scholarly journals Characterization of prenylated protein methyltransferase in Leishmania

1999 ◽  
Vol 342 (3) ◽  
pp. 513-518 ◽  
Author(s):  
Marie-Pierre HASNE ◽  
Françoise LAWRENCE
Keyword(s):  
Protein Kinase C ◽  
Leishmania Donovani ◽  
Post Translational Modification ◽  
Methyltransferase Activity ◽  
Kinase C ◽  
Protein Methyltransferase ◽  
Molecular Masses ◽  
Endogenous Substrates

Prenylated protein methyltransferase, an enzyme involved in the post-translational modification of many signalling proteins, has been characterized in a parasitic flagellated protozoan, Leishmania donovani. The activity of this enzyme was monitored by the methylation of an artificial substrate, an S-prenylated cysteine analogue, with S-adenosyl-L-[methyl-3H]methionine as methyl donor. More than 85% of the methyltransferase activity was associated with membranes. The enzyme methylates N-acetyl-S-trans,trans-farnesyl-L-cysteine and N-acetyl-S-all-trans-geranylgeranyl-L-cysteine, but N-acetyl-S-trans,trans-geranyl-L-cysteine only very weakly. In contrast with the enzyme from mammals, the leishmanial enzyme had a greater affinity for the farnesylated substrate than for the geranylgeranylated one. Activity in vitro was not modulated by cAMP, protein kinase C activator or guanosine 5′-[γ-thio]triphosphate. An analysis of the endogenous substrates showed that the carboxymethylated proteins were also isoprenylated. The main carboxymethylated proteins have molecular masses of 95, 68, 55, 46, 34-23, 18 and less than 14 kDa. Treatment of cells with N-acetyl-S-trans,trans-farnesyl-L-cysteine decreased the carboxymethylation level, whereas treatment with guanosine 5′-[γ-thio]triphosphate increased the carboxymethylation of various proteins, particularly those of molecular masses 30-20 kDa.

scholarly journals Regulation of Impaired Protein Kinase C Signaling by Chemokines in Murine Macrophages during Visceral Leishmaniasis

2005 ◽  
Vol 73 (12) ◽  
pp. 8334-8344 ◽  
Author(s):  
Ranadhir Dey ◽  
Arup Sarkar ◽  
Nivedita Majumder ◽  
Suchandra Bhattacharyya (Majumdar) ◽  
Kaushik Roychoudhury ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Leishmania Donovani ◽  
Disease Process ◽  
Kinase C ◽  
Parasitic Burden ◽  
Infected Cells

ABSTRACT The protein kinase C (PKC) family regulates macrophage function involved in host defense against infection. In the case of Leishmania donovani infection, the impairment of PKC-mediated signaling is one of the crucial events for the establishment of parasite into the macrophages. Earlier reports established that C-C chemokines mediated protection against leishmaniasis via the generation of nitric oxide after 48 h. In this study, we investigated the role of MIP-1α and MCP-1 in the regulation of impaired PKC activity in the early hours (6 h) of infection. These chemokines restored Ca2+-dependent PKC activity and inhibited Ca2+-independent atypical PKC activity in L. donovani-infected macrophages under both in vivo and in vitro conditions. Pretreatment of macrophages with chemokines induced superoxide anion generation by activating NADPH oxidase components in infected cells. Chemokine administration in vitro induced the migration of infected macrophages and triggered the production of reactive oxygen species. In vivo treatment with chemokines significantly restricted the parasitic burden in livers as well as in spleens. Collectively, these results indicate a novel regulatory role of C-C chemokines in controlling the intracellular growth and multiplication of L. donovani, thereby demonstrating the antileishmanial properties of C-C chemokines in the disease process.

scholarly journals Characterization of prenylcysteine methyltransferase in insulin-secreting cells

1996 ◽  
Vol 316 (1) ◽  
pp. 345-351 ◽  
Author(s):  
Guodong LI ◽  
Anjaneyulu KOWLURU ◽  
Stewart A. METZ
Keyword(s):  
Specific Activity ◽  
Post Translational Modification ◽  
Methyltransferase Activity ◽  
Intact Cells ◽  
Kinase C ◽  
Insulin Secreting Cells ◽  
Normal Rat ◽  
Cellular Organelles

Prenylcysteine carboxymethyltransferase, an enzyme involved in the post-translational modification of many signalling proteins, was characterized in insulin-secreting INS-1 cells and normal rat pancreatic islets. The activity of this enzyme was monitored by the methylation of an artificial substrate (a prenylated cysteine analogue) with S-adenosyl[methyl-3H]methionine as methyl donor. More than 95% of the methyltransferase activity was associated with the membranes, and high-salt treatment only partially extracted the enzyme from the membranes. The highest specific activity was in the insulin-granule-enriched 25000 g pellet obtained by differential centrifugation. However, a highly purified insulin-enriched fraction obtained by density centrifugation in Percoll did not exhibit methyltransferase activity. The analyses of marker enzymes for cellular organelles revealed that the methyltransferase was co-localized with the plasma membrane and probably the endoplasmic reticulum, but not with the mitochondria or lysosomes. Guanosine 5′-[γ-thio]triphosphate failed to increase methyltransferase activity directly, although it promotes the methylation of GTP-binding proteins. Mastoparan, Ca2+, cAMP and the protein kinase C activator phorbol 12-myristate 13-acetate did not alter enzyme activity. In addition, methyltransferase activity was not stably modified by stimulation of intact cells using glucose or other agents. However, the carboxymethylation of certain low-molecular-mass G-proteins is increased by glucose stimulation; conversely, treatment of cells with N-acetyl-S-trans, trans-farnesyl-L-cysteine inhibited glucose- and forskolin-induced insulin secretion. These results suggest that the membrane-associated prenylcysteine carboxymethyltransferase may be constitutively active and that the methylation of target proteins in vivo is regulated by the access of these proteins to the methyltransferase, as well as by their active (GTP-liganded) configuration.

scholarly journals Characterization and properties of protein kinase C from the filamentous fungus Trichoderma reesei

1998 ◽  
Vol 330 (2) ◽  
pp. 689-694 ◽  
Author(s):  
Thomas LENDENFELD ◽  
P. Christian KUBICEK
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Trichoderma Reesei ◽  
Fold Increase ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Ligand Affinity ◽  
Kinase C ◽  
Endogenous Substrates

The Trichoderma reesei pkc1 gene encodes a fungal homologue of the protein kinase C (PKC) family. Using antibodies directed against the nt-sequence-deduced pseudosubstrate domain for identification, Pkc1p was purified by dye-ligand affinity chromatography and Mono Q anion-exchange chromatography. Both the denatured as well as the native enzyme showed an Mr of 116-118 kDa, indicating that Pkc1p is a monomer. The enzyme phosphorylates the mutated (A → S) pseudosubstrate peptide and myelin basic protein, but not histone. Replacing three of the five basic amino acids around the serine acceptor residue resulted in a 25-fold increase in the Km. Pkc1p activity was stimulated by phospholipids, but this stimulation was counteracted by micromolar concentrations of Ca2+. Three proteins (85, 48 and 45 kDa) were identified as preferred endogenous substrates of Pkc1p in vitro. The enzyme was capable of autophosphorylation, and neither phosphorylation nor dephosphorylation in vitro affected the activity of the enzyme. A 116 kDa protein of T. reesei was demonstrated to bind to the N-terminal C2-region of Pkc1p in vitro. These data define Pkc1p as a unique member of the PKC family.

scholarly journals Inhibitory action of polyamines on protein kinase C association to membranes

1987 ◽  
Vol 247 (1) ◽  
pp. 175-180 ◽  
Author(s):  
M Moruzzi ◽  
B Barbiroli ◽  
M G Monti ◽  
B Tadolini ◽  
G Hakim ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Activation Process ◽  
Dependent Manner ◽  
Kinase C ◽  
Association Reaction ◽  
Dose Dependent ◽  
Inside Out

Physiological activation of protein kinase C requires the interaction of this enzyme with cellular membranes [Nishizuka (1986) Science 233, 305-312]. In the present work a reconstituted system of protein kinase C and human inside-out erythrocyte vesicles was utilized to study the effect in vitro of naturally occurring polyamines on the activation process of protein kinase C. The active membrane-associated complex was conveniently determined by its ability to bind radioactive phorbol ester with an exact 1:1 stoichiometry. The association reaction of the enzyme to membrane was rapid, being complete within 1 min at 25 degrees C. The addition of polyamines, particularly spermine, greatly decreased in a dose-dependent manner the amount of protein kinase C bound to membranes (i.e. in the activated form). The effect observed was quite specific, since it was dependent on the chemical structure of the polyamine and it was manifest at micromolar concentrations of the polycation; the order of potency was spermine greater than spermidine greater than putrescine. A characterization of this effect is presented and possible physiological implications are discussed.

scholarly journals Lambda-interacting protein, a novel protein that specifically interacts with the zinc finger domain of the atypical protein kinase C isotype lambda/iota and stimulates its kinase activity in vitro and in vivo.

1996 ◽  
Vol 16 (1) ◽  
pp. 105-114 ◽  
Author(s):  
M T Diaz-Meco ◽  
M M Municio ◽  
P Sanchez ◽  
J Lozano ◽  
J Moscat
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Zinc Finger ◽  
Zinc Finger Domain ◽  
Interacting Protein ◽  
Kinase C ◽  
Novel Protein

The members of the atypical subfamily of protein kinase C (PKC) show dramatic structural and functional differences from other PKC isotypes. Thus, in contrast to the classical or novel PKCs, they are not activated by diacylglycerol or phorbol esters. However, the atypical PKCs are the target of important lipid second messengers such as ceramide, phosphatidic acid, and 3'-phosphoinositides. The catalytic and pseudosubstrate sequences in the two atypical PKCs (lambda/iota PKC and zeta PKC) are identical but are significantly different from those of conventional or novel PKCs. It has been shown that microinjection of a peptide with the sequence of the pseudosubstrate of the atypical PKC isotypes but not of alpha PKC or epsilon PKC dramatically inhibited maturation and NF-kappa B activation in Xenopus oocytes, as well as reinitiation of DNA synthesis in quiescent mouse fibroblasts. This indicates that either or both atypical isoforms are important in cell signalling. Besides the pseudosubstrate, the major differences in the sequence between lambda/iota PKC and zeta PKC are located in the regulatory domain. Therefore, any functional divergence between the two types of atypical PKCs will presumably reside in that region. We report here the molecular characterization of lambda-interacting protein (LIP), a novel protein that specifically interacts with the zinc finger of lambda/iota PKC but not zeta PKC. We show in this paper that this interaction is detected not only in vitro but also in vivo, that LIP activates lambda/iota PKC but not zeta PKC in vitro and in vivo, and that this interaction is functionally relevant. Thus, expression of LIP leads to the transactivation of a kappa B-dependent promoter in a manner that is dependent on lambda/iota PKC. To our knowledge, this is the first report on the cloning and characterization of a protein activator of a PKC that binds to the zinc finger domain, which has so far been considered a site for binding of lipid modulators. The fact that LIP binds to lambda/iota PKC but not to the highly related zeta PKC isoform suggests that the specificity of the activation of the members of the different PKC subfamilies will most probably be accounted for by proteins like LIP rather than by lipid activators.

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