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Fumio Sakane - One of the best experts on this subject based on the ideXlab platform.
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the Pleckstrin Homology Domain of diacylglycerol kinase η strongly and selectively binds to phosphatidylinositol 4 5 bisphosphate
Journal of Biological Chemistry, 2016Co-Authors: Aiko Kume, Koki Kawase, Suguru Komenoi, Takako Usuki, Ena Takeshita, Hiromichi Sakai, Fumio SakaneAbstract:Type II diacylglycerol kinase (DGK) isozymes (δ, η, and κ) have a Pleckstrin Homology Domain (PH) at their N termini. Here, we investigated the lipid binding properties of the PHs of type II DGK isozymes using protein-lipid overlay and liposome binding assays. The PH of DGKη showed the most pronounced binding activity to phosphatidylinositol (PI) 4,5-bisphosphate (PI(4,5)P2) among the various glycero- and sphingolipids including PI 3,4,5-trisphosphate, PI 3,4-bisphosphate, PI 3-phosphate, PI 4-phosphate, and PI 5-phosphate. Moreover, the PI(4,5)P2binding activity of the DGKη-PH was significantly stronger than that of other type II DGK isozymes. Notably, compared with the PH of phospholipase C (PLC) δ1, which is generally utilized as a cellular PI(4,5)P2- probe, the DGKη-PH is equal to or superior than the PLCδ1-PH in terms of affinity and selectivity for PI(4,5)P2 Furthermore, in COS-7 cells, GFP-fused wild-type DGKη1 and its PH partly translocated from the cytoplasm to the plasma membrane where the PLCδ1-PH was co-localized in response to hyperosmotic stress in an inositol 5-phosphatase-sensitive manner, whereas a PH deletion mutant did not. Moreover, K74A and R85A mutants of DGKη-PH, which lack the conserved basic amino acids thought to ligate PI(4,5)P2, were indeed unable to bind to PI(4,5)P2and co-localize with the PLCδ1-PH even in osmotically shocked cells. Overexpression of wild-type DGKη1 enhanced EGF-dependent phosphorylation of ERK, whereas either K74A or R85A mutant did not. Taken together, these results indicate that the DGKη-PH preferentially interacts with PI(4,5)P2and has crucial roles in regulating the subcellular localization and physiological function of DGKη. Moreover, the DGKη-PH could serve as an excellent cellular sensor for PI(4,5)P2.
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The plasma membrane translocation of diacylglycerol kinase delta1 is negatively regulated by conventional protein kinase C-dependent phosphorylation at Ser-22 and Ser-26 within the Pleckstrin Homology Domain.
The Biochemical journal, 2004Co-Authors: Shin-ichi Imai, Masahiro Kai, Keiko Yamada, Hideo Kanoh, Fumio SakaneAbstract:DGK (diacylglycerol kinase) regulates the concentration of two bioactive lipids, diacylglycerol and phosphatidic acid. DGKdelta1 or its PH (Pleckstrin Homology) Domain alone has been shown to be translocated to the plasma membrane from the cytoplasm in PMA-treated cells. In the present study, we identified Ser-22 and Ser-26 within the PH Domain as the PMA- and epidermal-growth-factor-dependent phosphorylation sites of DGKdelta1. Experiments in vitro and with intact cells suggested that the cPKC (conventional protein kinase C) phosphorylated these Ser residues directly. Puzzlingly, alanine/asparagine mutants at Ser-22 and Ser-26 of DGKdelta1 and its PH Domain are still persistently translocated by PMA treatment, suggesting that the PH Domain phosphorylation is not responsible for the enzyme translocation and that the translocation was caused by a PMA-dependent, but cPKC-independent, process yet to be identified. Interestingly, the aspartate mutation, which mimics phosphoserine, at Ser-22 or Ser-26, inhibited the translocation of full-length DGKdelta1 and the PH Domain markedly, suggesting that the phosphorylation regulates negatively the enzyme translocation. Our results provide evidence of the phosphorylation of the DGKdelta1 PH Domain by cPKC, and suggest that the phosphorylation is involved in the control of subcellular localization of DGKdelta1.
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The plasma membrane translocation of diacylglycerol kinase δ1 is negatively regulated by conventional protein kinase C-dependent phosphorylation at Ser-22 and Ser-26 within the Pleckstrin Homology Domain
Biochemical Journal, 2004Co-Authors: Shin-ichi Imai, Masahiro Kai, Keiko Yamada, Hideo Kanoh, Fumio SakaneAbstract:DGK (diacylglycerol kinase) regulates the concentration of two bioactive lipids, diacylglycerol and phosphatidic acid. DGKdelta1 or its PH (Pleckstrin Homology) Domain alone has been shown to be translocated to the plasma membrane from the cytoplasm in PMA-treated cells. In the present study, we identified Ser-22 and Ser-26 within the PH Domain as the PMA- and epidermal-growth-factor-dependent phosphorylation sites of DGKdelta1. Experiments in vitro and with intact cells suggested that the cPKC (conventional protein kinase C) phosphorylated these Ser residues directly. Puzzlingly, alanine/asparagine mutants at Ser-22 and Ser-26 of DGKdelta1 and its PH Domain are still persistently translocated by PMA treatment, suggesting that the PH Domain phosphorylation is not responsible for the enzyme translocation and that the translocation was caused by a PMA-dependent, but cPKC-independent, process yet to be identified. Interestingly, the aspartate mutation, which mimics phosphoserine, at Ser-22 or Ser-26, inhibited the translocation of full-length DGKdelta1 and the PH Domain markedly, suggesting that the phosphorylation regulates negatively the enzyme translocation. Our results provide evidence of the phosphorylation of the DGKdelta1 PH Domain by cPKC, and suggest that the phosphorylation is involved in the control of subcellular localization of DGKdelta1.
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molecular cloning of a novel diacylglycerol kinase isozyme with a Pleckstrin Homology Domain and a c terminal tail similar to those of the eph family of protein tyrosine kinases
Journal of Biological Chemistry, 1996Co-Authors: Fumio Sakane, Shin-ichi Imai, Masahiro Kai, Ikuo Wada, Hideo KanohAbstract:Abstract A fourth member of the diacylglycerol kinase (DGK) gene family termed DGK was cloned from the human testis cDNA library. The cDNA sequence contains an open reading frame of 3,507 nucleotides encoding a putative DGK protein of 130,006 Da. Interestingly, the new DGK isozyme contains a Pleckstrin Homology Domain found in a number of proteins involved in signal transduction. Furthermore, the C-terminal tail of this isozyme is very similar to those of the EPH family of receptor tyrosine kinases. The primary structure of the -isozyme also has two cysteine-rich zinc finger-like structures (C3 region) and the C-terminal C4 region, both of which have been commonly found in the three isozymes previously cloned (DGKs α, β and ). However, DGK lacks the EF-hand motifs (C2) and contains a long Glu- and Ser-rich insertion (317 residues), which divides the C4 region into two portions. Taken together, these structural features of DGK indicate that this isozyme belongs to a DGK subfamily distinct from that consisting of DGKs α, β, and . Inc reased DGK activity without marked preference to arachidonoyl type of diacylglycerol was detected in the particulate fraction of COS-7 cells expressing the transfected DGK cDNA. The enzyme activity was independent of phosphatidylserine, which is a common activator for the previously sequenced DGKs. Northern blot analysis showed that the DGK mRNA (6.3 kilobases) is most abundant in human skeletal muscle but undetectable in the brain, thymus, and retina. This expression pattern is different from those of the previously cloned DGKs. Our results show that the DGK gene family consists of at least two subfamilies consisting of enzymes with distinct structural characteristics and that each cell type probably expresses its own characteristic repertoire of DGKs whose functions may be regulated through different signal transduction pathways.
Hartmut Oschkinat - One of the best experts on this subject based on the ideXlab platform.
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automated noesy interpretation with ambiguous distance restraints the refined nmr solution structure of the Pleckstrin Homology Domain from beta spectrin
Journal of Molecular Biology, 1997Co-Authors: Michael Nilges, Maria J Macias, Sean I Odonoghue, Hartmut OschkinatAbstract:We have used a novel, largely automated, calculation method to refine the NMR solution structure of the Pleckstrin Homology Domain of β-spectrin. The method is called ARIA for Ambiguous Restraints for Iterative Assignment. The starting point for ARIA is an almost complete assignment of the proton chemical shifts, and a list of partially assigned NOEs, mostly sequential and secondary structure NOEs. The restraint list is then augmented by automatically interpreting peak lists generated by automated peak-picking. The central task of ARIA is the assignment of ambiguous NOEs during the structure calculation using a combination of ambiguous distance restraints and an iterative assignment strategy. In addition, ARIA calibrates ambiguous NOEs to derive distance restraints, merges overlapping data sets to remove duplicate information, and uses empirical rules to identify erroneous peaks. While the distance restraints for the structure calculations were exclusively extracted from homonuclear 2D experiments, ARIA is especially suited for the analysis of multidimensional spectra. Applied to the Pleckstrin Homology Domain, ARIA generated structures of good quality, and of sufficiently high accuracy to solve the X-ray crystal structure of the same Domain by molecular replacement. The comparison of the free NMR solution structure to the X-ray structure, which is complexed to d-myo-inositol-1,4,5-triphosphate, shows that the ligand primarily induces a disorder-order transition in the binding loops, which are disordered in the NMR ensemble but well ordered in the crystal. The structural core of the protein is unaffected, as evidenced by a backbone root-mean-square difference between the average NMR coordinates and the X-ray crystal structure for the secondary structure elements of less than 0.6 A.
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automated noesy interpretation with ambiguous distance restraints the refined nmr solution structure of the Pleckstrin Homology Domain from beta spectrin
Journal of Molecular Biology, 1997Co-Authors: Michael Nilges, Maria J Macias, Sean I Odonoghue, Hartmut OschkinatAbstract:We have used a novel, largely automated, calculation method to refine the NMR solution structure of the Pleckstrin Homology Domain of beta-spectrin. The method is called ARIA for Ambiguous Restraints for Iterative Assignment. The starting point for ARIA is an almost complete assignment of the proton chemical shifts, and a list of partially assigned NOEs, mostly sequential and secondary structure NOEs. The restraint list is then augmented by automatically interpreting peak lists generated by automated peak-picking. The central task of ARIA is the assignment of ambiguous NOEs during the structure calculation using a combination of ambiguous distance restraints and an iterative assignment strategy. In addition, ARIA calibrates ambiguous NOEs to derive distance restraints, merges overlapping data sets to remove duplicate information, and uses empirical rules to identify erroneous peaks. While the distance restraints for the structure calculations were exclusively extracted from homonuclear 2D experiments, ARIA is especially suited for the analysis of multidimensional spectra. Applied to the Pleckstrin Homology Domain, ARIA generated structures of good quality, and of sufficiently high accuracy to solve the X-ray crystal structure of the same Domain by molecular replacement. The comparison of the free NMR solution structure to the X-ray structure, which is complexed to D-myo-inositol-1,4,5-triphosphate, shows that the ligand primarily induces a disorder-order transition in the binding loops, which are disordered in the NMR ensemble but well ordered in the crystal. The structural core of the protein is unaffected, as evidenced by a backbone root-mean-square difference between the average NMR coordinates and the X-ray crystal structure for the secondary structure elements of less than 0.6 A.
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structure of the Pleckstrin Homology Domain from beta spectrin
Nature, 1994Co-Authors: Maria J Macias, Michael Nilges, Andrea Musacchio, Hannes Ponstingl, Matti Saraste, Hartmut OschkinatAbstract:THE ‘Pleckstrin Homology’ or PH Domain is a 100-residue protein module. It is present in many kinases, different isoforms of phospholipase C, GTPase-activating proteins and nucleotide-exchange factors1–4. Its function is not known, but many proteins that contain a PH Domain interact with GTP-binding proteins5. The PH Domain in β-adrenergic receptor kinase may be involved in binding to the βγ subunits of a trimeric G-protein3, 4, 6, 7. We report here the three-dimensional structure of the PH Domain of the cytoskeletal protein spectrin using homonuclear nuclear magnetic resonance. The core of the molecule is an antiparallel β-sheet consisting of seven strands. The C terminus is folded into a long α-helix, and another helix is present in one of the surface loops. The molecule is electrostatically polarized and contains a pocket which may be involved in the binding of a ligand. There is a distant relationship to the peptidyl-prolyl-cis-trans-isomerase FKBP in which this pocket is involved in the binding of the macrocyclic compound FK506(refs 8–11).
Richard A Roth - One of the best experts on this subject based on the ideXlab platform.
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akt a Pleckstrin Homology Domain containing kinase is activated primarily by phosphorylation
Journal of Biological Chemistry, 1996Co-Authors: Aimee D Kohn, Fumito Takeuchi, Richard A RothAbstract:Abstract Akt is a serine/threonine kinase that is stimulated by receptor tyrosine kinases and contains a Pleckstrin Homology Domain. One model proposed to explain this activation suggests that receptor tyrosine kinases stimulate a phosphatidylinositol 3-kinase whose lipid products directly activate Akt kinase by interacting with its Pleckstrin Homology Domain. In the present study, we show, in three cell types, that Akt does not require its Pleckstrin Homology Domain to respond to either insulin or platelet-derived growth factor. Moreover, attachment of the src myristoylation signal to target Akt, without its Pleckstrin Homology Domain, to the membrane constitutively activates Akt by causing an increase in its basal level of phosphorylation. This constitutively active form of Akt can also activate p70S6K, indicating that the Pleckstrin Homology Domain is not necessary for downstream interactions. Fusion of the inter src Homology 2 Domain from the p85 regulatory subunit of the phosphatidylinositol 3-kinase to Akt also constitutively activated Akt and induced an association with the lipid kinase. Phosphorylation of this fusion protein still critically contributes toward its increased activity. The sum of these results indicates that the primary mechanism of Akt activation is via protein phosphorylation.
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insulin stimulates the kinase activity of rac pk a Pleckstrin Homology Domain containing ser thr kinase
The EMBO Journal, 1995Co-Authors: Aimee D Kohn, Kristina S Kovacina, Richard A RothAbstract:In the present study, insulin is shown to rapidly stimulate by 8- to 12-fold the enzymatic activity of RAC-PK alpha, a Pleckstrin Homology Domain containing ser/thr kinase. In contrast, activation of protein kinase C by phorbol esters had almost no effect on the enzymatic activity of RAC-PK alpha. Insulin activation was accompanied by a shift in molecular weight of the RAC-PK alpha protein, and the activated kinase was deactivated by treatment with a phosphatase, indicating that insulin activated the enzyme by stimulating its phosphorylation. This insulin-induced shift in RAC-PK was also observed in primary rat epididymal adipocytes, as well as in a muscle cell line called C2C12 cells. The insulin-stimulated increase in RAC-PK alpha activity was inhibited by wortmannin (an inhibitor of phosphatidylinositol 3-kinase) in a dose-dependent manner with a half-maximal inhibition of 10 nM, but not by 20 ng/ml of rapamycin. Activation of RAC-PK alpha activity was also observed in a variant RAC lacking the Pleckstrin Homology Domain. These results indicate that RAC-PK alpha activity can be regulated by the insulin receptor. RAC-PK alpha may therefore play a general role in intracellular signaling mediated by receptor tyrosine kinases.
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Insulin stimulates the kinase activity of RAC‐PK, a Pleckstrin Homology Domain containing ser/thr kinase.
The EMBO journal, 1995Co-Authors: Aimee D Kohn, Kristina S Kovacina, Richard A RothAbstract:In the present study, insulin is shown to rapidly stimulate by 8- to 12-fold the enzymatic activity of RAC-PK alpha, a Pleckstrin Homology Domain containing ser/thr kinase. In contrast, activation of protein kinase C by phorbol esters had almost no effect on the enzymatic activity of RAC-PK alpha. Insulin activation was accompanied by a shift in molecular weight of the RAC-PK alpha protein, and the activated kinase was deactivated by treatment with a phosphatase, indicating that insulin activated the enzyme by stimulating its phosphorylation. This insulin-induced shift in RAC-PK was also observed in primary rat epididymal adipocytes, as well as in a muscle cell line called C2C12 cells. The insulin-stimulated increase in RAC-PK alpha activity was inhibited by wortmannin (an inhibitor of phosphatidylinositol 3-kinase) in a dose-dependent manner with a half-maximal inhibition of 10 nM, but not by 20 ng/ml of rapamycin. Activation of RAC-PK alpha activity was also observed in a variant RAC lacking the Pleckstrin Homology Domain. These results indicate that RAC-PK alpha activity can be regulated by the insulin receptor. RAC-PK alpha may therefore play a general role in intracellular signaling mediated by receptor tyrosine kinases.
Aimee D Kohn - One of the best experts on this subject based on the ideXlab platform.
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akt a Pleckstrin Homology Domain containing kinase is activated primarily by phosphorylation
Journal of Biological Chemistry, 1996Co-Authors: Aimee D Kohn, Fumito Takeuchi, Richard A RothAbstract:Abstract Akt is a serine/threonine kinase that is stimulated by receptor tyrosine kinases and contains a Pleckstrin Homology Domain. One model proposed to explain this activation suggests that receptor tyrosine kinases stimulate a phosphatidylinositol 3-kinase whose lipid products directly activate Akt kinase by interacting with its Pleckstrin Homology Domain. In the present study, we show, in three cell types, that Akt does not require its Pleckstrin Homology Domain to respond to either insulin or platelet-derived growth factor. Moreover, attachment of the src myristoylation signal to target Akt, without its Pleckstrin Homology Domain, to the membrane constitutively activates Akt by causing an increase in its basal level of phosphorylation. This constitutively active form of Akt can also activate p70S6K, indicating that the Pleckstrin Homology Domain is not necessary for downstream interactions. Fusion of the inter src Homology 2 Domain from the p85 regulatory subunit of the phosphatidylinositol 3-kinase to Akt also constitutively activated Akt and induced an association with the lipid kinase. Phosphorylation of this fusion protein still critically contributes toward its increased activity. The sum of these results indicates that the primary mechanism of Akt activation is via protein phosphorylation.
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insulin stimulates the kinase activity of rac pk a Pleckstrin Homology Domain containing ser thr kinase
The EMBO Journal, 1995Co-Authors: Aimee D Kohn, Kristina S Kovacina, Richard A RothAbstract:In the present study, insulin is shown to rapidly stimulate by 8- to 12-fold the enzymatic activity of RAC-PK alpha, a Pleckstrin Homology Domain containing ser/thr kinase. In contrast, activation of protein kinase C by phorbol esters had almost no effect on the enzymatic activity of RAC-PK alpha. Insulin activation was accompanied by a shift in molecular weight of the RAC-PK alpha protein, and the activated kinase was deactivated by treatment with a phosphatase, indicating that insulin activated the enzyme by stimulating its phosphorylation. This insulin-induced shift in RAC-PK was also observed in primary rat epididymal adipocytes, as well as in a muscle cell line called C2C12 cells. The insulin-stimulated increase in RAC-PK alpha activity was inhibited by wortmannin (an inhibitor of phosphatidylinositol 3-kinase) in a dose-dependent manner with a half-maximal inhibition of 10 nM, but not by 20 ng/ml of rapamycin. Activation of RAC-PK alpha activity was also observed in a variant RAC lacking the Pleckstrin Homology Domain. These results indicate that RAC-PK alpha activity can be regulated by the insulin receptor. RAC-PK alpha may therefore play a general role in intracellular signaling mediated by receptor tyrosine kinases.
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Insulin stimulates the kinase activity of RAC‐PK, a Pleckstrin Homology Domain containing ser/thr kinase.
The EMBO journal, 1995Co-Authors: Aimee D Kohn, Kristina S Kovacina, Richard A RothAbstract:In the present study, insulin is shown to rapidly stimulate by 8- to 12-fold the enzymatic activity of RAC-PK alpha, a Pleckstrin Homology Domain containing ser/thr kinase. In contrast, activation of protein kinase C by phorbol esters had almost no effect on the enzymatic activity of RAC-PK alpha. Insulin activation was accompanied by a shift in molecular weight of the RAC-PK alpha protein, and the activated kinase was deactivated by treatment with a phosphatase, indicating that insulin activated the enzyme by stimulating its phosphorylation. This insulin-induced shift in RAC-PK was also observed in primary rat epididymal adipocytes, as well as in a muscle cell line called C2C12 cells. The insulin-stimulated increase in RAC-PK alpha activity was inhibited by wortmannin (an inhibitor of phosphatidylinositol 3-kinase) in a dose-dependent manner with a half-maximal inhibition of 10 nM, but not by 20 ng/ml of rapamycin. Activation of RAC-PK alpha activity was also observed in a variant RAC lacking the Pleckstrin Homology Domain. These results indicate that RAC-PK alpha activity can be regulated by the insulin receptor. RAC-PK alpha may therefore play a general role in intracellular signaling mediated by receptor tyrosine kinases.
Shin-ichi Imai - One of the best experts on this subject based on the ideXlab platform.
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The plasma membrane translocation of diacylglycerol kinase delta1 is negatively regulated by conventional protein kinase C-dependent phosphorylation at Ser-22 and Ser-26 within the Pleckstrin Homology Domain.
The Biochemical journal, 2004Co-Authors: Shin-ichi Imai, Masahiro Kai, Keiko Yamada, Hideo Kanoh, Fumio SakaneAbstract:DGK (diacylglycerol kinase) regulates the concentration of two bioactive lipids, diacylglycerol and phosphatidic acid. DGKdelta1 or its PH (Pleckstrin Homology) Domain alone has been shown to be translocated to the plasma membrane from the cytoplasm in PMA-treated cells. In the present study, we identified Ser-22 and Ser-26 within the PH Domain as the PMA- and epidermal-growth-factor-dependent phosphorylation sites of DGKdelta1. Experiments in vitro and with intact cells suggested that the cPKC (conventional protein kinase C) phosphorylated these Ser residues directly. Puzzlingly, alanine/asparagine mutants at Ser-22 and Ser-26 of DGKdelta1 and its PH Domain are still persistently translocated by PMA treatment, suggesting that the PH Domain phosphorylation is not responsible for the enzyme translocation and that the translocation was caused by a PMA-dependent, but cPKC-independent, process yet to be identified. Interestingly, the aspartate mutation, which mimics phosphoserine, at Ser-22 or Ser-26, inhibited the translocation of full-length DGKdelta1 and the PH Domain markedly, suggesting that the phosphorylation regulates negatively the enzyme translocation. Our results provide evidence of the phosphorylation of the DGKdelta1 PH Domain by cPKC, and suggest that the phosphorylation is involved in the control of subcellular localization of DGKdelta1.
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The plasma membrane translocation of diacylglycerol kinase δ1 is negatively regulated by conventional protein kinase C-dependent phosphorylation at Ser-22 and Ser-26 within the Pleckstrin Homology Domain
Biochemical Journal, 2004Co-Authors: Shin-ichi Imai, Masahiro Kai, Keiko Yamada, Hideo Kanoh, Fumio SakaneAbstract:DGK (diacylglycerol kinase) regulates the concentration of two bioactive lipids, diacylglycerol and phosphatidic acid. DGKdelta1 or its PH (Pleckstrin Homology) Domain alone has been shown to be translocated to the plasma membrane from the cytoplasm in PMA-treated cells. In the present study, we identified Ser-22 and Ser-26 within the PH Domain as the PMA- and epidermal-growth-factor-dependent phosphorylation sites of DGKdelta1. Experiments in vitro and with intact cells suggested that the cPKC (conventional protein kinase C) phosphorylated these Ser residues directly. Puzzlingly, alanine/asparagine mutants at Ser-22 and Ser-26 of DGKdelta1 and its PH Domain are still persistently translocated by PMA treatment, suggesting that the PH Domain phosphorylation is not responsible for the enzyme translocation and that the translocation was caused by a PMA-dependent, but cPKC-independent, process yet to be identified. Interestingly, the aspartate mutation, which mimics phosphoserine, at Ser-22 or Ser-26, inhibited the translocation of full-length DGKdelta1 and the PH Domain markedly, suggesting that the phosphorylation regulates negatively the enzyme translocation. Our results provide evidence of the phosphorylation of the DGKdelta1 PH Domain by cPKC, and suggest that the phosphorylation is involved in the control of subcellular localization of DGKdelta1.
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molecular cloning of a novel diacylglycerol kinase isozyme with a Pleckstrin Homology Domain and a c terminal tail similar to those of the eph family of protein tyrosine kinases
Journal of Biological Chemistry, 1996Co-Authors: Fumio Sakane, Shin-ichi Imai, Masahiro Kai, Ikuo Wada, Hideo KanohAbstract:Abstract A fourth member of the diacylglycerol kinase (DGK) gene family termed DGK was cloned from the human testis cDNA library. The cDNA sequence contains an open reading frame of 3,507 nucleotides encoding a putative DGK protein of 130,006 Da. Interestingly, the new DGK isozyme contains a Pleckstrin Homology Domain found in a number of proteins involved in signal transduction. Furthermore, the C-terminal tail of this isozyme is very similar to those of the EPH family of receptor tyrosine kinases. The primary structure of the -isozyme also has two cysteine-rich zinc finger-like structures (C3 region) and the C-terminal C4 region, both of which have been commonly found in the three isozymes previously cloned (DGKs α, β and ). However, DGK lacks the EF-hand motifs (C2) and contains a long Glu- and Ser-rich insertion (317 residues), which divides the C4 region into two portions. Taken together, these structural features of DGK indicate that this isozyme belongs to a DGK subfamily distinct from that consisting of DGKs α, β, and . Inc reased DGK activity without marked preference to arachidonoyl type of diacylglycerol was detected in the particulate fraction of COS-7 cells expressing the transfected DGK cDNA. The enzyme activity was independent of phosphatidylserine, which is a common activator for the previously sequenced DGKs. Northern blot analysis showed that the DGK mRNA (6.3 kilobases) is most abundant in human skeletal muscle but undetectable in the brain, thymus, and retina. This expression pattern is different from those of the previously cloned DGKs. Our results show that the DGK gene family consists of at least two subfamilies consisting of enzymes with distinct structural characteristics and that each cell type probably expresses its own characteristic repertoire of DGKs whose functions may be regulated through different signal transduction pathways.