Identification and Cloning of Centaurin-α

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As part of the growing effort to understand the role inositol phosphates and inositol lipids play in the regulation of vesicle traffic within nerve terminals, we determined whether or not the synapse-specific clathrin assembly protein AP-3 can interact with inositol lipids. We found that soluble dioctanoyl-phosphatidylinositol 3,4,5-trisphosphate (DiC 8 PtdIns(3,4,5)P 3 ) was only 7.5-fold weaker a ligand than D-myo-inositol hexakisphosphate in assays that measured the displacement of D-myo-[3 H]inositol hexakisphosphate. In functional assays we found that both of these ligands inhibited clathrin assembly, but DiC 8 -PtdIns(3,4,5)P 3 was more potent and exhibited a larger maximal effect. We also examined the structural features of DiC 8 -PtdIns(3,4,5)P 3 that establish specificity. 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 DiC 8 -PtdIns(3,4,5)P 3 as inhibitors of clathrin assembly. Deacylation of DiC 8 -PtdIns(3,4,5)P 3 reduced its affinity for AP-3 almost 20-fold, and also dramatically lowered 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.There is increasing evidence that certain inositides (members of a large family of inositol phosphates and inositol lipids) act at several levels to regulate both the recruitment and the activation of proteins involved in vesicular transport. PtdIns(3,4,5)P 3 , 1 and the polyphosphoinositide 3-kinase that synthesizes this lipid both feature prominently in this area (1, 2). For example, intracellular trafficking of tyrosine kinase receptors depends upon interactions between the internalized receptors and the 3-kinase (3). Additionally, PtdIns(3,4,5)P 3 interacts either directly or indirectly with several GTPases that play important roles in vesicle traffic (1). Finally, in yeast, the Vps34 protein that is essential for Golgi to vacuole transport is a phosphoinositide 3-kinase (4).Interest in this field is not restricted to the inositol lipids. Other studies into the roles of the inositides in protein traffic have ascertained that certain inositol polyphosphates can bind with high affinity to the clathrin assembly proteins, AP-2 and AP-3 (5-9). AP-2 is believed to play a general role in endocytosis, since it has been found on vesicles budding from the plasma membranes of all types of cells (10). AP-3 is believed to play a more specialized role in synaptic vesicle biogenesis and recycling...

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Regulation of AP-3 Function by Inositides

Hao

Tan

Prasad

et al. 1997

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“…This observation in fact enabled the isolation and purification of centaurin-␣, a novel multi-domain protein with both homology regions, suggesting its role in communication of membrane lipid changes to the cytoskeleton (21). Very recently, the Nterminal region of the ␣ subunit of AP-2 was found to have high affinity for PIP 3 (38), a finding foreshadowed by the highly selective and efficient photoaffinity labeling of AP-2 by [ 3 H]BZDC-IP 4 (31,32).…”

Section: Photolabeling Of Different Domains Of Syt II and Syt Iii Witmentioning

confidence: 99%

“…For example, four IP 4 binding proteins were affinity-purified from rat brain (30) and photoaffinity labeled with [ 3 H]BZDC-IP 4 (22,31). Two have been further characterized; the ␣ subunit of clathrin assembly protein (AP-2) (32) had the highest affinity for IP 6 , whereas the novel cytoskeletal linker protein centaurin-␣ had the highest affinity for PIP 3 (21). The IP 3 binding site of the affinity-purified rat brain IP 3 receptor (19) has been identified by sequencing a proteolytic fragment of the [ 3 H]BZDC-IP 3 photoaffinity-labeled protein (33).…”

Section: Photolabeling Of Different Domains Of Syt II and Syt Iii Witmentioning

confidence: 99%

Selective Photoaffinity Labeling of the Inositol Polyphosphate Binding C2B Domains of Synaptotagmins

Mehrotra

Elliott

Chen

et al. 1997

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. Although the CD spectrum of this 32-mer at two pH values showed a random coil, the photoaffinity analogue of IP 6 appeared to induce a binding-compatible structure in the short peptide.The synaptotagmins (Syts) 1 are synaptic vesicle proteins that play essential roles in nucleating the clathrin coat during endocytosis and in acting as Ca 2ϩ sensors (1-3) and phosphoinositide sensors (4) during exocytosis. They are a critical part of a complex machinery of intracellular protein transport (5, 6) and the synaptic vesicle cycle (7). In addition to a short Nterminal intravesicular region and single transmembrane domain, Syts have two copies of highly conserved repeats, known as the C2A and C2B domains, which are homologous to the C2 regulatory region of protein kinase C (8). In particular, the C2B domain appears to play several roles. First, the C2B domain of mouse Syt II shows specific binding to high polyphosphate inositols (IP n s) (9), a feature that is not shared by the highly homologous C2A domain nor with the C2 domains of other proteins such as rabphilin. Moreover, the C2B domain is necessary but not sufficient for IP n binding. Although Syt II and IV show high affinity binding of IP 4 and IP 6 , the Syt III-C2B domain shows negligible binding, despite a high sequence identity with the Syt II-C2B domain, including the 32-residue region examined by mutational analysis (10). Inhibition of C2B function in the squid giant axon disrupts synaptic vesicle release and recycling (11). Similarly, Caenorhabditis elegans mutants lacking Syt or simply the C2B domain cannot recycle synaptic vesicles (12), and the presence of Syt I in cerebrospinal fluid of Alzheimer's patients (13) may provide clues to the disruption of synaptic function in humans. The C2B domain acts as a high affinity receptor for clathrin assembly protein AP-2 (14), and Ca 2ϩ appears to mediate Syt dimerization via the C2B domain (15).Synaptotagmin II isolated from mouse cerebellum shows high affinity binding to Ins(1,3,4,5)P 4 with a K D of 30 M (16) and 117 nM for the GST-Syt II-C2B construct (9). Curiously, the rank order of IP n s binding for native Syt II was Ins-(1,3,4,5,6)-P 5 Ͼ (1,3,4,5)-P 4 Ͼ Ins(1,2,3,4,5,6)-P 6 Ͼ Ins(1,4,5)-P 3 , whereas for the GST-Syt II-C2B domain the order was IP 6 Ͼ IP 5 Ͼ IP 4 Ͼ IP 3 . Deletion mutants allowed mapping of the IP n binding site to the central region of the mouse Syt II-C2B domain, specifically residues 315-346 (IHLMQNGKRLKKKK-TTVKKKTLNPYFNESFSF) (9). In order to obtain direct evidence for the binding site of IP n s in the C2B domain, to examine the InsP n selectivity of the domains, to explore the Ca 2ϩ dependence of binding, to determine the role of the central 315-346 peptide, and to evaluate the reason for the failure of Syt III-C2B domain to bind IP n s, we undertook a series of photoaffinity labeling experiments using four tritium-labeled, benzophenone-containing derivatives of IP 3 , IP 4 , and IP 6 (17). EXPERIMENTAL PROCEDURES Chemicals-P-1-Tethered

“…The resulting PtdIns(3,4,5)P 3 has been shown to increase rapidly and transiently in many cells in response to a wide variety of stimuli and since there are no known phospholipases that can metabolize this lipid (13), it has been suggested that PtdIns(3,4,5)P 3 itself is a second messenger. At present there are reports that PtdIns(3,4,5)P 3 can activate atypical forms of protein kinase C (14) and can bind to protein kinase B (also named Akt or Rac) (15)(16)(17) and the recently cloned centaurin (18), encoding for a novel gene, which is highly expressed in brain and which shows homology to yeast and mammalian Arf GTPase-activating proteins.…”

mentioning

confidence: 99%

A Novel, Rapid, and Highly Sensitive Mass Assay for Phosphatidylinositol 3,4,5-Trisphosphate (PtdIns(3,4,5)P3) and Its Application to Measure Insulin-stimulated PtdIns(3,4,5)P3 Production in Rat Skeletal Muscle in Vivo

Kaay¹,

Batty²,

Cross³

et al. 1997

The pivotal role of phosphatidylinositol 3-kinase (PI 3-kinase) in signal transduction has been well established in recent years. Receptor-regulated forms of PI 3-kinase are thought to phosphorylate phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P 2 ) at the 3-position of the inositol ring to give the putative lipid second messenger, phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P 3 ). Cellular levels of PtdIns(3,4,5)P 3 are currently measured by time-consuming procedures involving radiolabeling with high levels of 32 PO 4 , extraction, and multiple chromatography steps. To avoid these lengthy and hazardous procedures, many laboratories prefer to assay PI 3-kinase activity in cell extracts and/or appropriate immunoprecipitates. Such approaches are not readily applied to measurements of PtdIns(3,4,5)P 3 in extracts of animal tissues. Moreover, they can be misleading since the association of PI 3-kinases in molecular complexes is not necessarily correlated with the enzyme's activity state. Direct measurements of PtdIns(3,4,5)P 3 would also be desirable since its concentration may be subject to additional control mechanisms such as activation or inhibition of the phosphatases responsible for PtdIns(3,4,5)P 3 metabolism. We now report a simple, reproducible isotope dilution assay which detects PtdIns(3,4,5)P 3 at subpicomole sensitivity, suitable for measurements of both basal and stimulated levels of PtdIns(3,4,5)P 3 obtained from samples containing approximately 1 mg of cellular protein. Total lipid extracts, containing PtdIns(3,4,5)P 3 , are first subjected to alkaline hydrolysis which results in the release of the polar head group Ins(1,3,4,5)P 4 . The latter is measured by its ability to displace [ 32 P]Ins(1,3,4,5)P 4 from a highly specific binding protein present in cerebellar membrane preparations. We show that this assay solely detects PtdIns(3,4,5)P 3 and does not suffer from interference by other compounds generated after alkaline hydrolysis of total cellular lipids. Measurements on a wide range of cells, including rat-1 fibroblasts, 1321N1 astrocytoma cells, HEK 293 cells, and rat adipocytes, show wortmannin-sensitive increased levels of PtdIns(3,4,5)P 3 upon stimulation with appropriate agonists. The enhanced utility of this procedure is further demonstrated by measurements of PtdIns(3,4,5)P 3 levels in tissue derived from whole animals. Specifically, we show that stimulation with insulin increases PtdIns(3,4,5)P 3 levels in rat skeletal muscle in vivo with a time course which parallels the activation of protein kinase B in the same samples.PI 3-kinases 1 represent a family of enzymes which phosphorylate phosphoinositides on the 3-position of the inositol ring (1). The role of PI 3-kinases in mitogenic signaling, membrane ruffling, trafficking, cell motility, inflammatory and immune cell responses, activation of neutrophils, and the metabolic effects of insulin (2-8) has been well established using biochemical, pharmacological, and genetic approaches. At least two major classes of PI 3-kinas...