pocket predicted by molecular modelling and NMR spectroscopy. In contrast, the Bruton's tyrosine kinase (Btk)PH domain specifically binds liposomes containing phosphatidylinositol-3,4,5-trisphosphate [PI(3,4,5)P3]: an interaction requiring Arg28, a residue found to be mutated in some X-linked agammaglobulinaemia patients. A rational explanation for these different specificities is proposed through modelling of candidate binding pockets and is supported by NMR spectroscopy (Salim, 1996).

As part of an effort to understand the role inositol phosphates and inositol lipids play in the regulation of vesicle traffic within nerve terminals, a determination was made of whether or not the synapse-specific clathrin assembly protein AP-3 can interact with inositol lipids. Soluble dioctanoyl-phosphatidylinositol 3,4,5-trisphosphate (DiC8PtdIns(3,4, 5)P3) is only 7.5-fold weaker a ligand than D-myo-inositol hexakisphosphate in assays that measured the displacement of D-myo-[3H]inositol hexakisphosphate. Both of these ligands inhibit clathrin assembly, but DiC8-PtdIns(3,4,5)P3 is more potent and exhibits a larger maximal effect. The structural features of DiC8-PtdIns(3,4, 5)P3 that establish specificity were examined. Dioctanoyl-phosphatidylinositol 3, 4-bisphosphate, which does not have a 5-phosphate, and 4, 5-O-bisphosphoryl-D-myo-inosityl 1-O-(1, 2-O-diundecyl)-sn-3-glycerylphosphate, which does not have a 3-phosphate, were, respectively, 2-fold and 4-fold less potent than DiC8-PtdIns(3,4,5)P3 as inhibitors of clathrin assembly. Deacylation of DiC8-PtdIns(3,4,5)P3 reduces its affinity for AP-3 almost 20-fold, and also dramatically lowers its ability to inhibit clathrin assembly. The deacylated products of the soluble derivatives of phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 4, 5-bisphosphate were both not significant inhibitors of clathrin assembly. It therefore appears that the interactions of inositides with AP-3 should not be considered simply in terms of electrostatic effects of the highly charged phosphate groups. Ligand specificity appears also to be mediated by hydrophobic interactions with the fatty-acyl chains of the inositol lipids (Hao, 1997).

The clathrin-associated AP-2 adaptor protein is a major polyphosphoinositide-binding protein in mammalian cells. A high affinity binding site has been localized to the NH(2)-terminal region of the AP-2 alpha subunit. Deletion and site-directed mutagenesis have been used to determine that alpha residues 21-80 comprise a discrete folding and inositide-binding domain. Further, positively charged residues located within this region are involved in binding, with a lysine triad at positions 55-57 particularly critical. Mutant peptides and protein in which these residues were changed to glutamine retain wild-type structural and functional characteristics by several criteria including circular dichroism spectra, resistance to limited proteolysis, and clathrin binding activity. When expressed in intact cells, mutated alpha subunit show defective localization to clathrin-coated pits; at high expression levels, the appearance of endogenous AP-2 in coated pits is also blocked consistent with a dominant-negative phenotype. These results, together with recent work indicating that phosphoinositides are also critical to ligand-dependent recruitment of arrestin-receptor complexes to coated pits, suggest that phosphoinositides play a critical and general role in adaptor incorporation into plasma membrane clathrin-coated pits (Gaidarov, 1999).

Adaptors appear to control clathrin-coat assembly by determining the site of lattice polymerization but the nucleating events that target soluble adaptors to an appropriate membrane are poorly understood. Using an in vitro model system that allows AP-2-containing clathrin coats to assemble on lysosomes, it has been shown that adaptor recruitment and coat initiation requires phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] synthesis. PtdIns(4,5)P2 is generated on lysosomes by the sequential action of a lysosome-associated type II phosphatidylinositol 4-kinase and a soluble type I phosphatidylinositol 4-phosphate 5-kinase. Phosphatidic acid, which potently stimulates type I phosphatidylinositol 4-phosphate 5-kinase activity, is generated on the bilayer by a phospholipase D1-like enzyme located on the lysosomal surface. Quenching phosphatidic acid function with primary alcohols prevents the synthesis of PtdIns(4, 5)P2 and blocks coat assembly. Generating phosphatidic acid directly on lysosomes with exogenous bacterial phospholipase D in the absence of ATP still drives adaptor recruitment and limited coat assembly, indicating that PtdIns(4,5)P2 functions, at least in part, to activate the PtdIns(4,5)P2-dependent phospholipase D1. These results provide the first direct evidence for the involvement of anionic phospholipids in clathrin-coat assembly on membranes and define the enzymes responsible for the production of these important lipid mediators (Arneson, 1999).

Actin polymerization occurs downstream of phosphatidylinositol-4-phosphate 5-kinases

Bursts of actin polymerization in vivo involve the transient appearance of free barbed ends. To determine how rapidly barbed ends might appear and how long they might remain free in vivo, the kinetics of capping protein (the major barbed end capper) binding to barbed ends were studied in vitro; four observations have been noted:

    (1) the off-rate constant for capping protein leaving a barbed end is slow, predicting a half-life for a capped barbed end of approximately 30 min. This half-life implies that cells cannot wait for capping protein to spontaneously dissociate from capped barbed ends in order to create free barbed ends. Phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylinositol 4-mono-phosphate (PIP) cause rapid and efficient dissociation of capping protein from capped filaments. PIP2 is a strong candidate for a second messenger regulating actin polymerization; therefore, the ability of PIP2 to remove capping protein from barbed ends is a potential mechanism for stimulating actin polymerization in vivo.
    (2) The on-rate constant for capping protein binding to free barbed ends predicts that actin filaments could grow to the length of filaments observed in vivo during one lifetime.
    (3) Capping protein beta-subunit isoforms did not differ in their actin binding properties, even in tests with different actin isoforms. A major hypothesis for why capping protein beta-subunit isoforms exist is thereby excluded.
    (4) The proposed capping protein regulators, Hsc70 and S100, have no effect on capping protein binding to actin in vitro (Schafer, 1996).

Phosphatidylinositol 4,5-bisphosphate (PIP2) dramatically increases the gelating activity of smooth muscle alpha-actinin, and the hydrolysis of PIP2 on alpha-actinin by tyrosine kinase activation may be important in cytoskeletal reorganization. A proteolytic fragment with lysylendopeptidase comprising amino acids 168-184 (TAPYRNVNIQNFHLSWK) from striated muscle alpha-actinin contains a PIP2-binding site. A synthetic peptide composed of the 17 amino acids remarkably inhibits the activities of phospholipase C (PLC)-gamma 1 and -delta 1. An interaction between PIP2 and a bacterially expressed alpha-actinin fragment (amino acids 137-259) has been detected by PLC inhibition assay. Point mutants in which arginine 172 or lysine 184 of alpha-actinin are replaced by isoleucine reduce the inhibitory effect on PLC activity by nearly half. Direct interactions between PIP2 and the peptide (amino acids 168-184) or the bacterially expressed protein (amino acids 137-259) were confirmed by enzyme-linked immunosorvent assay. This region homologous to the sequence of the PIP2-binding site in spectrin and the pleckstrin homology domains of PLC-delta 1 and Grb7. Synthetic peptides from the homologous regions in spectrin and PLC-delta 1 inhibit PLC activities. These results indicate that residues 168-184 comprise a binding site for PIP2 in alpha-actinin and that similar sequences found in spectrin and PLC-delta 1 may be involved in the interaction with PIP2 (Fukami, 1996).

Mutation of mammalian phosphatidylinositol-4-phosphate 5-kinases

The STM7 gene on chromosome 9 was recently 'excluded' as a candidate for Friedreich's ataxia following the identification of an expanded intronic GAA triplet repeat in the adjacent gene, X25, in patients with the disease. Using RT-PCR, northern and sequence analyses, it is demonstrated that X25 comprises part of the STM7 gene, contributing to at least four splice variants, and the identification of new coding sequences is reported. Functional analysis of the STM7 recombinant protein corresponding to the reported 2.7-kilobase transcript demonstrates PtdlnsP 5-kinase activity, supporting the idea that the disease is caused by a defect in the phosphoinositide pathway, possibly affecting vesicular trafficking or synaptic transmission (Carvajal, 1996).

Phosphatidylinositol-4-phosphate 5-kinases and response to gravistimulation in plants

The internodal maize pulvinus responds to gravistimulation with differential cell elongation on the lower side. As the site of both graviperception and response, the pulvinus is an ideal system to study how organisms sense changes in orientation. A transient 5-fold increase in inositol 1,4,5-trisphosphate (IP3) is observed within 10 seconds of gravistimulation in the lower half of the pulvinus, indicating that the positional change is sensed immediately. Over the first 30 min, rapid IP3 fluctuations are observed between the upper and lower halves. Maize plants require a presentation time of between 2 and 4 h before the cells on the lower side of the pulvinus are committed to elongation. After 2 h of gravistimulation, the lower half consistently has higher IP3, and IP3 levels on the lower side continue to increase up to approximately 5-fold over basal levels before visible growth. As bending becomes visible after 8-10 h, IP3 levels return to basal values. Additionally, phosphatidylinositol 4-phosphate 5-kinase activity in the lower pulvinus half increases transiently within 10 min of gravistimulation, suggesting that the increased IP3 production is accompanied by an up-regulation of phosphatidylinositol 4, 5-bisphosphate biosynthesis. Neither IP3 levels nor phosphatidylinositol 4-phosphate 5-kinase activity change in pulvini halves from vertical control plants. These data indicate the involvement of IP3 and inositol phospholipids in both short- and long-term responses to gravistimulation. As a diffusible second messenger, IP3 provides a mechanism to transmit and amplify the signal from the perceiving to the responding cells in the pulvinus, coordinating a synchronized growth response (Perera, 1999).

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