A functional study of nucleocytoplasmic transport signals of the EhNCABP166 protein from Entamoeba histolytica.

EhNCABP166 is an Entamoeba histolytica actin-binding protein that localizes to the nucleus and cytoplasm. Bioinformatic analysis of the EhNCABP166 amino acid sequence shows the presence of 3 bipartite nuclear localization signals (NLS) and a nuclear export signal (NES). The present study aimed to investigate the functionality of these signals in 3 ways. First, we fused each potential NLS to a cytoplasmic domain of ehFLN to determine whether the localization of this domain could be altered by the presence of the NLSs. Furthermore, the localization of each domain of EhNCABP166 was determined. Similarly, we generated mutations in the first block of bipartite signals from the domains that contained these signals. Additionally, we added an NES to 2 constructs that were then evaluated. We confirmed the intranuclear localization of EhNCABP166 using transmission electron microscopy. Fusion of each NLS resulted in shuttling of the cytoplasmic domain to the nucleus. With the exception of 2 domains, all of the evaluated domains localized within the nucleus. A mutation in the first block of bipartite signals affected the localization of the domains containing an NLS. The addition of an NES shifted the localization of these domains to the cytoplasm. The results presented here establish EhNCABP166 as a protein containing functional nuclear localization signals and a nuclear export signal.

Leishmania mexicana lipophosphoglycan differentially regulates PKCalpha-induced oxidative burst in macrophages of BALB/c and C57BL/6 mice.

Leishmania are protozoan parasites that infect macrophages and their survival is partially achieved through inhibition of the cellular oxidative burst by parasite lipophosphoglycan (LPG). PKCalpha is the predominant PKC isoenzyme required for macrophage oxidative burst, yet it is not known if different susceptibility of BALB/c and C57BL/6 mice to Leishmania mexicana could be related to PKCalpha. We analysed the effect of L. mexicana promastigotes and parasite LPG on expression of PKCalpha and on its activity in macrophages of both mouse strains. Our data show that expression of the isoenzyme was not altered either by LPG or by L. mexicana promastigotes. Yet LPG exerted opposing effects on PKCalpha activity of macrophages between both strains: in susceptible BALB/c cells, it inhibited PKCalpha activity, whereas in the more resistant strain it augmented enzymatic activity 2.8 times. In addition, LPG inhibited oxidative burst only in susceptible BALB/c macrophages and the degree of inhibition correlated with parasite survival. Promastigotes also inhibited PKCalpha activity and oxidative burst in macrophages of BALB/c mice, whereas in C57BL/6, they enhanced PKCalpha activity and oxidative burst inhibition was less severe. Our data indicate that control of PKCalpha-induced oxidative burst by L. mexicana LPG relates with its success to infect murine macrophages.

Tight-junction protein zonula occludens 2 is a target of phosphorylation by protein kinase C.

Zonula occludens 2 (ZO-2) protein is a tight-junction phos phorylated protein that belongs to the membrane-associated guanylate kinase ('MAGUK') family. Here we study the interaction between ZO-2 and protein kinase C (PKC). We have constructed two ZO-2 fusion proteins of the middle (3PSG) and C-terminal (AP) regions of the molecule and demonstrate that they are phosphorylated by PKC isoenzymes beta, epsilon, lambda and zeta. To understand the physiological significance of the interaction between ZO-2 and PKC, we analysed the phosphorylation state of ZO-2 immunoprecipitated from monolayers with mature tight junctions or from cells that either lack them or have them disassembled through Ca(2+) chelation. We found that in the latter condition the phosphorylation level of ZO-2 is significantly higher and is due to the action of both PKC and cAMP-dependent protein kinase. These results therefore suggest that the phosphorylated state of ZO-2 restrains its capacity to operate at the junctional complex.

Characterization of calreticulin as a protein interacting with protein kinase C.

A protein kinase C (PKC)-binding protein was purified to homogeneity from the Triton-insoluble fraction from rat hepatocytes homogenates. The protein was identified as the mature calreticulin chain by N-terminal amino acid sequencing and by its immunoreactivity with anti-calreticulin antibody raised against the C-terminal KDEL (single-letter code) sequence. The calculated molecular mass was 46. 6 kDa but the protein migrates in SDS/PAGE as a doublet with apparent molecular masses of 60 and 55 kDa. Studies in vitro with purified calreticulin with the use of an overlay assay approach demonstrated that it binds to activated PKC isoenzymes expressed in rat hepatocytes. Phosphorylation of purified calreticulin with a PKC isoenzyme-specific immune complex kinase assay showed that it is also a very good substrate for all PKC isoforms in vitro. The treatment of intact cells with phorbol ester or with adrenaline (epinephrine) plus propranolol increased calreticulin phosphorylation, which was blocked by the pretreatment of cells with the PKC-specific inhibitor Ro 31-8220. The analysis of calreticulin immunoprecipitates from control or treated cells indicated that PKCalpha, PKCbeta, PKCtheta;, PKCzeta and PKCmu, but not PKCdelta or PKCepsilon, co-immunoprecipitated with calreticulin. Taken together, our results indicate that PKC interacts in vivo with calreticulin and suggest that they can operate in common signalling pathways.

Purification and characterization of receptors for activated protein kinase C from rat hepatocytes.

It has been proposed that protein kinase C may be targeted to specific locations via interactions with anchoring proteins located at various subcellular sites. A group of proteins collectively termed RACKs (Receptors for Activated C-Kinase) have been identified. Here, we made use of a rapid and simple method to purify several RACKs from rat hepatocytes, taking advantage of the ability of these proteins to be precipitated with Triton X-100. The method can be used for the isolation of other proteins that share these properties. Four proteins were purified to apparent homogeneity with M(r) values of 14, 15, 16, and 34 kDa. Amino acid composition and biochemical characteristics of these proteins are presented.

Cross-talk between glucagon- and adenosine-mediated signalling systems in rat hepatocytes: effects on cyclic AMP-phosphodiesterase activity.

The effect of adenosine analogues on glucagon-stimulated cyclic AMP accumulation in rat hepatocytes was explored. N6-Cyclopentyladenosine (CPA), 5'-N-ethylcarboxamidoadenosine and N6-(R-phenylisopropyl)adenosine inhibited in a dose-dependent manner the cyclic AMP accumulation induced by glucagon. This effect seems to be mediated through A1 adenosine receptors. Pertussis toxin completely abolished the effect of CPA on glucagon-stimulated cyclic AMP accumulation in whole cells which suggested that a pertussis-toxin-sensitive G-protein was involved. On the other hand, this action of adenosine analogues on glucagon-induced cyclic AMP accumulation was reverted by the selective low-Km cyclic AMP-phosphodiesterase inhibitor Ro 20-1724. Analysis of cyclic AMP-phosphodiesterase activity in purified hepatocyte plasma membranes showed that glucagon in the presence of GTP inhibited basal PDE activity by 45% and that CPA reverted this inhibition in dose-dependent manner. In membranes derived from pertussis-toxin-treated rats, we observed no inhibition of cyclic AMP-phosphodiesterase activity by glucagon in the absence or presence of CPA. Our results indicate that in hepatocyte plasma membranes, stimulation of adenylate cyclase activity and inhibition of a low-Km cyclic AMP phosphodiesterase activity are co-ordinately regulated by glucagon, and that A1 adenosine receptors can inhibit glucagon-stimulated cyclic AMP accumulation by blocking glucagon's effect on phosphodiesterase activity.

Immunological crossreactivity of G-protein beta subunit and receptors for activated C-kinase.

Selective antisera against: 1) RACKs, 2) a putative common PKC-binding domain (peptide I), and 3) G-protein subunits, were used for immunoblot analysis with recombinant purified beta 1 gamma 2 subunits and a crude preparation of RACKs. Antipeptide I recognized not only RACKs but also the G-protein beta subunit. In addition, two RACK-specific antisera immunoreacted with the G-protein beta subunit. Similarly, anti-G beta comm and S-217 (beta gamma-specific antiserum) crossreacted with RACKs. Using an overlay assay, it was observed that proteins immunoprecipitated with anti-RACKs or with anti-G beta antisera bound activated PKC.

Activated protein kinase C binds to intracellular receptors in rat hepatocytes.

The aim of this study was to identify in rat hepatocytes cellular polypeptides that bind protein kinase C (PKC) and may influence its activity and its compartmentation. At least seven proteins, with apparent M(r) values between 12,000 and 36,000, that behave like Receptors for Activated C-Kinase (RACKs) were found in the Triton-X-100-insoluble fraction of these cells; i.e. PKC bound to these polypeptides when it was in its active form. RACKS seem to be PKC substrates. Studies using isotype-specific PKC antibodies suggested some selectivity of RACKs, i.e. RACKs in the M(r) approximately 28,000-36,000 region bound PKC-alpha and PKC-beta in the presence of phosphatidylserine, diolein and Ca2+, whereas those of M(r) approximately 12,000-14,000 bound all isoforms studied, and, in contrast with the other RACKs, they did this even in the absence of Ca2+. Peptide I (KGDYEKILVALCGGN), which has a sequence suggested to be involved in the PKC-RACKs interaction [Mochly-Rosen, Khaner, Lopez and Smith (1991) J. Biol. Chem. 266, 14866-14868], inhibited PKC activity. Preincubation of RACKs with antisera directed against peptide I prevented PKC binding to them. The data suggest that peptide I blocks PKC binding to RACKs by two mechanisms: inhibition of PKC activity and competition with a putative binding site.

Staurosporine and calphostin-C inhibit the phorbol ester-induced decrease of protein kinase C activity in rat hepatocytes.

Two main forms of protein kinase C (PKC 1 and PKC 2) are detected in homogenates of rat hepatocytes using DEAE-cellulose column chromatography. The activity of these forms paradoxically, is rapidly decreased by treatment in vivo with phorbol myristate acetate (PMA). The dose-response curves to PMA for decreasing the activities of PKC 1 and 2 were shifted to the right by the potent and selective PKC inhibitors, staurosporine and calphostin-C. The decreases induced by 100 nM PMA were dose-dependently blocked by these inhibitors. It is concluded that activation of PKC is required and precedes such decreases in activity induced by the active phorbol ester.

Modulation by protein kinase C of the hormonal responsiveness of hepatocytes from lean (Fa/fa?) and obese (fa/fa) Zucker rats.

The effect of phorbol myristate acetate (PMA) on the hormonal responsiveness of hepatocytes from lean and obese Zucker rats was studied. Phenylephrine-stimulated phosphatydylinositol labeling and phosphorylase activation were antagonized by PMA in cells from obese and lean animals; bigger residual effects were observed in cells from obese animals even at high PMA concentrations. Cyclic AMP accumulation induced by isoproterenol, glucagon, forskolin and cholera toxin was higher in cells from lean animals than in those from obese rats. PMA diminished glucagon- and cholera toxin-induced cyclic AMP accumulation; cells from lean animals were more sensitive to PMA. Two groups of isoforms of protein kinase C (PKC) were observed in hepatocytes from Zucker rats using DEAE-cellulose column chromatography: PKC 1 and PKC 2. The PKC 1 isozymes were separated into four peaks using hydroxylapatite: aa, 1a (PKC-beta), 1b (PKC-alpha) and 1c. Short treatment with PMA decreased the activity of PKC 1 (peaks 1b (PKC-alpha) and 1c) and to a lesser extent of PKC 2; cells from lean animals were more sensitive to PMA than those obtained from obese rats. Our results indicate that cells from genetically obese Zucker rats are in general less sensitive to this activator of protein kinase C than those from their lean littermates. The possibility that alterations in the phosphorylation/dephosphorylation cycles, that control metabolism and hormonal responsiveness, may contribute to this obese state is suggested.

Differences in phorbol ester-induced decrease of the activity of protein kinase C isozymes in rat hepatocytes.

Two main forms of protein kinase C (PKC) activity were found in rat hepatocytes using DEAE-cellulose chromatography: PKC 1 and PKC 2. Treatment of cells with 1 microM 12-O-tetradecanoylphorbol 13-acetate (TPA) for 15 min caused a marked loss of PKC 1 activity and only a small loss of PKC 2 activity. Hydroxyapatite column chromatography resolved PKC 1 into three distinct peaks 1a, 1b and 1c, and PKC 2 into four peaks 2a, 2b, 2c and 2d. Immunoblot analysis with isozyme-specific monoclonal antibodies identified peak 1a as PKC-beta and peak 1b as PKC-alpha; the other peaks of activity were not identified. Treatment with TPA provoked a loss of activity of peaks 1b (PKC-alpha) and 1c, whereas peak 1a (PKC-beta) activity was not affected. The peaks of activity corresponding to PCK 2 did not show any major change due to TPA treatment except peak 2d that decreased. The apparent disappearance of PKC histone-kinase activity induced by TPA was also observed using other substrates (protamine or vinculin). The TPA-induced decrease in activity occurs in a time-dependent and dose-dependent fashion. However, the time-courses, the extent of depletion and the potency order of phorbol esters in induction of an activity decrease in the two groups of isoforms exhibited substantial differences.