Biomedical subjects

Y S Bae

Publications and source records attributed to Y S Bae.

At least 37 records · Page 2Linked to original sources

PubMed2000

Platelet-derived growth factor-induced H(2)O(2) production requires the activation of phosphatidylinositol 3-kinase.

Autophosphorylation of the platelet-derived growth factor (PDGF) receptor triggers intracellular signaling cascades as a result of recruitment of Src homology 2 domain-containing enzymes, including phosphatidylinositol 3-kinase (PI3K), the GTPase-activating protein of Ras (GAP), the protein-tyrosine phosphatase SHP-2, and phospholipase C-gamma1 (PLC-gamma1), to specific phosphotyrosine residues. The roles of these various effectors in PDGF-induced generation of H(2)O(2) have now been investigated in HepG2 cells expressing various PDGF receptor mutants. These mutants included a kinase-deficient receptor and receptors in which various combinations of the tyrosine residues required for the binding of PI3K (Tyr(740) and Tyr(751)), GAP (Tyr(771)), SHP-2 (Tyr(1009)), or PLC-gamma1 (Tyr(1021)) were mutated to Phe. PDGF failed to increase H(2)O(2) production in cells expressing either the kinase-deficient mutant or a receptor in which the two Tyr residues required for the binding of PI3K were replaced by Phe. In contrast, PDGF-induced H(2)O(2) production in cells expressing a receptor in which the binding sites for GAP, SHP-2, and PLC-gamma1 were all mutated was slightly greater than that in cells expressing the wild-type receptor. Only the PI3K binding site was alone sufficient for PDGF-induced H(2)O(2) production. The effect of PDGF on H(2)O(2) generation was blocked by the PI3K inhibitors LY294002 and wortmannin or by overexpression of a dominant negative mutant of Rac1. These results suggest that a product of PI3K is required for PDGF-induced production of H(2)O(2) in nonphagocytic cells, and that Rac1 mediates signaling between the PI3K product and the putative NADPH oxidase.

Binding Sites↗

PubMed2000

In vitro binding analysis of hepatitis B virus preS-derived putative helper T-cell epitopes to MHC class II molecules using stable HLA-DRB10405/DRA0101 transfected cells.

In designing epitope-based vaccines, the inclusion of a helper T-lymphocyte (HTL) epitope is necessary to elicit both humoral and cellular immune responses. Whereas the preS region of the hepatitis B virus (HBV) surface antigen is well-known to raise protective immunity, the epitopes for activating HTLs are poorly characterized. In an attempt to identify such epitopes, the HBV-preS region was screened for peptide sequences with HLA-DR4 binding motifs, and putative HTL candidate peptides were synthesized in a biotinylated form. Using L929 mouse fibroblasts stably transfected with HLA-DRB1*0405 and HLA-DRA*0101 cDNA, specific binding of the peptides was then detected using fluorescence-conjugated streptavidin. The cell-surface expression of HLA-DR molecules on transfectants was confirmed by confocal microscopy, and quantitative analysis of candidate peptide binding was performed by fluorescence activated cell sorting. Among eight preS-derived peptides, three candidate peptides-namely preS1(23-33), preS1(62-72), and preS1(76-86)-showed good binding characteristics to HLA-DR4 molecules, among which the preS1(23-33) epitope was regarded as the most promising HTL epitope. Further studies with these candidate HTL stimulatory peptides will show their ability to activate the human immune system against HBV.

Animals↗

PubMed2000

Cotransformation of Trichoderma harzianum with beta-glucuronidase and green fluorescent protein genes provides a useful tool for monitoring fungal growth and activity in natural soils.

Trichoderma harzianum was cotransformed with genes encoding green fluorescent protein (GFP), beta-glucuronidase (GUS), and hygromycin B (hygB) resistance, using polyethylene glycol-mediated transformation. One cotransformant (ThzID1-M3) was mitotically stable for 6 months despite successive subculturing without selection pressure. ThzID1-M3 morphology was similar to that of the wild type; however, the mycelial growth rate on agar was reduced. ThzID1-M3 was formed into calcium alginate pellets and placed onto buried glass slides in a nonsterile soil, and its ability to grow, sporulate, and colonize sclerotia of Sclerotinia sclerotiorum was compared with that of the wild-type strain. Wild-type and transformant strains both colonized sclerotia at levels above those of indigenous Trichoderma spp. in untreated controls. There were no significant differences in colonization levels between wild-type and cotransformant strains; however, the presence of the GFP and GUS marker genes permitted differentiation of introduced Trichoderma from indigenous strains. GFP activity was a useful tool for nondestructive monitoring of the hyphal growth of the transformant in a natural soil. The green color of cotransformant hyphae was clearly visible with a UV epifluorescence microscope, while indigenous fungi in the same samples were barely visible. Green-fluorescing conidiophores and conidia were observed within the first 3 days of incubation in soil, and this was followed by the formation of terminal and intercalary chlamydospores and subsequent disintegration of older hyphal segments. Addition of 5-bromo-4-chloro-3-indolyl-beta-D-glucuronic acid (X-Gluc) substrate to recovered glass slides confirmed the activity of GUS as well as GFP in soil. Our results suggest that cotransformation with GFP and GUS can provide a valuable tool for the detection and monitoring of specific strains of T. harzianum released into the soil.

Anti-Bacterial Agents↗

PubMed2000

Prevention of encephalomyocarditis virus-induced diabetes by live recombinant Mycobacterium bovis bacillus Calmette-Guérin in susceptible mice.

The D variant of encephalomyocarditis (EMC-D) virus causes diabetes in susceptible mice by direct cytolysis of pancreatic beta-cells. cDNA covering the major outer capsid protein (VP1) of the EMC-D virus was cloned into Mycobacterium bovis bacillus Calmette-Guerin (BCG). None of the SJL/J mice immunized with live recombinant BCG-VP1 (rBCG-VP1) became diabetic when challenged with the highly diabetogenic EMC-D virus, but the control mice inoculated with normal BCG developed diabetes during the same challenge. VP1-specific antibodies (including neutralizing antibodies) were markedly increased over time and reached the maximum titer at week 10 after a single immunization. The plateau of the titer lasted longer than 4 weeks. Mice and guinea pigs immunized with live rBCG-VP1 showed strong delayed-type hypersensitivity to the VP1 of the EMC-D virus. The preventive immunity still worked effectively 10 months after the primary immunization. At that time, the VP1-specific antibody was almost undetectable in the bloodstream, but a large number of VP1-specific lymphocytes was found in the spleen of the immunized mice. Our results show that live rBCG-VP1 elicits effective humoral and long-lasting cellular immune responses against EMC-D virus infection that results in the prevention of virus-induced diabetes in susceptible mice.

Animals↗

PubMed1999

Protein kinase CKII interacts with and phosphorylates the SAG protein containing ring-H2 finger motif.

To investigate the biological function of CKII, we have identified proteins that interact with the subunits of CKII using the yeast two-hybrid system. Here we report that SAG, an antioxidant protein containing Ring-H2 finger motif, is a cellular partner associating with the beta subunit of CKII. SAG does not interact with the alpha subunit of CKII. Analysis of SAG deletion mutants indicates that the Ring-H2 motif of SAG is necessary and sufficient for its binding to the beta subunit of CKII. Recombinant SAG can be phosphorylated by CKII in vitro, providing evidence that the beta subunit mediates the interaction of CKII enzyme with substrate proteins. Overlay experiment shows that SAG and the beta subunit of CKII associate directly in vitro and that CKII-mediated phosphorylation of SAG does not affect the interaction between SAG and the beta subunit of CKII. Northern blot analysis indicates that both SAG and the beta subunit of CKII were relatively rich in human heart, liver, skeletal muscle, and pancreas, but were detected in only trace amounts in brain, placenta, and lung. Our present results suggest that CKII may play a role in the regulation of SAG function.

Calcium-Calmodulin-Dependent Protein Kinase Type 2↗

PubMed1999

Phosphorylation of ribosomal protein L5 by protein kinase CKII decreases its 5S rRNA binding activity.

We have recently reported that ribosomal protein L5 associates with the beta subunit of protein kinase CKII (CKII) (Kim, J.-M., Cha, J. -Y., Marshak, D. R., and Bae, Y.-S. (1996) Biochem. Biophys. Res. Commun. 226, 180-186). In this study, we demonstrate that CKII is able to catalyze the phosphorylation of the human L5 protein in vitro, which results in a decrease in 5S rRNA binding activity. Phosphoamino acid analysis indicated that the phosphorylation occurs on serine residues. Sequence analysis of cyanogen bromide-digested phosphopeptides and analysis of L5 deletion mutants indicates that the main phosphorylated residues are located within two fragments corresponding of residues 142-200 and residues 272-297 of the human L5. Based on our present results, we suggest that the phosphorylation of L5 by CKII is one of the mechanisms that regulates nucleolar targeting of 5S rRNA and/or ribosome assembly in the cell.

Amino Acid Sequence↗

PubMed1999

Effects of a water-soluble antitumor ether phosphonoinositide, D-myo-inositol 4-(hexadecyloxy)-3(S)-methoxybutanephosphonate (C4-PI), on inositol lipid metabolism in breast epithelial cancer cell lines.

We have demonstrated previously that D-myo-inositol 4-(hexadecyloxy)-3(S)-methoxybutanephosphonate (C4-PI), an isosteric phosphonate analog of phosphatidylinositol developed to inhibit inositol lipid metabolism, was unable to inhibit phosphatidylinositol (PI) 3-kinase activity. We now report the effects of the compound on other aspects of inositol metabolism. We demonstrated that C4-PI inhibits the activity of purified recombinant PI-phospholipase C-beta (PLC-beta) at all concentrations tested; it enhanced the activity of PI-PLC-gamma and PI-PLC-delta at low concentrations (10 microM), while severely inhibiting their activities at higher concentrations. In the breast cancer cell lines MCF-7 (estrogen receptor positive) and MDA-MB-468 (estrogen receptor negative), C4-PI had no effect on the uptake of D-myo-inositol but severely inhibited its incorporation into PI. In spite of the drastic decrease in PI synthesis, C4-PI did not affect the levels of inositol incorporated into phosphatidylinositol 4,5-bisphosphate (PIP2) in the cells. In vitro assays showed that C4-PI inhibited PI synthase activity (inhibition of 35% at 50 microM) but had little effect on PI 4-kinase activity (inhibition of 13% at 150 microM). C4-PI inhibited the proliferation of MCF-7 and MDA-MB-468 cell lines with IC(50) values of 12 and 18 microM. Taken together, the results suggest that the accumulation of [3H]inositol in PIP2 in cells incubated with C4-PI may be due to the inhibition of PIP2 hydrolysis in the cells with no effect on its synthesis. The role of these C4-PI-induced effects in the mechanism of growth inhibition by C4-PI remains to be established.

Antineoplastic Agents↗

PubMed1999

AHNAK, a protein that binds and activates phospholipase C-gamma1 in the presence of arachidonic acid.

We have recently shown that phospholipase C-gamma (PLC-gamma) is activated by tau, a neuronal cell-specific microtubule-associated protein, in the presence of arachidonic acid. We now report that non-neuronal tissues also contain a protein that can activate PLC-gamma in the presence of arachidonic acid. Purification of this activator from bovine lung cytosol yielded several proteins with apparent molecular sizes of 70-130 kDa. They were identified as fragments derived from an unusually large protein (approximately 700 kDa) named AHNAK, which comprises about 30 repeated motifs each 128 amino acids in length. Two AHNAK fragments containing one and four of the repeated motifs, respectively, were expressed as glutathione S-transferase fusion proteins. Both recombinant proteins activated PLC-gamma1 at nanomolar concentrations in the presence of arachidonic acid, suggesting that an intact AHNAK molecule contains multiple sites for PLC-gamma activation. The role of arachidonic acid was to promote a physical interaction between AHNAK and PLC-gamma1, and the activation by AHNAK and arachidonic acid was mainly attributable to reduction in the enzyme's apparent Km toward the substrate phosphatidylinositol 4,5-bisphosphate. Our results suggest that arachidonic acid liberated by phospholipase A2 can act as an additional trigger for PLC-gamma activation, constituting an alternative mechanism that is independent of tyrosine phosphorylation.

Amino Acid Sequence↗

PubMed1999

Trp-Lys-Tyr-Met-Val-D-Met stimulates superoxide generation and killing of Staphylococcus aureus via phospholipase D activation in human monocytes.

Among the phagocytic leukocytes, monocytes have the important role of clearing out parasitic microorganisms. They accomplish this through production of toxic metabolites of oxygen. Trp-Lys-Tyr-Met-Val-D-Met (WKYMVm), a peptide that stimulates phosphoinositide (PI) hydrolysis in human leukocytes, including monocytes, binds to a unique cell surface receptor and stimulates superoxide generation, killing of Staphylococcus aureus, and activation of phospholipase D (PLD) in human monocytes. Preincubation of the cells with a PI-specific phospholipase C (PLC) inhibitor (U-73122), protein kinase C inhibitor (GF109203X), or intracellular Ca2+ chelator (BAPTA/AM) before the peptide stimulus totally inhibits the peptide-induced PLD activation and superoxide generation. On the other hand, tyrosine kinase inhibitor genistein only partially inhibits the peptide-induced processes. The peptide-induced bacteria killing activity shares regulatory mechanisms for PLD activation with the superoxide generation, which is inhibited in the presence of 1-butanol. We suggest that the peptide stimulates PLD downstream of PLC activation and PLD activation in turn is essential for the peptide-induced immunological functions such as the superoxide generation and killing of bacteria by human monocytes.

Calcium↗

PubMed1999

Trp-Lys-Tyr-Met-Val-D-Met is a chemoattractant for human phagocytic cells.

Trp-Lys-Tyr-Met-Val-D-Met (WKYMVm) is a synthetic peptide that stimulates phosphoinositide (PI) hydrolysis in human leukocytes. The peptide binds to a unique cell surface receptor(s). Recently we had demonstrated that human neutrophils, monocytes, and B lymphocytes express this peptide-specific receptor and that stimulation of human leukocytes with the peptide leads to activation of the oxidative respiratory system and the bactericidal activity of neutrophils or monocytes. In this study we showed that the peptide induces chemotaxis of phagocytic leukocytes and studied the signaling pathway leading to chemotaxis in human monocytes. The peptide-induced monocyte chemotaxis is pertussis toxin (PTX)-sensitive. This fact correlates with the peptide's stimulation of PI hydrolysis and intracellular Ca2+ ([Ca2+]i) release, which is also PTX-sensitive. We demonstrate that the peptide-specific receptor is different from receptor(s) for monocyte chemoattractant protein-1 (MCP-1). We also show that intracellular signaling of WKYMVm leading to monocyte chemotaxis is different from that of MCP-1. The peptide-mediated monocyte chemotaxis is insensitive to protein kinase C (PKC) inhibitor (GF109203X) and butan-1-ol, ruling out PKC and phospholipase D participation in this process. On the other hand, a tyrosine kinase inhibitor (genistein) and RhoA inhibitor (C3 transferase) curtailed the peptide-induced chemotaxis in a concentration-dependent manner, implying the involvement of tyrosine kinase and RhoA, respectively. Treatment of human monocytes with the peptide stimulates tyrosine phosphorylation of several cellular proteins, including p125FAK and Pyk2 and translocation of RhoA from the cytosol to the membrane. We conclude that WKYMVm induces chemotaxis of human phagocytic leukocytes via unique receptors and signaling.

1-Butanol↗

PubMed1999

Regulation of phospholipase C isozymes: activation of phospholipase C-gamma in the absence of tyrosine-phosphorylation.

Activation of PLC-gamma isozymes in response to various agonists involves tyrosine phosphorylation of the effector enzymes. Recent evidence indicates that PLC-gamma isozymes are additionally activated by phosphatidic acid, phosphatidylinositol 3,4,5-trisphosphate and arachidonic acid in the absence of PLC-gamma tyrosine phosphorylation. These lipid-derived messengers are the immediate products of phospholipase D, phosphatidylinositol 3-kinase, and phospholipase A2, enzymes which are often stimulated along with PLC-gamma in response to an agonist. Furthermore, phosphatidylinositol 4,5-bisphosphate acts as a substrate for both PLC-gamma and phosphatidylinositol 3-kinase and as an activator for phospholipase D and phospholipase A2. These results reveal an elaborate mechanism of cross-talk and mutual regulation between four effector enzymes that participate in receptor signaling by acting on phospholipids.

Animals↗

PubMed1999

Trp-Lys-Tyr-Met-Val-Met activates mitogen-activated protein kinase via a PI-3 kinase-mediated pathway independent of PKC.

Trp-Lys-Tyr-Met-Val-Met (WKYMVM) is a novel potent peptide which can stimulate phosphoinositide hydrolysis in U937 as well as U266 and HL-60 cells (Baek et al., J. Biol. Chem. 271, 8170 (1996)). The peptide also induces superoxide generation in human neutrophils (Seo et al., J. Immunol. 158, 1896 (1997)). However, the signaling pathway down-stream of PLC set in motion by the peptide is not yet completely understood. We studied the signaling pathway of the peptide with the goal of elucidating the mechanism of the peptide's action. WKYMVM induced a rapid and transient activation of the ERKs in human histiocytic lymphoma cells, U937. The ERK1 activation peaked at 5 min and returned to the basal level after 30 min. The ERK1 stimulation by the peptide was partially inhibited by pretreatment of the cells with pertussis toxin (PTX), implicating G-protein involvement in the peptide's action. Pretreatment of staurosporine, protein kinase C (PKC) inhibitor, or PKC down-regulating PMA had no impact on the ERK1 activation by the peptide, indicating that the signaling pathway is independent of PKC activation. Pretreatment of the cells with neomycin and intracellular Ca2+ mobilizing reagents had also no effect on the ERK1 activation by the peptide. However, pretreatment with wortmannin or LY294002, the inhibitor of phosphatidylinositol 3 kinase (PI-3K), strongly inhibited peptide-stimulated ERK1 activation. Our results suggest that PI-3K may be an important participant in the ERK cascade induced by the peptide. Furthermore, the treatment of U937 cells with the peptide activated p74Raf-1, an upstream kinase of ERK. Taken together, our results suggest that the peptide activate ERK via a G-protein/PI-3K/Ras/Raf-1 mediated signaling pathway in U937 cells.

Blotting, Western↗

PubMed1998

Characterization of protein interaction among subunits of protein kinase CKII in vivo and in vitro.

Protein kinase CKII (CKII) is a ubiquitous protein serine/threonine kinase. CKII usually exists in tetrameric complexes composed of two catalytic (CKII alpha and/or CKII alpha') and two regulatory (CKII beta) subunits. In the present study, using a combined in vivo and in vitro approach, we have investigated the role of CKII subunits in the formation of the tetrameric structure of CKII and the formation of the polymeric structure of CKII holoenzyme. Our in vivo experiments show that CKII beta interacts with either another CKII beta or CKII alpha and that CKII alpha does not interact with another CKII alpha (or CKII alpha'). Our in vitro experiments also show that CKII beta is able to associate with both CKII alpha and another CKII beta and that CKII alpha exists as a monomeric form in solution. These data indicate that CKII beta mediates the formation of a tetramer by both the dimerization of CKII beta and the interaction of CKII beta with CKII alpha. The results of this study also suggest that CKII beta may be involved in the formation of the polymeric structure of the CKII holoenzyme.

Casein Kinase II↗

PubMed1998

Activation of phospholipase C-gamma by phosphatidylinositol 3,4,5-trisphosphate.

Signal transduction across cell membranes often involves the activation of both phosphatidylinositol (PI)-specific phospholipase C (PLC) and phosphoinositide 3-kinase (PI 3-kinase). Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), a substrate for both enzymes, is converted to phosphatidylinositol 3,4, 5-trisphosphate (PI(3,4,5)P3) by the action of PI 3-kinase. Here, we show that PI(3,4,5)P3 activates purified PLC-gamma isozymes by interacting with their Src homology 2 domains. Furthermore, the expression of an activated catalytic subunit of PI 3-kinase in COS-7 cells resulted in an increase in inositol phosphate formation, whereas platelet-derived growth factor-induced PLC activation in NIH 3T3 cells was markedly inhibited by the specific PI 3-kinase inhibitor LY294002. These results suggest that receptors coupled to PI 3-kinase may activate PLC-gamma isozymes indirectly, in the absence of PLC-gamma tyrosine phosphorylation, through the generation of PI(3,4,5)P3.

Animals↗

PubMed1998

Distribution of the receptor for a novel peptide stimulating phosphoinositide hydrolysis in human leukocytes.

OBJECTIVES: Previously we reported that a synthetic peptide, WKYMVm, stimulates phosphoinositide (PI) hydrolysis in several hematopoietic cell lines and human neutrophils. In addition, the peptide induces superoxide generation in human neutrophils. The biochemical stimulation appeared to be mediated by specific receptors on leukocytes. In this report we try to find whether the specific receptor(s) exists on specific types of cells in human leukocytes. DESIGN AND METHODS: To study distribution of the peptide-specific receptors, we prepared a probe, biotin-labeled WKYMVm, and analyzed distribution of the peptide receptor using a biochemical study, cytochemical staining, and flow cytometric analysis. RESULTS: The peptide-specific receptors are expressed in several human leukocytes including granulocytes, monocytes, and B-lymphocytes but not T-lymphocytes and erythrocytes. CONCLUSIONS: These data suggest that the receptors for the peptide restricted to certain types of leukocytes and this specificity is probably related to the specific functions of the stimulated leukocytes.

Amino Acid Sequence↗

PubMed1997

The highly basic ribosomal protein L41 interacts with the beta subunit of protein kinase CKII and stimulates phosphorylation of DNA topoisomerase IIalpha by CKII.

Protein kinase CKII (CKII) is a heterotetramer composed of two catalytic (alpha or alpha') and two regulatory (beta) subunits. Using the yeast two-hybrid system, we have identified the highly basic, ribosomal protein L41 as a cellular protein capable of interacting with the beta subunit of CKII. We show, furthermore, using purified proteins, that L41 protein and CKIIbeta associate directly in vitro. L41 protein is not a substrate for CKII phosphorylation, and it does not stimulate CKII activity with either beta-casein or synthetic peptide substrate (RRREEETEEE). However, L41 protein stimulates the phosphorylation of DNA topoisomerase IIalpha by CKII by 2.5 times. Additionally, L41 protein enhances the autophosphorylation of CKIIalpha. The data indicate that L41 protein associates with CKII and can modulate its activity toward a specific substrate or substrates. The direct interaction of CKIIbeta with ribosomal proteins also suggests that CKIIbeta itself or CKII holoenzyme may be involved in ribosome assembly or translational control.

Amino Acid Sequence↗

PubMed1997

Synaptojanin inhibition of phospholipase D activity by hydrolysis of phosphatidylinositol 4,5-bisphosphate.

A 150-kDa protein that inhibits phospholipase D (PLD) activity stimulated by ADP-ribosylation factor and phosphatidylinositol 4, 5-bisphosphate (PI(4,5)P2) was previously purified from rat brain. The sequences of peptides derived from the purified PLD inhibitor now identify it as synaptojanin, a nerve terminal protein that has been implicated in the endocytosis of fused synaptic vesicles and shown to be a member of the inositol polyphosphate 5-phosphatase family. Further characterization of the enzymatic properties of synaptojanin now shows that it hydrolyzes only the 5-phosphate from inositol 1,4,5-trisphosphate (I(1,4,5)P3) and that it does not catalyze the dephosphorylation of either I(1,3,4)P3 or inositol 1, 4-bisphosphate. However, synaptojanin hydrolyzes both the 4- and 5-phosphates of PI(4,5)P2 and the 4-phosphate of phosphatidylinositol 4-phosphate, converting both compounds to phosphatidylinositol. Magnesium is required for the hydrolysis of I(1,4,5)P3, but not for that of phosphoinositides, by synaptojanin. The inhibition of PLD by synaptojanin is attributable to its ability to hydrolyze PI(4,5)P2. Synaptojanin did not inhibit PLD in the absence of PI(4,5)P2, and the extent of PLD inhibition was related to the extent of PI(4,5)P2 hydrolysis in substrate vesicles. It has been proposed that the biosynthesis of PI(4,5)P2 and the activation of PLD by ADP-ribosylation factor constitute a positive loop to increase rapidly the concentrations of PI(4,5)P2 and phosphatidic acid (PA) during membrane vesiculation. The PA thus produced, probably together with PI(4,5)P2, facilitates vesicle coat assembly. The hydrolysis of PI(4,5)P2, and consequent inhibition of PLD, by synaptojanin might therefore constitute a mechanism to halt the positive loop connecting PI(4,5)P2 and PA during the endocytotic cycle of synaptic vesicles and serve as a signal for uncoating.

Amino Acid Sequence↗

PubMed1997

Inhibition of phospholipase D by clathrin assembly protein 3 (AP3).

In the accompanying paper (Chung, J.-K., Sekiya, F., Kang, H.-S., Lee, C., Han, J.-S., Kim, S. R., Bae, Y. S., Morris, A. J., and Rhee, S. G. (1997) J. Biol. Chem. 272, 15980-15985), synaptojanin is identified as a protein that inhibits phospholipase D (PLD) activity stimulated by ADP-ribosylation factor and phosphatidylinositol 4, 5-bisphosphate (PI(4,5)P2). Here, the purification from rat brain cytosol of another PLD-inhibitory protein that is immunologically distinct from synaptojanin is described, and this protein is identified as clathrin assembly protein 3 (AP3) by peptide sequencing and immunoblot analysis. AP3 binds both inositol hexakisphosphate and preassembled clathrin cages with high affinity. However, neither inositol hexakisphosphate binding nor clathrin cage binding affected the ability of AP3 to inhibit PLD. AP3 also binds to PI(4,5)P2 with low affinity. But the PI(4,5)P2 binding was not responsible for PLD inhibition, because the potency and efficacy of AP3 as an inhibitor of PLD were similar in the absence and presence of PI(4,5)P2. A bacterially expressed fusion protein, glutathione S-transferase-AP3 (GST-AP3), also inhibited PLD with a potency equal to that of brain AP3. The inhibitory effect of AP3 appeared to be the result of direct interaction between AP3 and PLD because PLD bound GST-AP3 in an in vitro binding assay. Using GST fusion proteins containing various AP3 sequences, we found that the sequence extending from residues Pro-290 to Lys-320 of AP3 is critical for both inhibition of and binding to PLD. The fact that AP3 is a synapse-specific protein indicates that the AP3-dependent inhibition of PLD might play a regulatory role that is restricted to the rapid cycling of synaptic vesicles.

Adaptor Protein Complex 3↗