Casein kinase I associates with members of the centaurin-alpha family of phosphatidylinositol 3,4,5-trisphosphate-binding proteins.

Mammalian casein kinases I (CKI) belong to a family of serine/threonine protein kinases involved in diverse cellular processes including cell cycle progression, membrane trafficking, circadian rhythms, and Wnt signaling. Here we show that CKIalpha co-purifies with centaurin-alpha(1) in brain and that they interact in vitro and form a complex in cells. In addition, we show that the association is direct and occurs through the kinase domain of CKI within a loop comprising residues 217-233. These residues are well conserved in all members of the CKI family, and we show that centaurin-alpha(1) associates in vitro with all mammalian CKI isoforms. To date, CKIalpha represents the first protein partner identified for centaurin-alpha(1). However, our data suggest that centaurin-alpha(1) is not a substrate for CKIalpha and has no effect on CKIalpha activity. Centaurin-alpha(1) has been identified as a phosphatidylinositol 3,4,5-trisphosphate-binding protein. Centaurin-alpha(1) contains a cysteine-rich domain that is shared by members of a newly identified family of ADP-ribosylation factor guanosine trisphosphatase-activating proteins. These proteins are involved in membrane trafficking and actin cytoskeleton rearrangement, thus supporting a role for CKIalpha in these biological events.

Cytohesins and centaurins: mediators of PI 3-kinase-regulated Arf signaling.

Receptor-activated phosphoinositide (PI) 3-kinases produce PtdIns(3, 4,5)P(3) and its metabolite PtdIns(3,4)P(2) that function as second messengers in membrane recruitment and activation of target proteins. The cytohesin and centaurin protein families are potential targets for PtdIns(3,4,5)P(3) that also regulate and interact with Arf GTPases. Consequently, these families are poised to transduce PI 3-kinase activation into coordinated control of Arf-dependent pathways. Proposed downstream events in PI 3-kinase-regulated Arf cascades include modulation of vesicular trafficking and the actin cytoskeleton.

Restructuring of focal adhesion plaques by PI 3-kinase. Regulation by PtdIns (3,4,5)-p(3) binding to alpha-actinin.

Focal adhesions are an elaborate network of interconnecting proteins linking actin stress fibers to the extracellular matrix substrate. Modulation of the focal adhesion plaque provides a mechanism for the regulation of cellular adhesive strength. Using interference reflection microscopy, we found that activation of phosphoinositide 3-kinase (PI 3-kinase) by PDGF induces the dissipation of focal adhesions. Loss of this close apposition between the cell membrane and the extracellular matrix coincided with a redistribution of alpha-actinin and vinculin from the focal adhesion complex to the Triton X-100-soluble fraction. In contrast, talin and paxillin remained localized to focal adhesions, suggesting that activation of PI 3-kinase induced a restructuring of the plaque rather than complete dispersion. Furthermore, phosphatidylinositol (3,4, 5)-trisphosphate (PtdIns (3,4,5)-P(3)), a lipid product of PI 3-kinase, was sufficient to induce restructuring of the focal adhesion plaque. We also found that PtdIns (3,4,5)-P(3) binds to alpha-actinin in PDGF-treated cells. Further evidence demonstrated that activation of PI 3-kinase by PDGF induced a decrease in the association of alpha-actinin with the integrin beta subunit, and that PtdIns (3,4,5)-P(3) could disrupt this interaction in vitro. Modification of focal adhesion structure by PI 3-kinase and its lipid product, PtdIns (3,4,5)-P(3), has important implications for the regulation of cellular adhesive strength and motility.

Cocaine inhibition of neuronal differentiation in NGF-induced PC12 cells is independent of ras signaling.

In utero exposure to cocaine may result in altered neuronal development. Our previous studies demonstrated cocaine inhibits neurite outgrowth in NGF-induced PC12 cells through dopamine, by activation of D1 receptors. This study examined where cocaine interferes in the NGF signaling cascade. GSrasl cells that inducibly express activated forms of Ras upon treatment with dexamethasone were used. Morphological differentiation was quantified by counting cells bearing neurite-like processes after 72 h exposure to either dexamethasone or NGF alone, or with cocaine, dopamine or SKF-38393. Cocaine, dopamine, and the D1 agonist inhibited neurite-like process outgrowth in both dexamethasone and NGF-induced GSras1 cells. GAP-43 expression, used as a measure for biochemical differentiation was severely diminished in NGF and dexamethasone-induced GSras1 cells treated with cocaine. These results suggest that cocaine, dopamine and activation of D1 receptors affect the NGF signaling downstream, independent of ras expression, leading to altered neuronal differentiation.

Regulation of AMP deaminase by phosphoinositides.

AMP deaminase (AMPD) converts AMP to IMP and is a diverse and highly regulated enzyme that is a key component of the adenylate catabolic pathway. In this report, we identify the high affinity interaction between AMPD and phosphoinositides as a mechanism for regulation of this enzyme. We demonstrate that endogenous rat brain AMPD and the human AMPD3 recombinant enzymes specifically bind inositide-based affinity probes and to mixed lipid micelles that contain phosphatidylinositol 4,5-bisphosphate. Moreover, we show that phosphoinositides specifically inhibit AMPD catalytic activity. Phosphatidylinositol 4,5-bisphosphate is the most potent inhibitor, effecting pure noncompetitive inhibition of the wild type human AMPD3 recombinant enzyme with a K(i) of 110 nM. AMPD activity can be released from membrane fractions by in vitro treatment with neomycin, a phosphoinositide-binding drug. In addition, in vivo modulation of phosphoinositide levels leads to a change in the soluble and membrane-associated pools of AMPD activity. The predicted human AMPD3 sequence contains pleckstrin homology domains and (R/K)X(n)(R/K)XKK sequences, both of which are characterized phosphoinositide-binding motifs. The interaction between AMPD and phosphoinositides may mediate membrane localization of the enzyme and function to modulate catalytic activity in vivo.

GCS1, an Arf guanosine triphosphatase-activating protein in Saccharomyces cerevisiae, is required for normal actin cytoskeletal organization in vivo and stimulates actin polymerization in vitro.

Recent cloning of a rat brain phosphatidylinositol 3,4, 5-trisphosphate binding protein, centaurin alpha, identified a novel gene family based on homology to an amino-terminal zinc-binding domain. In Saccharomyces cerevisiae, the protein with the highest homology to centaurin alpha is Gcs1p, the product of the GCS1 gene. GCS1 was originally identified as a gene conditionally required for the reentry of cells into the cell cycle after stationary phase growth. Gcs1p was previously characterized as a guanosine triphosphatase-activating protein for the small guanosine triphosphatase Arf1, and gcs1 mutants displayed vesicle-trafficking defects. Here, we have shown that similar to centaurin alpha, recombinant Gcs1p bound phosphoinositide-based affinity resins with high affinity and specificity. A novel GCS1 disruption strain (gcs1Delta) exhibited morphological defects, as well as mislocalization of cortical actin patches. gcs1Delta was hypersensitive to the actin monomer-sequestering drug, latrunculin-B. Synthetic lethality was observed between null alleles of GCS1 and SLA2, the gene encoding a protein involved in stabilization of the actin cytoskeleton. In addition, synthetic growth defects were observed between null alleles of GCS1 and SAC6, the gene encoding the yeast fimbrin homologue. Recombinant Gcs1p bound to actin filaments, stimulated actin polymerization, and inhibited actin depolymerization in vitro. These data provide in vivo and in vitro evidence that Gcs1p interacts directly with the actin cytoskeleton in S. cerevisiae.

C-fos mediates cocaine inhibition of NGF-induced PC12 cell differentiation.

In utero cocaine exposure can affect CNS development. Previous studies showed that cocaine inhibits neuronal differentiation in a dose-dependent fashion, in nerve growth factor (NGF)-stimulated PC12 cells, without affecting cell viability. NGF activates intracellular signaling proteins, specific immediate-early genes (IEG) including a transient peak of c-fos expression, and induction of late genes expression, leading to the neuronal phenotype. We hypothesized that cocaine interferes with NGF signaling. Therefore, we examined the pattern of c-fos expression in our cellular model. Time course of c-fos expression up to 72 h was determined in cells treated with NGF 20 ng/ml and cocaine 10 microgram/ml (a moderately toxic level) by RT-PCR analysis. Total RNA was isolated from cells, and levels of c-fos mRNA were estimated using gene-specific primers. In both control and experimental conditions, c-fos level was maximal at 0.5 h. In the control cells, c-fos expression declined rapidly to less than 5% of the 0.5h value, while in the cocaine-treated cells, c-fos level persisted through the 72-h exposure. Adding c-fos antisense to cells treated with NGF and cocaine resulted in significant improvement of neurite out-growth, from 28% (NGF + cocaine) to 89% (NGF + cocaine + c-fos antisense) of control differentiation after 72 h of exposure (Dunnet's T < 3.24). Inhibitory effects of cocaine on NGF-induced PC12 differentiation may be attributed to alteration of c-fos expression. Further studies will be required to examine the role of D1 receptor activation in mediating c-fos expression and to explore the effects of cocaine on other IEGs.

Novel developmentally regulated phosphoinositide binding proteins from soybean whose expression bypasses the requirement for an essential phosphatidylinositol transfer protein in yeast.

Phosphatidylinositol transfer proteins (PITPs) have been shown to play important roles in regulating a number of signal transduction pathways that couple to vesicle trafficking reactions, phosphoinositide-driven receptor-mediated signaling cascades, and development. While yeast and metazoan PITPs have been analyzed in some detail, plant PITPs remain entirely uncharacterized. We report the identification and characterization of two soybean proteins, Ssh1p and Ssh2p, whose structural genes were recovered on the basis of their abilities to rescue the viability of PITP-deficient Saccharomyces cerevisiae strains. We demonstrate that, while both Ssh1p and Ssh2p share approximately 25% primary sequence identity with yeast PITP, these proteins exhibit biochemical properties that diverge from those of the known PITPs. Ssh1p and Ssh2p represent high-affinity phosphoinositide binding proteins that are distinguished from each other both on the basis of their phospholipid binding specificities and by their substantially non-overlapping patterns of expression in the soybean plant. Finally, we show that Ssh1p is phosphorylated in response to various environmental stress conditions, including hyperosmotic stress. We suggest that Ssh1p may function as one component of a stress response pathway that serves to protect the adult plant from osmotic insult.

Thrombospondin signaling of focal adhesion disassembly requires activation of phosphoinositide 3-kinase.

Thrombospondin is an extracellular matrix protein involved in modulating cell adhesion. Thrombospondin stimulates a rapid loss of focal adhesion plaques and reorganization of the actin cytoskeleton in cultured bovine aortic endothelial cells. The focal adhesion labilizing activity of thrombospondin is localized to the amino-terminal domain, specifically amino acids 17-35. Use of a synthetic peptide (hep I), containing amino acids 17-35 of thrombospondin, enables us to examine the signaling mechanisms specifically involved in thrombospondin-induced disassembly of focal adhesions. We tested the hypothesis that activation of phosphoinositide 3-kinase is a necessary step in the thrombospondin-induced signaling pathway regulating focal adhesion disassembly. Both wortmannin and LY294002, membrane permeable inhibitors of phosphoinositide 3-kinase activity, blocked hep I-induced disassembly of focal adhesions. Similarly, wortmannin inhibited hep I-mediated actin microfilament reorganization and the hep I-induced translocation of alpha-actinin from focal adhesion plaques. Hep I also stimulated phosphoinositide 3-kinase activity approximately 2-3-fold as measured in anti-phosphoinositide 3-kinase and anti-phosphotyrosine immunoprecipitates. Increased immunoreactivity for the 85-kDa regulatory subunit in anti-phosphotyrosine immunoprecipitates suggests that the p85/p110 form of phosphoinositide 3-kinase is involved in this pathway. In 32Pi-labeled cells, hep I increased levels of phosphatidylinositol (3,4,5)-trisphosphate, the major product of phosphoinositide 3-kinase phosphorylation. These results suggest that thrombospondin signals the disassembly of focal adhesions and reorganization of the actin cytoskeleton by a pathway involving stimulation of phosphoinositide 3-kinase activity.

Elevated adenosine monophosphate deaminase activity in Alzheimer's disease brain.

Abnormal elevations in ammonia have been implicated in the pathogenesis of Alzheimer's disease. However, the biochemical mechanism(s) leading to increased ammonia in Alzheimer's disease have not yet been identified. A potential source of increased ammonia production is adenosine monophosphate (AMP) deaminase, an important enzyme in the regulation of the purine nucleotide cycle and adenylate energy charge. AMP deaminase activity is expressed in human brain and converts AMP to inosine monophosphate with the release of ammonia. We have investigated AMP deaminase activity in postmortem brain tissue from Alzheimer's disease subjects and age-matched controls. Compared to control brain, Alzheimer's disease brain AMP deaminase activity is 1.6- to 2.4-fold greater in the regions examined--the cerebellum, occipital cortex, and temporal cortex. Similar increases in AMP deaminase protein and mRNA levels are observed in Alzheimer's disease brain. These results suggest that increased AMP deaminase activity may augment ammonia levels in the brain in Alzheimer's disease.

Identification and cloning of centaurin-alpha. A novel phosphatidylinositol 3,4,5-trisphosphate-binding protein from rat brain.

Using an affinity resin and photoaffinity label based on phospholipid analogs of inositol 1,3,4,5-tetrakisphosphate (InsP4), we have isolated, characterized, and cloned a 46-kDa protein from rat brain, which we have named centaurin-alpha. Binding specificity was determined using displacement of 1-O-[3H](3-[4-benzoyldihydrocinnamidyl]propyl)-InsP4 photoaffinity labeling. Centaurin-alpha displayed highest affinity for phosphatidylinositol 3,4,5-trisphosphate (PtdInsP3) (IC50 = 120 nM), whereas InsP4, PtdInsP2, and InsP3 bound with 5-, 12-, and >50-fold lower affinity, respectively. Screening a rat brain cDNA library with a polymerase chain reaction product, generated using partial amino acid sequence from tryptic peptides, yielded a full-length clone. The 2,450-base pair cDNA contained an open reading frame (ORF) encoding a novel protein of 419 amino acids. Northern analysis revealed a 2.5-kilobase transcript that is highly expressed in brain. The deduced sequence contains a novel putative zinc finger motif, 10 ankyrin-like repeats, and shows homology to recently identified yeast and mammalian Arf GTPase-activating proteins. Given the specificity of binding and enrichment in brain, centaurin-alpha is a candidate PtdInsP3 receptor that may link the activation of phosphoinositide 3-kinase to downstream responses in the brain.

Initiation and maintenance of NGF-stimulated neurite outgrowth requires activation of a phosphoinositide 3-kinase.

Application of nerve growth factor (NGF) to PC12 cells stimulates a programme of physiological changes leading to the development of a sympathetic neuron like phenotype, one aspect of which is the development of a neuronal morphology characterised by the outgrowth of neuritic processes. We have investigated the role of phosphoinositide 3-kinase in NGF-stimulated morphological differentiation through two approaches: firstly, preincubation with wortmannin, a reputedly specific inhibitor of phosphoinositide kinases, completely inhibited initial morphological responses to NGF, the formation of actin filament rich microspikes and subsequent neurite outgrowth. This correlated with wortmannin inhibition of NGF-stimulated phosphatidylinositol(3,4,5)trisphosphate (PtdInsP3) and phosphatidylinositol(3,4)bisphosphate (PtdIns(3,4)P2) production and with inhibition of NGF-stimulated phosphoinositide 3-kinase activity in anti-phosphotyrosine immunoprecipitates. Secondly, the overexpression of a mutant p85 regulatory subunit of the phosphoinositide 3-kinase, which cannot interact with the catalytic p110 subunit, also substantially inhibited the initiation of NGF-stimulated neurite outgrowth. In addition, we found that wortmannin caused a rapid collapse of more mature neurites formed following several days exposure of PC12 cells to NGF. These results indicate that NGF-stimulated neurite outgrowth requires the activity of a tyrosine kinase regulated PI3-kinase and suggest that the primary product of this enzyme, PtdInsP3, is a necessary second messenger for the cytoskeletal and membrane reorganization events which occur during neuronal differentiation.

Inositol hexakisphosphate receptor identified as the clathrin assembly protein AP-2.

To clarify the function of the receptor binding protein for inositol hexakisphosphate (IP6), we obtained a partial amino acid sequence from the purified protein and a partial nucleotide sequence from a cDNA clone of the gene. The sequences are essentially identical to those of the alpha-subunit of the clathrin assembly protein AP-2. The IP6 receptor protein analyzed by SDS-PAGE contains a series of subunits which are the same as those of AP-2. Antibodies to AP-2 react with the IP6 receptor protein in immunoblot analysis.

Photoaffinity labeling and characterization of isolated inositol 1,3,4,5-tetrakisphosphate- and inositol hexakisphosphate-binding proteins.

We have isolated high affinity inositol (1,3,4,5)-tetrakisphosphate (IP4)- and inositol hexakisphosphate (IP6)-binding proteins from detergent-solubilized rat brain membranes using a P1-tethered IP4 derivative linked to an Affi-Gel support. To determine the identity, binding characteristics, and distribution of the individual IP4 recognition sites, we have synthesized an IP4 photoaffinity label probe, 125I-(D,L)-1-O-[N-(4-azidosalicyloxy)-3-aminopropyl-1-phospho]- IP4 (125I-ASA-IP4). Two apparently distinct IP4-binding proteins (IP4BP), isolated with the IP4 affinity column, display high affinity and selectivity for IP4 over inositol trisphosphate (IP3), inositol pentakisphosphate (IP5), and IP6. The first IP4-binding protein (IP4BP1) which has a KD for IP4 of 4 nM, is comprised of a protein at 182 kDa which is specifically photolabeled with high affinity by 125I-ASA-IP4. The second, IP4BP2, has an affinity for IP4 of 1.5 nM and contains proteins at 84 and 174 kDa, both of which are specifically photoaffinity labeled. A putative IP6-binding protein (IP6BP), also isolated with the IP4 affinity column, binds IP6 with a KD of 14 nM and comprises three proteins of 115, 105, and 50 kDa. The 115- and 105-kDa subunits, but not the 50-kDa subunit, specifically incorporate the photolabel. The IP4BP (182, 174, and 84 kDa) and IP6BP (115 and 105 kDa) proteins are specifically photolabeled in the crude membrane, partially purified, and purified fractions. These receptor-binding proteins vary in inositol phosphate specificity and in the effects of pH, Ca2+, and heparin on IP4 photoaffinity labeling. In addition, IP4BP and IP6BP are enriched in the brain but differ in their regional localizations within the brain.

Inositol 1,3,4,5-tetrakisphosphate and inositol hexakisphosphate receptor proteins: isolation and characterization from rat brain.

High-affinity, membrane-associated inositol 1,3,4,5-tetrakisphosphate (IP4) and inositol hexakisphosphate (IP6) binding proteins were solubilized and isolated utilizing a heparin-agarose resin followed by an IP4 affinity resin. The IP6 receptor comprises a protein complex of 115-, 105-, and 50-kDa subunits, all of which comigrate under native conditions. The Kd of the receptor for IP6 is 12 nM, whereas inositol 1,3,4,5,6-pentakisphosphate (IP5), IP4, and inositol 1,4,5-trisphosphate (IP3) are 50%, 30%, and 15%, respectively, as potent. Two protein complexes copurify with the IP4 receptor fraction. A 182/123-kDa complex elutes first from the affinity column followed by a 174/84-kDa protein complex, which elutes at higher salt. Both complexes show high affinity for IP4 (Kd = 3-4 nM). IP5, IP6, and IP3 display approximately 25%, 10%, and 0.1%, respectively, the affinity of IP4. Ligand binding to IP6 and IP4 receptors is inhibited 50% by heparin at 0.1 microgram/ml. IP4 receptor proteins are stoichiometrically phosphorylated by cyclic AMP-dependent protein kinase and protein kinase C, whereas negligible phosphorylation is observed for the IP6 receptor.

Solubilization and separation of inositol 1,3,4,5-tetrakisphosphate- and inositol 1,4,5-trisphosphate-binding proteins and metabolizing enzymes in rat brain.

The two inositol phosphate-binding proteins, the Ins(1,4,5)P3 (InsP3) and Ins(1,3,4,5)P4 (InsP4) receptors, and the two particulate InsP3-metabolizing enzymes, InsP3 5-phosphatase and InsP3 3-kinase, were solubilized with detergent from rat cerebellar membranes. These four activities are shown to be distinct molecular species by separation using a variety of protein chromatographic steps. The pharmacology of the partially purified InsP4-binding site indicates that the binding has a high affinity and selectivity for InsP4 over InsP3. These results suggest the existence of a distinct specific InsP4-binding protein which may represent the receptor for this putative second messenger.

Demonstration of inositol 1,3,4,5-tetrakisphosphate receptor binding.

Inositol 1,3,4,5-tetrakisphosphate (InsP4) is produced rapidly upon stimulation of the phosphoinositide system and may serve as a second messenger in hormone and neurotransmitter action. In this report we demonstrate specific binding sites for [3H]InsP4 in rat tissue membranes. In cerebellar membranes, [3H]InsP4 binding sites are displaced both by InsP4 and inositol 1,4,5-trisphosphate (InsP3) with similar potency (IC50 approximately equal to 300 nM) whereas several other inositol phosphates are much weaker. We have distinguished the InsP4 binding site from the InsP3 receptor binding site by differences in brain regional and tissue distribution, affinity for InsP4 and InsP3, and sensitivity to calcium.