Involvement of protein kinase C in the response of Neurospora crassa to blue light

As a first step towards understanding the process of blue light perception, and the signal transduction mechanisms involved, in Neurospora crassa we have used a pharmacological approach to screen a wide range of second messengers and chemical compounds known to interfere with the activity of well-known signal transducing molecules in vivo. We tested the influence of these compounds on the induction of the al-3 gene, a key step in light-induced carotenoid biosynthesis. This approach has implicated protein kinase C (PKC) as a component of the light transduction machinery. The conclusion is based on the effects of specific inhibitors (calphostin C and chelerythrine chloride) and activators of PKC (1,2-dihexanoyl-sn-glycerol). During vegetative growth PKC may be responsible for desensitization to light because inhibitors of the enzyme cause an increase in the total amount of mRNA transcribed after illumination. PKC is therefore proposed here to be an important regulator of transduction of the blue light signal, and may act through modification of the protein White Collar-1, which we show to be a substrate for PKC in N. crassa. Received: 4 December 1998 / Accepted: 21 May 1999     

Selective and transient activation of protein kinase C alpha by fumonisin B1, a ceramide synthase inhibitor mycotoxin, in cultured porcine renal cells

Fumonisin B₁ (FB₁), a potent and naturally occurring mycotoxin produced by the fungus Fusarium verticillioides, has been implicated in fatal and debilitating diseases in animals and humans. FB₁ affects a variety of cell signaling proteins including protein kinase C (PKC); a serine/threonine kinase, involved in a number of signal transduction pathways that include cytokine induction, carcinogenesis and apoptosis. The aim of this study was to investigate the short-term temporal and concentration-dependent effects of FB₁ on PKC isoforms present in LLC-PK₁ cells in relation to the FB₁-induced accumulation of sphinganine and sphingosine utilizing various inhibitors and activators. Our studies demonstrated that FB₁ (0.1-1 μM) selectively and transiently activated PKCα at 5 min, without affecting PKC-δ, -ε and -ζ isoforms. At higher FB₁ concentrations and later time points (15-120 min), PKCα membrane concentrations declined to untreated levels. The observed increase in cytosol PKCα protein expression at 15 min was not associated with an increase in its activity or protein biosynthesis. Calphostin C, a PKC inhibitor, abrogated the FB₁-induced translocation of PKCα. Pre-incubation with the PKC activator, phorbol 12-myristate 13-acetate, resulted in an additive effect on membrane translocation of PKCα. Intracellular sphinganine and sphingosine concentrations were unaltered at the time points tested. Myriocin, a specific inhibitor of serine palmitoyltransferase, the first enzyme in de novo sphingolipid biosynthesis, did not prevent the FB₁-induced PKCα cytosol to membrane redistribution. Altering PKCα and its signal transduction pathways may be of importance in the ability of FB₁ to exert its toxicity via apoptosis and/or carcinogenesis.     

Involvement of Calmodulin and Calmodulin-Dependent Myosin Light Chain Kinase in Blue Light-Dependent H Pumping by Guard Cell Protoplasts from Vicia faba L

Signal transduction processes involved in blue light-dependent proton pumping were investigated using guard cell protoplasts from Vicia faba. N-[2-(Methylamino)ethyl]-5-isoquinolinesulfonamide, an inhibitor of cyclic AMP- and cyclic GMP-dependent protein kinases, had no effect. 1-(5-Isoquinolinesulfonyl)-2-methylpiperazine (H-7) and calphostin C, inhibitors of protein kinase C, produced slight inhibition of the blue light-dependent proton pumping. 1-[N, O-Bis(5-isoquinolinesulfonyl)-N-methyl-l-tyrosyl] -4-phenylpiperazine, a specific inhibitor of Ca²⁺/calmodulin (CaM)-dependent protein kinase II, did not inhibit the proton pumping, but 1-(5-iodonaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine and 1-(5-chloro-naphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine (ML-9), inhibitors of Ca²⁺/CaM-dependent myosin light chain kinase, strongly suppressed the proton pumping. A CaM antagonist, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), inhibited blue light-dependent proton pumping, whereas its less active structural analog, N-(6-aminohexyl)-1-naphthalenesulfonamide (W-5), had little effect on the response. Other CaM antagonists, trifluoperazine, compound 48/80, prenylamine, and 3-(2-benzothiazolyl)-4,5-dimethoxy-N-[3-(4-phenyl-piperidinyl)- propylbenzenesulfonamide inhibited the proton pumping. In accord with these results, light-induced stomatal opening in the epidermis of Commelina benghalensis ssp. was inhibited by ML-9 and W-7, but not by H-7 and W-5. Thus, it is concluded that CaM and Ca²⁺/CaM-dependent myosin light chain kinase are the components of the signal transduction process in blue light-dependent proton pumping in guard cells.     

Binding of curcumin and its long chain derivatives to the activator binding domain of novel protein kinase C

Protein kinase C (PKC) is a family of serine/threonine kinases that play a central role in cellular signal transduction. The second messenger diacylglycerol having two long carbon chains acts as the endogenous ligand for the PKCs. Polyphenol curcumin, the active constituent of Curcuma longa is an anti-cancer agent and modulates PKC activity. To develop curcumin derivatives as effective PKC activators, we synthesized several long chain derivatives of curcumin, characterized their absorption and fluorescence properties and studied their interaction with the activator binding second cysteine-rich C1B subdomain of PKCδ, PKCε and PKCθ. Curcumin (1) and its C16 long chain analog (4) quenched the intrinsic fluorescence of PKCδC1B, PKCεC1B and PKCθC1B in a manner similar to that of PKC activator 12-O-tetradecanoylphorbol 13-acetate (TPA). The EC₅₀s of the curcumin derivatives for fluorescence quenching varied in the range of 4–11 μM, whereas, EC₅₀s for TPA varied in the range of 3–6 μM. Fluorescence emission maxima of 1 and 4 were blue shifted and the fluorescence anisotropy values were increased in the presence of the C1B domains in a manner similar to that shown by the fluorescent analog of TPA, sapintoxin-D, confirming that they were bound to the proteins. Molecular docking of 1 and 4 with novel PKC C1B revealed that both the molecules form hydrogen bonds with the protein residues. The present result shows that curcumin and its long chain derivatives bind to the C1B subdomain of novel PKCs and can be further modified structurally to improve its binding and activity.     

Opposite Ends of the Spectrum: Plant and Animal G-Protein Signaling

Different classes of biotic (e.g., plant hormones) and abiotic (e.g., different wavelengths of light) signals act through specific signal transduction mechanisms to coordinate all aspects of plant development. Full signal transduction chains have not yet been described for most light or hormonal-mediated events despite the wide range of events early in development which are dependent upon hormonal and light signals. We recently reported a single signal transduction chain which can be initiated by both blue light (BL) and ABA, and which leads to the expression of specific members of the Lhcb gene family in the apical bud of etiolated Arabidopsis seedlings. The signal transduction chain consists of GCR1 (one of two Arabidopsis proteins coding for a potential G-protein coupled receptor), GPA1 (the sole Arabidopsis Ga subunit), PRN1 (Pirin1, one of four members of an iron-containing subgroup of the cupin superfamily), and a Nuclear Factor -Y (NF-Y) heterotrimer comprised of A5, B9 and possibly C9. The same signaling proteins control ABA-mediated delay of germination.Key Words: blue light, G-protein coupled receptor, G-protein sub unit, abscisic acid (ABA)     

Role of protein kinase C, G-protein coupled receptors, and calcium flux during metamorphosis of the sea urchin Strongylocentrotus purpuratus

Artificial inducers have been used to study signal-transduction pathways involved in metamorphosis of some marine invertebrates. However, the transduction mechanisms for echinoderms have been less explored. In the present study, participation of protein kinase C (PKC), G-protein-coupled receptors (GPCRs), and calcium has been investigated during metamorphosis of the sea urchin Stronglylocentrotus purpuratus. Competent larvae were induced with different drugs that activate (PKC and GP activators, Ca2+ ionophores, and inhibitors of Ca2+ ATPase) or inhibit (PKC, G-protein, and Ca2+ flux inhibitors) metamorphosis. Six of the compounds were effective: the PKC activators TPA and indolactam; the G-protein inhibitors suramin and guanosine; the calcium ionophore A23187, and the calcium ATPase inhibitor thapsigargin. TPA was effective at 0.001 microM; indolactam was effective at 0.001 microM. In the presence of KCl as inducer, the G-protein inhibitor suramin was effective at 10 microM and guanosine at 0.001 microM. In the presence of a bacterial film as inducer, suramin was effective at 50 microM, and guanosine inhibited metamorphosis at 1 microM. A23187 was effective at 5 and 10 microM and thapsigargin at 50 and 100 microM. Our results indicate that GPCRs, protein kinase C, and calcium participate in the metamorphosis of S. purpuratus. These elements of the transduction pathways triggered during metamorphosis may be part of a cascade of signal transduction routes that interact from induction to the end of the morphogenetic events that shape the postlarval form. In addition, according to the results obtained with G-protein inhibitors, the GPCRs may be shared between the artificial (KCl) and natural (biofilm) inducers.     

Genetic Analysis of Phototropism of Neurospora crassa Perithecial Beaks Using White Collar and Albino Mutants

Positive phototropism of perithecial beaks in the fungus Neurospora crassa has been demonstrated. The effect was shown to be mediated by blue light. When mutants (white collar-1 and white collar-2) which are blocked in the light induction of enzymes in the carotenoid biosynthetic pathway were used as the protoperithecial parent in crosses, the resulting perithecial beaks did not show a phototropic response. However, when wild type, albino-1, albino-2, or albino-3 strains were used as the protoperithecial parent, phototropism occurred.

The results show that both photoinduced carotenogenesis and phototropism in N. crassa are controlled by the white collar-1 and white collar-2 loci. Thus, the sensory transduction pathways for the two photoresponses must have some steps in common. The results further support the proposal that the white collar strains are regulatory mutants blocked in the light induction process, whereas the albino-1, albino-2, and albino-3 strains can carry out light induction but have the albino phenotype because they are each defective for a different enzyme in the carotenoid biosynthetic pathway.

     

Signal transduction controlling the blue- and red-light mediated gene expression of S-adenosylmethionine decarboxylase in Pharbitis nil

The signal transduction processes involved in the regulation of SAMDC gene expression by blue and red light were examined using pharmacological inhibitors of signalling pathways. Calcium and calmodulin positively regulated SAMDC gene expression in red light, whereas in blue light they regulated negatively. These results indicate that calcium homeostasis is involved in both red and blue light induction of SAMDC expression. Both signal transduction pathways also require new protein synthesis.     

Protein kinase C modulates light responses in Neurospora by regulating the blue light photoreceptor WC-1

The Neurospora protein kinase C (NPKC) is a regulator of light responsive genes. We have studied the function of NPKC in light response by investigating its biochemical and functional interaction with the blue light photoreceptor white-collar 1 (WC-1), showing that activation of NPKC leads to a significant decrease in WC-1 protein levels. Furthermore, we show that WC-1 and NPKC interact in a light-regulated manner in vivo, and that protein kinase C (PKC) phosphorylates WC-1 in vitro. We designed dominant negative and constitutively active forms of PKC which are able to induce either a large increase of WC-1 protein level or a strong reduction respectively. Moreover, these changes in PKC activity result in an altered light response. As WC-1 is a key component of Neurospora circadian clock and regulates the clock oscillator component FRQ we investigated the effect of NPKC-mutated forms on FRQ levels. We show that changes in PKC activity affect FRQ levels and the robustness of the circadian clock. Together these data identify NPKC as a novel component of the Neurospora light signal transduction pathway that modulates the circadian clock.     

Regulation of transplasmalemma electron transport in oat mesophyll cells by sphingoid bases and blue light

Long-chain sphingoid bases inhibit transplasmalemma electron transport in certain animal cells in part by inhibiting protein phosphorylation. As a first step in determining whether similar regulatory processes exist for cell surface redox activity in plants, peeled leaf segments of Avena sativa L. cv Garry were exposed to sphingoid bases and other long chain lipids. Sphingoid bases which are the most active inhibitors of protein kinase C in animal cells inhibit transplasmalemma electron transport by mesophyll cells in the dark as measured by reduction of exogenous ferricyanide. In white light, however, the same compounds markedly stimulate redox activity. The stimulation by sphingoid bases in the light is not eliminated by the inhibitor of photosynthesis, 3-(3,4-dichlorophenyl)-1,1 dimethylurea (DCMU). Redox activity remaining in the presence of DCMU and sphingoid bases can be observed in blue but not red light. A tentative hypothesis considering the involvement of two separate redox systems is presented in an attempt of explain the disparate action of sphingoid bases on electron transport across the plasmalemma.     

Proteomic analysis of blue light-induced twining response in Cuscuta australis

The parasitic plant Cuscuta australis (dodder) invades a variety of species by entwining the stem and leaves of a host and developing haustoria. The twining response prior to haustoria formation is regarded as the first sign for dodders to parasitize host plants, and thus has been the focus of studies on the host-parasite interaction. However, the molecular mechanism is still poorly understood. In the present work, we have investigated the different effects of blue and white light on the twining response, and identified a set of proteins that were differentially expressed in dodder seedlings using a proteomic approach. Approximately 1,800 protein spots were detected on each 2-D gel, and 47 spots with increased or decreased protein levels were selected and analyzed with MALDI-TOF-MS. Peptide mass fingerprints (PMFs) obtained for these spots were used for protein identification through cross-species database searches. The results suggest that the blue light-induced twining response in dodder seedlings may be mediated by proteins involved in light signal transduction, cell wall degradation, cell structure, and metabolism.     

Biosynthesis of phytoene from isopentenyl pyrophosphate by a Neurospora enzyme system.

An enzyme system catalyzing the conversion of isopentenyl pyrophosphate to phytoene has been isolated from Neurospora crassa mycelia. This enzyme system shows an absolute requirement for Mg^?, but no other cofactors. Cultures of N. crassa exhibit a low level of phytoene synthesizing activity when grown in the dark. A 2-min in vivo blue light irradiation results in a ninefold increase in activity after 24 h. This increase is dependent on the duration of the light treatment and is inhibited by cycloheximide. A similar blue light-induced elevation of phytoene synthesizing activity was demonstrated in an albino-1 mutant. This enzyme activity was not found in either dark-grown or irradiated cultures of an albino-2 or an albino-3 mutant.     

A ras homologue of Neurospora crassa regulates morphology

In order to study the role of signal transduction pathways in the regulation of morphology in Neurospora crassa, we cloned and characterized a ras homologue, termed NC-ras2. The predicted protein product of this gene is composed of 229 amino acid residues and contains all the consensus sequences shared by the ras protein family. The gene is located in linkage group V. An NC-ras2 disruptant showed morphological characteristics very similar to those of the smco7 mutant, which also maps to linkage group V. Nucleotide sequence analysis revealed that the smco7 mutant harbored a single base deletion in the NC-ras2 gene, which is predicted to result in the truncation of the protein product. Introduction into the smco7 mutant of an NC-ras2 clone yielded stable transformants with a wild-type phenotype. The smco7 mutant exhibited very slow hyphal growth and the rate of conidial formation was approximately one two-hundredth of wild type. The smco7 mutation causes both the changes in the pattern of hyphal growth and the defects in cell wall synthesis. Both the diameter and the length of the apical compartment were shorter in the hyphae of the smco7 mutant. These results suggest that NC-ras2 is identical to smco7, and that the signal transduction pathway mediated by the NC-ras2 protein regulates the apical growth of hyphae, cell wall synthesis, and conidial formation in N. crassa.