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京都大学 / 新制・課程博士 / 博士(医学) / 甲第23805号 / 医博第4851号 / 新制||医||1058(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 長船 健二, 教授 寺田 智祐, 教授 稲垣 暢也 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Platelets are primary components of hemostasis. However, incongruous activation of platelets lead to thrombosis, which result in multiple cardio-vascular and cerebrovascular complications. Thus, platelet activation is tightly regulated. Molecular components that aid in activation of platelets have been extensively studied. However, molecular pathways that negatively regulate platelet activation and prevent accidental activation of platelets are poorly understood. In this study we investigated the molecular mechanisms that negatively regulate platelet activation. Protein Kinase C isforms (PKCs) are serine threonine kinases that regulate various platelet functional responses leading to hemostasis. Positive regulatory role of PKCs towards platelet aggregation and secretion has been extensively studied. However, we have recently demonstrated that PKCs negatively regulate ADP- induced thromboxane generation. The PKC isoforms and mechanism involved in this process have not been known. Thus, in this study we investigated the mechanism by which PKCs negatively regulate ADP-induced thromboxane generation and identified PKC isoforms that regulate thromboxane generation. Thromboxane generation in platelets is a multi-step process beginning with cPLA2 activation. cPLA2 activation is the rate limiting step in the process of thromboxane generation. Furthermore, cPLA2 activation is regulated by ERK and calcium in various cell systems including platelets. PKC inhibition potentiated both cPLA2 and ERK activation, suggesting that PKCs negatively regulate thromboxane generation by regulating ERK activation, which in turn regulates cPLA2 activation. Furthermore, we have also shown that PKCs negatively regulate ADP-induced calcium mobilization. ADP activates platelets via P2Y1 and P2Y12 receptors. P2Y12 receptor-mediated signaling is shown to positively regulate P2Y1-mediated calcium mobilization in platelets. Furthermore, PKCs are shown to negatively regulate P2Y12 receptor desensitization in platelets. Thus, we investigated if PKCs regulate calcium mobilization indirectly by regulating P2Y12 receptor function. However, PKCs regulate calcium mobilization independent of P2Y12 receptor signaling. In summary we have shown that PKC isoforms negatively regulate ADP-induced thromboxane generation by regulating calcium mobilization and ERK activation that in turn regulates cPLA2 activity. We further investigated the PKC isoforms involved in this process. Based on our results with Go-6976, a classical PKC inhibitor and GF109203X, a pan PKC inhibitor, we identified that that novel or atypical PKC isoforms are involved in negative regulation of ADP-induced thromboxane generation. Thus, we investigated the role of various novel class of PKC isoforms (nPKCs) in platelets. We first investigated the nPKCs activated by ADP. In aspirin-treated platelets, ADP failed to activate nPKC θ and δ non-stirring conditions. Thus, we conclude that these isoforms are not involved in negative regulation of thromboxane generation. We further investigated if other non-classical PKC isoforms i. e nPKC η and ε or atypical PKC isoforms could be involved in this process. We began our investigation with the mechanism of activation and functional role of nPKC η in platelets. The mechanism of activation of PKCs has been extensively studied in various cell systems including platelets. However, the mechanism by which they are inactivated is not completely understood. In this study, we demonstrate a novel mechanism of inactivation of nPKC η isoform by integrin associated serine/threonine phosphatase. we demonstrated that ADP activates nPKC η via P2Y1 receptor coupled to Gq. As expected, Gi pathway, which does not generate DAG or mobilize calcium, has no role in regulation of nPKC η. Interestingly, we show that upon activation of platelets, αIIbβ3 mediated outside-in signaling dephosphorylates nPKCη through PP1γ phosphatase. We have also evaluated the role of nPKC η using η-RACK antagonistic peptides that interfere with enzyme-substrate interaction. Similar antagonistic peptides have been successfully used in various cell systems such as cardiomyocytes and neuronal cell. Using η-RACK antagonists we have demonstrated that nPKC η positively regulates agonist- induced thromboxane generation with no effect on agonist- induced platelet aggregation. The peptides were targeted in to the cell using TAT carrier protein, which is also used as a negative control for these experiments. The specificity of η-RACK antagonistic peptides is further elucidated by the fact that they do not affect the platelet aggregation. In summary, nPKC η is activated by ADP via P2Y1 receptor and is dephosphorylated by integrin αIIbβ3 via PP1γ phosphatase. Furthermore, activated nPKC η positively regulates ADP- induced thromboxane generation with no effect on aggregation. Since, our aim was to investigate the nPKC isoforms that negatively regulate ADPinduced thromboxane generation we investigated if nPKC ε is involved in this process. We made use of PKC ε knockout mice (PKC ε KO) for this process. We observed potentiated thromboxane generation in ADP-induced PKC ε murine platelets compared to witd type murine platelets. Thus, PKC ε might be one of the PKC isoforms involved in negative regulation of ADP-induced thromboxane generation. However, we failed to detect PKC ε in human platelets using western blot analysis. Since, PKC ε has been reported to be a part of platelet kinase repertoire, it could be limitation of our technique that we failed to detect it in western blot analysis. Since, PKCs negatively regulate ADP-induced thromboxane generation, we also investigated if PKCs also regulate PAR-mediated thromboxane generation. Similar to ADP, PAR-mediated thromboxane generation is not affected by Classical PKC isoforms. Furthermore, although non-classical PKC isoforms negatively regulate thromboxane generation, the extent of negative regulation is smaller and non-significant compared to ADP. Thus, we investigated if activation of nPKC isoforms were different between ADP and AYPGKF (PAR4 agonist). While, ADP fails to activate nPKC δ and θ, PARs activate Them. Furthermore, we have recently demonstrated that nPKC δ and θ are positive regulators of PAR-mediated platelet functional responses. Therefore, PKCinduced potentiation of thromboxane generation by ADP and PAR agonist are different due to differential activation of PKCs. This data lead to our final project, where we investigated the reason for differential activation of nPKC isoforms by various platelet agonists. Using strong and weak platelet agonists and DAG analogue, DiC8, we demonstrated that different platelet agonists differentially regulate nPKC activation due to variable amounts of DAG generated by them. Furthermore, we also have demonstrated that nPKC η and ε have higher affinities to DAG compared to nPKC δ and θ. / Molecular and Cellular Physiology

Protein Kinase C delta (PKCδ) is expressed in platelets and activated downstream of protease-activated receptors (PAR)s and glycoprotein VI (GPVI) receptors. We evaluated the role of PKCδ in platelets using two approaches - pharmacological and molecular genetic approach. In human platelets pretreated with isoform selective antagonistic RACK peptide (δV1-1)TAT, and in the murine platelets lacking PKCδ, PAR4-mediated dense granule secretion was inhibited, whereas GPVI-mediated dense granule secretion was potentiated. These effects were statistically significant in the absence and presence of thromboxane A2 (TXA2). Furthermore, TXA2 generation was differentially regulated by PKCδ. However, PKCδ had a small effect on platelet P-selectin expression. Calcium- and PKC-dependent pathways independently activate fibrinogen receptor in platelets. When calcium pathways are blocked by dimethyl-BAPTA, AYPGKF-induced aggregation in PKCδ null mouse platelets and in human platelets pretreated with (δV1-1)TAT, was inhibited. In a FeCl3-induced injury in vivo thrombosis model, PKCδ-/- mice occluded similar to their wild-type littermates. Hence, we conclude that PKCδ differentially regulates platelet functional responses such as dense granule secretion and TXA2 generation downstream of PARs and GPVI receptors, but PKCδ deficiency does not affect the thrombus formation in vivo. We further investigated the mechanism of such differential regulation of dense granule release by PKCδ in platelets. SH2 domain-containing Inositol Phosphatase (SHIP)-1 is phosphorylated on Y1020, a marker for its activation, upon stimulation of human platelets with PAR agonists, SFLLRN and AYPGKF, or GPVI agonist, convulxin. GPVImediated SHIP-1 phosphorylation occurred rapidly at 15 sec whereas PAR-mediated phosphorylation was delayed, occurring at 1 min. Lyn and SHIP-1, but not SHIP-2 or Shc, preferentially associated with PKCδ upon stimulation of platelets with a GPVI agonists, but not with a PAR agonist. In PKCδ null murine platelets, convulxin-induced SHIP-1 phosphorylation was inhibited, suggesting that PKCδ regulates the phosphorylation of SHIP-1. Furthermore, in Lyn null murine platelets, GPVI-mediated phosphorylations on Y-1020 of SHIP-1, Y311 and Y155 of PKCδ were inhibited. In murine platelets lacking Lyn, or SHIP-1, GPVI-mediated dense granule secretions were potentiated, whereas PAR-mediated dense granule secretions were inhibited. Phosphorylated SHIP-1 associated with phosphorylated-Y155 PKCδ peptide. Therefore, we conclude that Lyn-mediated phosphorylations of PKCδ and SHIP-1 and their associations negatively regulate GPVI-mediated dense granule secretion in platelets. / Physiology

The bacteriophage T7 protein kinase enhances T7 growth under suboptimal growth conditions, including elevated temperature or limiting carbon source. T7PK phosphorylates numerous E. coli proteins, and it has been proposed that phosphorylation of these proteins is responsible for supporting T7 replication under stressful growth conditions. How the phosphorylation of host proteins supports T7 growth is not understood. Escherichia coli (Ec) RNase III is phosphorylated on serine in bacteriophage T7-infected cells. Phosphorylation of Ec-RNase III induces a ~4-fold increase in catalytic activity in vitro. Ec-RNase III is involved in the maturation of several T7 mRNAs, and it has been shown that RNase III processing controls the translational activity and stability of the T7 mRNAs. Perhaps T7PK phosphorylation of Ec- RNase III ensures optimal processing of T7 mRNAs under suboptimal growth conditions. In this study a biochemical analysis was performed on the N-terminal portion of the 0.7 gene (T7PK), exhibiting only the protein kinase activity. In addition to phosphotransferase activity, T7PK also undergoes self-phosphorylation on serine, which down-regulates catalytic activity by an unknown mechanism. Mass spectral analysis revealed that Ser216 is the autophosphorylation site in T7PK. The serine residue is highly conserved, which in turn suggests that autophosphorylation is a conserved reaction with functional importance. Phosphorylated T7PK exhibits reduced phosphotransferase activity, compared to its dephosphorylated counterpart (dT7PK). The dT7PK exhibits enhanced ability to phosphorylate proteins, as well as undergo autophosphorylation. The mechanism by which autophosphorylation inhibits T7PK activity is unknown. An in vitro phosphorylation assay revealed that T7PK directly phosphorylates RNase III. Ec-RNase III processing activity is stimulated from two to ten-fold upon phosphorylation by the T7PK. The primary site of phosphorylation in RNase III is found to be Ser33, and Ser34 may act as the recognition determinant for T7PK. This was established by Ser →Ala mutations at the concerned site. The enhancement of catalytic activity is primarily due to a larger turnover number (kcat), with some additional contribution from a greater substrate binding affinity, as revealed by lower Km and K‟D values. Substrate cleavage assays under single turn over conditions established that the product release is the rate limiting step. Since there is no significant increase in the kcat as measured under single-turnover (enzyme excess) conditions, the increase in the kcat in the steady-state is due to enhancement of the product release step, and not due to an enhancement of the hydrolysis (chemical) step. / Chemistry