Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.11.13 (protein kinase C)
 49,245 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Fc receptors play a pivotal role linking the cellular and humoral arms of the immune system [1-3]. Our previous studies have shown that the human high-affinity immunoglobulin G receptor Fc(gamma)RI couples to a novel intracellular signaling pathway requiring phospholipase D activation [4]. The mechanisms that regulate receptor coupling to phospholipase D in intact cells are poorly understood but involve small molecular weight GTPases and protein kinase C [5-7]. Here, we show that immune complex aggregation of Fc(gamma)RI stimulates the association of phospholipase D1 with ARF6 and protein kinase Calpha. Surprisingly, PKCalpha activity per se is not required. Rather, all of the Fc(gamma)RI-mediated increase in PKC activity requires phospholipase D1, as treatment of cells with butan-1-ol (0.3%) or specific downregulation of phospholipase D1 using antisense oligonucleotides inhibits Fc(gamma)RI-coupled PKC activation. Moreover, treatment of cells with butan-1-ol or phospholipase D1 antisense oligonucleotides inhibits translocation of PKCdelta, -epsilon, and -zeta but had no effect on the association of PKCalpha or ARF6 with phospholipase D1. These data indicate that association with ARF6 and PKCalpha plays a role in coupling Fc(gamma)RI to phospholipase D1 activation and that PLD1 lies upstream of all Fc(gamma)RI-mediated PKC activity.
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PMID:Crosstalk between ARF6 and protein kinase Calpha in Fc(gamma)RI-mediated activation of phospholipase D1. 1151 49

Differential effects of acetyl(C2-) ceramide (N-acetylsphingosine) were studied on coated vesicle formation from Golgi-enriched membranes of Chinese hamster ovary (CHO) and Madin-Darby canine kidney (MDCK) cells. C2-ceramide blocked the translocation of ADP-ribosylation factor-1 (ARF-1) and protein kinase C-alpha (PKC-alpha) to the membranes from CHO cells, but not those of MDCK cells. Consequently, C2-ceramide blocked the stimulation of phospholipase D1 (PLD1) by the cytosol and guanosine 5'-[gamma-thio]triphosphate (GTP[S]) in membranes from CHO cells. Basal specific activity of PLD1 and the concentration of ARF-1 were 3-4 times higher in Golgi-enriched membranes from MDCK cells compared with CHO cells. Moreover, PLD1 activity in MDCK cells was stimulated less by cytosol and GTP[S]. PLD2 was not detectable in the Golgi-enriched membranes. Incubation of intact CHO cells or their Golgi-enriched membranes with C2-ceramide also inhibited COP1 vesicle formation by membranes from CHO, but not MDCK, cells. Specificity was demonstrated, since dihydro-C2-ceramide had no significant effect on ARF-1 translocation, PLD1 activation or vesicle formation in membranes from both cell types. C2-ceramide also decreased the secretion of virus-like particles to a greater extent in CHO compared with MDCK cells, whereas dihydro-C2-ceramide had no significant effect. The results demonstrate a biological effect of C2-ceramide in CHO cells by decreasing ARF-1 and PKC-alpha binding to Golgi-enriched membranes, thereby preventing COP1 vesicle formation.
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PMID:Cell-permeable ceramides preferentially inhibit coated vesicle formation and exocytosis in Chinese hamster ovary compared with Madin-Darby canine kidney cells by preventing the membrane association of ADP-ribosylation factor. 1180 96

A 66-kDa molecular weight protein with phospholipase D activity was solubilized and partially purified from rat liver plasma membrane. The activity and regulation of this phospholipase D have been characterized. Immunoblot analyses indicated that the enzyme was distinct from hPLD1 and PLD2, but was recognized by an antibody to the 12 terminal amino acids of PLD1. PLD activity was stimulated by 1-100 microM Ca(2+) and Mg(2+) and displayed a pH optimum of 7.5. Activity was inhibited by both saturated and unsaturated fatty acids. This PLD was activated in an ATP-independent manner by the PKC isozymes alpha and betaII but not activated by other PKC isozymes. It was also stimulated by the small G-proteins RhoA and ARF. RhoA stimulated the greatest activation, followed by ARF and PKC(alpha). This enzyme was further activated in a synergistic manner when combinations of PKC(alpha) and RhoA or ARF were used. This enzyme displayed a greater response activation by RhoA than to activation by ARF. While a potential breakdown product of PLD1, activation by RhoA indicates that the PLD characterized here is distinct from the other PLDs cloned or isolated to date.
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PMID:Isolation and characterization of a 66-kDa protein from rat liver plasma membrane with RhoA-stimulated phospholipase D activity. 1181 16

Our earlier studies of rat brain phospholipase D1 (rPLD1) showed that the enzyme could be activated in cells by alpha subunits of the heterotrimeric G proteins G(13) and G(q). Recently, we showed that rPLD1 is modified by Ser/Thr phosphorylation and palmitoylation. In this study, we first investigated the roles of these post-translational modifications on the activation of rPLD1 by constitutively active Galpha(13)Q226L and Galpha(q)Q209L. Mutations of Cys(240) and Cys(241) of rPLD1, which abolish both post-translational modifications, did not affect the ability of either Galpha(13)Q226L or Galpha(q)Q209L to activate rPLD1. However, the RhoA-insensitive mutants, rPLD1(K946A,K962A) and rPLD1(K962Q), were not activated by Galpha(13)Q226L, although these mutant enzymes responded to phorbol ester and Galpha(q)Q209L. On the contrary, the PKC-insensitive mutant rPLD1(DeltaN168), which lacks the first 168 amino acids of rPLD1, responded to Galpha(13)Q226L but not to Galpha(q)Q209L. In addition, we found that rPLD2 was strongly activated by Galpha(q)Q209L and phorbol ester. However, surprisingly, the enzymatic activity of rPLD2 was suppressed by Galpha(13)Q226L and constitutively active V14RhoA in COS-7 cells. Abolition of the post-translational modifications of rPLD2 did not alter the effects of Galpha(q)Q209L or Galpha(13)Q226L. The suppressive effect of Galpha(13)Q226L on rPLD2 was reversed by dominant negative N19RhoA and the C3 exoenzyme of Clostridium botulinum, further supporting a role for RhoA. In summary, Galpha(13) activation of rPLD1 in COS-7 cells is mediated by Rho, while Galpha(q) activation requires PKC. rPLD2 is activated by Galpha(q), but is inhibited by Galpha(13). Neither Ser/Thr phosphorylation nor palmitoylation is required for these effects.
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PMID:Mechanisms of regulation of phospholipase D1 and D2 by the heterotrimeric G proteins G13 and Gq. 1181 83

Our previous studies showed that truncation of the N-terminal 168 amino acids of rat brain phospholipase D1 (rPLD1) abolishes its response to protein kinase C (PKC) and greatly diminishes its palmitoylation and Ser/Thr phosphorylation. In this study, we show that the response to PKC as well as the palmitoylation and Ser/Thr phosphorylation were restored when the truncated rPLD1 mutant (rPLD1(DeltaN168)) was coexpressed with a fragment containing the N-terminal 168 amino acids. Immunoprecipitation experiments showed that the N-terminal fragment associated with rPLD1(DeltaN168) when coexpressed in COS 7 cells and that palmitoylation of Cys(240) and Cys(241) was not necessary for the association. In addition, we found that rat PLD2 (rPLD2) was palmitoylated on Cys(223) and Cys(224) in COS 7 cells. Mutation of both these cysteines reduced the basal activity of rPLD2, however its response to PMA stimulation in vivo was retained. As in the case of rPLD1, loss of palmitoylation weakened membrane association of rPLD2. In summary, the N-terminal 168-amino-acid fragment of rPLD1 can associate with truncated rPLD1(DeltaN168) to restore its palmitoylation, Ser/Thr phosphorylation and PKC response. Although rPLD2 differs from rPLD1 in many properties, it is palmitoylated at the corresponding conserved cysteine residues in COS 7 cells.
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PMID:Functional implications of post-translational modifications of phospholipases D1 and D2. 1192 96

The regulation of phospholipase D1 (PLD1), which has been shown to be activated by protein kinase C (PKC) alpha, was investigated in the human melanoma cell lines. In G361 cell line, which lacks PKCalpha, 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced PLD activation was potentiated by introducing PKCalpha by the adenovirus vector. The kinase-negative PKCalpha elevated TPA-induced PLD activity less significantly than the wild type. A PKC specific inhibitor GF109203X lowered PLD activation in the cells expressing PKCalpha, but did not prevent PLD potentiation induced by the kinase-negative PKCalpha. Expression of PKCbetaII and the kinase-negative PKCbetaII enhanced TPA-stimulated PLD activity moderately in MeWo cell line, in which PKCbetaII is absent. Furthermore, the TPA treatment increased the association of PKCalpha, PKCbetaII, and their kinase-negative mutants with PLD1 in melanoma cells. These results indicate that PLD1 is dually regulated through phosphorylation as well as through the protein-protein interaction by PKCalpha, and probably by PKCbetaII, in vivo.
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PMID:Dual regulation of phospholipase D1 by protein kinase C alpha in vivo. 1207 91

Phospholipase D1 and D2 (PLD1, PLD2) both have PX and PH domains in their N-terminal regions with these inositol lipid binding domains playing key roles in regulating PLD activity and localisation. The activity of PLD1 is also regulated by protein kinase C and members of the Rho and Arf families of GTPases. Each of these proteins binds to unique sites; however, there appears to be little in vitro discrimination between individual family members. In agonist-stimulated cells, however, there is specificity, with, for example in RBL-2H3 cells, antigen stimulating the activation of PLD1 by association with Arf6, Rac1 and protein kinase Calpha. PLD2 appears to be less directly regulated by GTPases and rather is primarily controlled through interaction with phosphatidylinositol 4-phosphate 5-kinase that generates the activating phosphatidylinositol 4,5-bisphosphate.
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PMID:The regulation of phospholipase D by inositol phospholipids and small GTPases. 1240 Dec 4

It has been suggested that protein-protein interaction is important for protein kinase C (PKC) alpha to activate phospholipase D1 (PLD1). To determine the one or more sites on PKCalpha that are involved in binding to PLD1, fragments containing the regulatory domain, catalytic domain, and C1-C3 domain of PKCalpha were constructed and shown to be functional, but they all failed to bind and activate PLD1 in vivo and in vitro. A C-terminal 23-amino acid (aa) deletion mutant of PKCalpha was also found to be inactive. To define the binding/activation site(s) in the C terminus of PKCalpha, 1- to 11-aa deletion mutants were made in this terminus. Deletion of up to 9 aa did not alter the ability of PKCalpha to bind and activate PLDl, whereas a 10-aa deletion was inactive. The residue at position 10 was Phe(663). Mutations of this residue (F663D and F663A) caused loss of binding, activation, and phosphorylation of PLD1, indicating that Phe(663) is essential for these activities. Time course experiments showed that the activation of PLD1 by PMA was much faster than its phosphorylation, and its activity decreased as phosphorylation increased with time. Staurosporine, a PKC inhibitor, completely inhibited PLD1 phosphorylation in response to 4beta-phorbol 12-myristate 13-acetate PMA and blocked the later decrease in PLD activity. The same results were found with the D481E mutant of PKCalpha, which is unable to phosphorylate PLD1. These results indicate that neither the regulatory nor catalytic domains of PKCalpha alone can bind to or activate PLD1 and that a residue in the C terminus of PKCalpha (Phe(663)) is required for these effects. The initial activation of PLD1 by PMA is highly correlated with the binding of PKCalpha. Although PKCalpha can phosphorylate PLD1, this is a relatively slow process and is associated with inactivation of the enzyme.
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PMID:Mechanisms of regulation of phospholipase D1 by protein kinase Calpha. 1243 82

The regulation of phospholipase D1 (PLD1) by protein kinase C (PKC) isoforms was analyzed in human melanoma cell lines. 12-O-Tetradecanoylphorbol-13-acetate (TPA)-induced PLD1 activation was suppressed by the introduction of PKCdelta as well as its kinase-negative mutant in MeWo cells, which contain PKCalpha but lack PKCbeta. PLD activity was not affected by PKCdelta in G361 cells, which have PKCbeta but are deficient in PKCalpha. In MeWo cells introduced by PKCalpha and PLD1, the association of these proteins was observed, which was enhanced by the TPA treatment. In cells overexpressing PKCdelta in addition to PKCalpha and PLD1, TPA treatment increased the association of PKCdelta and PLD1, while it attenuated the association of PKCalpha and PLD1. These results indicate that PKCdelta inhibits TPA-induced PLD1 activation mediated by PKCalpha through the association with PLD1.
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PMID:PKCdelta inhibits PKCalpha-mediated activation of phospholipase D1 in a manner independent of its protein kinase activity. 1459 36

The inhibition of phorbol ester activation of phospholipase D1 (PLD1) by protein kinase C (PKC) inhibitors has been considered proof of phosphorylation-dependent activation of PLD1 by PKCalpha. We studied the effect of the PKC inhibitors Ro-31-8220 and bisindolylmaleimide I on PLD1 activation and found that they inhibited the activation by interfering with PKCalpha binding to PLD1. Further studies showed that only unphosphorylated PKCalpha could bind to and activate PLD1 and that both inhibitors induced phosphorylation of PKCalpha. The phosphorylation status of either PLD1 or PKCalpha per se did not affect PLD1 activation in vitro. Immunofluorescence studies showed that PLD1 remained in the perinuclear region after phorbol ester treatment, whereas PKCalpha translocated from cytosol to both plasma membrane and perinuclear regions. Both Ro-31-8220 and bisindolylmaleimide I blocked the translocation of PKCalpha to the perinuclear region but not to the plasma membrane. Studies with okadaic acid suggested that phosphorylation regulated the relocation of PKCalpha from the plasma membrane to the perinuclear region. It is proposed that localization and interaction of PKCalpha with PLD1 in the perinuclear region is required for PLD1 activation and that PKC inhibitors inhibit this through phosphorylation of PKCalpha, which blocks its translocation.
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PMID:Protein kinase Calpha translocates to the perinuclear region to activate phospholipase D1. 1518 91


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