Slow and Prolonged Activation of the p47 Protein Kinase during Hypersensitive Cell Death in a Culture of Tobacco Cells

To investigate the involvement of protein kinases in the signaling cascade that leads to hypersensitive cell death, we used a previously established system in which a fungal elicitor, xylanase from Trichoderma viride (TvX), induces a hypersensitive reaction in tobacco (Nicotiana tabacum) cells in culture (line XD6S). The elicitor induced the slow and prolonged activation of a p47 protein kinase, which has the characteristics of a family member of the mitogen-activated protein kinases. An inhibitor of protein kinases, staurosporine, and a blocker of Ca channels, Gd3+ ions, both of which blocked the TvX-induced hypersensitive cell death, inhibited the TvX-induced activation of p47 protein kinase. Moreover, an inhibitor of serine/threonine protein phosphatase alone induced both rapid cell death and the persistent activation of the p47 protein kinase. Thus, the p47 protein kinase might be a component of the signal transduction pathway that leads to hypersensitive cell death, and the regulation of the duration of activation of the p47 protein kinase might be important in determining the destiny of tobacco cells.

Protein kinase Cdc15 activates the Dbf2-Mob1 kinase complex

Exit from mitosis in budding yeast requires inactivation of cyclin-dependent kinases through mechanisms triggered by the protein phosphatase Cdc14. Cdc14 activity, in turn, is regulated by a group of proteins, the mitotic exit network (MEN), which includes Lte1, Tem1, Cdc5, Cdc15, Dbf2/Dbf20, and Mob1. The direct biochemical interactions between the components of the MEN remain largely unresolved. Here, we investigate the mechanisms that underlie activation of the protein kinase Dbf2. Dbf2 kinase activity depended on Tem1, Cdc15, and Mob1 in vivo. In vitro, recombinant protein kinase Cdc15 activated recombinant Dbf2, but only when Dbf2 was bound to Mob1. Conserved phosphorylation sites Ser-374 and Thr-544 (present in the human, Caenorhabditis elegans, and Drosophila melanogaster relatives of Dbf2) were required for DBF2 function in vivo, and activation of Dbf2-Mob1 by Cdc15 in vitro. Although Cdc15 phosphorylated Dbf2, Dbf2–Mob1, and Dbf2(S374A/T544A)–Mob1, the pattern of phosphate incorporation into Dbf2 was substantially altered by either the S374A T544A mutations or omission of Mob1. Thus, Cdc15 promotes the exit from mitosis by directly switching on the kinase activity of Dbf2. We propose that Mob1 promotes this activation process by enabling Cdc15 to phosphorylate the critical Ser-374 and Thr-544 phosphoacceptor sites of Dbf2.

New Arabidopsis cue Mutants Suggest a Close Connection between Plastid- and Phytochrome Regulation of Nuclear Gene Expression1

We searched for new components that are involved in the positive regulation of nuclear gene expression by light by extending a screen for Arabidopsis cue (chlorophyll a/b-binding [CAB] protein-underexpressed) mutants (H.-M. Li, K. Culligan, R.A. Dixon, J. Chory [1995] Plant Cell 7: 1599–1610). cue mutants display reduced expression of the CAB3 gene, which encodes light-harvesting chlorophyll protein, the main chloroplast antenna. The new mutants can be divided into (a) phytochrome-deficient mutants (hy1 and phyB), (b) virescent or delayed-greening mutants (cue3, cue6, and cue8), and (c) uniformly pale mutants (cue4 and cue9). For each of the mutants, the reduction in CAB expression correlates with the visible phenotype, defective chloroplast development, and reduced abundance of the light-harvesting chlorophyll protein. Levels of protochlorophyllide oxidoreductase (POR) were reduced to varying degrees in etiolated mutant seedlings. In the dark, whereas the virescent mutants displayed reduced CAB expression and the lowest levels of POR protein, the other mutants expressed CAB and accumulated POR at near wild-type levels. All of the mutants, with the exception of cue6, were compromised in their ability to derepress CAB expression in response to phytochrome activation. Based on these results, we propose that the previously postulated plastid-derived signal is closely involved in the pathway through which phytochrome regulates the expression of nuclear genes encoding plastid proteins.

Guard Cells Possess a Calcium-Dependent Protein Kinase That Phosphorylates the KAT1 Potassium Channel1

Increasing evidence suggests that changes in cytosolic Ca2+ levels and phosphorylation play important roles in the regulation of stomatal aperture and as ion transporters of guard cells. However, protein kinases responsible for Ca2+ signaling in guard cells remain to be identified. Using biochemical approaches, we have identified a Ca2+-dependent protein kinase with a calmodulin-like domain (CDPK) in guard cell protoplasts of Vicia faba. Both autophosphorylation and catalytic activity of CDPK are Ca2+ dependent. CDPK exhibits a Ca2+-induced electrophoretic mobility shift and its Ca2+-dependent catalytic activity can be inhibited by the calmodulin antagonists trifluoperazine and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide. Antibodies to soybean CDPKα cross-react with CDPK. Micromolar Ca2+ concentrations stimulate phosphorylation of several proteins from guard cells; cyclosporin A, a specific inhibitor of the Ca2+-dependent protein phosphatase calcineurin enhances the Ca2+-dependent phosphorylation of several soluble proteins. CDPK from guard cells phosphorylates the K+ channel KAT1 protein in a Ca2+-dependent manner. These results suggest that CDPK may be an important component of Ca2+ signaling in guard cells.

Specific infection of CD4+ target cells by recombinant rabies virus pseudotypes carrying the HIV-1 envelope spike protein.

A recombinant rabies virus (RV) mutant deficient for the surface spike glycoprotein (G) gene was used to study the incorporation of envelope proteins from HIV-1 expressed from transfected plasmids. A hybrid HIV-1 protein in which the cytoplasmic domain was replaced with that of RV G was incorporated into the virus envelope and rescued the infectivity of the RV mutant. The RV(HIV-1) pseudotype viruses could infect only CD4+ cells, and their infectivity was neutralized specifically by anti-HIV-1 sera. In contrast to the chimeric protein, wild-type HIV-1 envelope protein or mutants with truncated cytoplasmic domains failed to produce pseudotyped particles. This indicates the presence of a specific signal in the RV G cytoplasmic domain, allowing correct incorporation of a spike protein into the envelope of rhabdovirus particles. The possibility of directing the cell tropism of RV by replacement of the RV G with proteins of defined receptor specificity should prove useful for future development of targetable gene delivery vectors.

Invasion of minor veins of tobacco leaves inoculated with tobacco mosaic virus mutants defective in phloem-dependent movement.

To fully understand vascular transport of plant viruses, the viral and host proteins, their structures and functions, and the specific vascular cells in which these factors function must be determined. We report here on the ability of various cDNA-derived coat protein (CP) mutants of tobacco mosaic virus (TMV) to invade vascular cells in minor veins of Nicotiana tabacum L. cv. Xanthi nn. The mutant viruses we studied, TMV CP-O, U1mCP15-17, and SNC015, respectively, encode a CP from a different tobamovirus (i.e., from odontoglossum ringspot virus) resulting in the formation of non-native capsids, a mutant CP that accumulates in aggregates but does not encapsidate the viral RNA, or no CP. TMV CP-O is impaired in phloem-dependent movement, whereas U1mCP15-17 and SNC015 do not accumulate by phloem-dependent movement. In developmentally-defined studies using immunocytochemical analyses we determined that all of these mutants invaded vascular parenchyma cells within minor veins in inoculated leaves. In addition, we determined that the CPs of TMV CP-O and U1mCP15-17 were present in companion (C) cells of minor veins in inoculated leaves, although more rarely than CP of wild-type virus. These results indicate that the movement of TMV into minor veins does not require the CP, and an encapsidation-competent CP is not required for, but may increase the efficiency of, movement into the conducting complex of the phloem (i.e., the C cell/sieve element complex). Also, a host factor(s) functions at or beyond the C cell/sieve element interface with other cells to allow efficient phloem-dependent accumulation of TMV CP-O.

Role of the regulatory subunit of bovine pyruvate dehydrogenase phosphatase.

Bovine pyruvate dehydrogenase phosphatase (PDP) is a Mg2+-dependent and Ca2+-stimulated heterodimer that is a member of the protein phosphatase 2C family and is localized to mitochondria. Insight into the function of the regulatory subunit of PDP (PDPr) has been gained. It decreases the sensitivity of the catalytic subunit of PDP (PDPc) to Mg2+. The apparent Km of PDPc for Mg2+ is increased about 5-fold, from about 0.35 mM to 1.6 mM. The polyamine spermine increases the sensitivity of PDP but not PDPc to Mg2+, apparently by interacting with PDPr. PDPc but not PDP can use the phosphopeptide RRAT(P)VA as a substrate. These observations are interpreted to indicate that PDPr blocks or distorts the active site of PDPc and that spermine produces a conformational change in PDPr that reverses its inhibitory effect. These findings suggest that PDPr may be involved in the insulin-induced activation of the mitochondrial PDP in adipose tissue, which is characterized by a decrease in its apparent Km for Mg2+.

Cutting activates a 46-kilodalton protein kinase in plants.

Using SDS/polyacrylamide gels that contained myelin basic protein, we identified a 46-kDa protein kinase in tobacco that is transiently activated by cutting. Although the activity of the kinase was rarely detectable in mature leaves, marked activity became apparent within several minutes after isolation of leaf discs and subsided within 30 min. In the presence of cycloheximide (CHX), the kinase activity did not diminish after the isolation over the course of 2 hr, suggesting that protein synthesis was not required for the activation of the kinase. A second cutting of leaf discs between 30 min and 60 min after the isolation failed to activate the kinase, whereas a second cutting given 3 hr after isolation apparently activated the kinase. These results suggest that the 46-kDa protein kinase is desensitized immediately after the first activation, which can be blocked by CHX, but the response ability recovers with time. When protein extracts containing the active kinase were treated with serine/threonine-specific or tyrosine-specific protein phosphatase, the kinase activity was abolished. After immunoprecipitation with antibody against phosphotyrosine, activity of the kinase was recovered in the immunoprecipitate. These results suggest that the active form of the kinase is phosphorylated at both serine/threonine and tyrosine residues. It seems likely that the 46-kDa protein kinase can be activated by dual phosphorylation. The activity of a 46-kDa protein kinase was also detected in leaves of a wide variety of plant species including dicotyledonous and monocotyledonous plants. We propose the name PMSAP (plant multisignal-activated protein) kinase for this kinase because the kinase was also activated by various signals other than cutting.

Antagonistic interaction between abscisic acid and jasmonate-ethylene signaling pathways modulates defense gene expression and disease resistance in Arabidopsis

The plant hormones abscisic acid (ABA), jasmonic acid (JA), and ethylene are involved in diverse plant processes, including the regulation of gene expression during adaptive responses to abiotic and biotic stresses. Previously, ABA has been implicated in enhancing disease susceptibility in various plant species, but currently very little is known about the molecular mechanisms underlying this phenomenon. In this study, we obtained evidence that a complex interplay between ABA and JA-ethylene signaling pathways regulate plant defense gene expression and disease resistance. First, we showed that exogenous ABA suppressed both basal and JA-ethylene-activated transcription from defense genes. By contrast, ABA deficiency as conditioned by the mutations in the ABA1 and ABA2 genes, which encode enzymes involved in ABA biosynthesis, resulted in upregulation of basal and induced transcription from JA-ethylene responsive defense genes. Second, we found that disruption of AtMYC2 (allelic to JASMONATE INSENSITIVE1 [JIN1]), encoding a basic helix-loop-helix Leu zipper transcription factor, which is a positive regulator of ABA signaling, results in elevated levels of basal and activated transcription from JA-ethylene responsive defense genes. Furthermore, the jin1/myc2 and aba2-1 mutants showed increased resistance to the necrotrophic fungal pathogen Fusarium oxysporum. Finally, using ethylene and ABA signaling mutants, we showed that interaction between ABA and ethylene signaling is mutually antagonistic in vegetative tissues. Collectively, our results indicate that the antagonistic interactions between multiple components of ABA and the JA-ethylene signaling pathways modulate defense and stress responsive gene expression in response to biotic and abiotic stresses.

Phosphate forms an unusual tripodal complex with the Fe-Mn center of sweet potato purple acid phosphatase

Purple acid phosphatases (PAPs) are a family of binuclear metalloenzymes that catalyze the hydrolysis of phosphoric acid esters and anhydrides. A PAP in sweet potato has a unique, strongly antiferromagnetically coupled Fe(III)-Mn(II) center and is distinguished from other PAPs by its increased catalytic efficiency for a range of activated and unactivated phosphate esters, its strict requirement for Mn(II), and the presence of a mu-oxo bridge at pH 4.90. This enzyme displays maximum catalytic efficiency (k(cat)/K-m) at pH 4.5, whereas its catalytic rate constant (k(cat)) is maximal at near-neutral pH, and, in contrast to other PAPs, its catalytic parameters are not dependent on the pK(a) of the leaving group. The crystal structure of the phosphate-bound Fe(III)-Mn(II) PAP has been determined to 2.5-Angstrom resolution (final R-free value of 0.256). Structural comparisons of the active site of sweet potato, red kidney bean, and mammalian PAPs show several amino acid substitutions in the sweet potato enzyme that can account for its increased catalytic efficiency. The phosphate molecule binds in an unusual tripodal mode to the two metal ions, with two of the phosphate oxygen atoms binding to Fe(III) and Mn(II), a third oxygen atom bridging the two metal ions, and the fourth oxygen pointing toward the substrate binding pocket. This binding mode is unique among the known structures in this family but is reminiscent of phosphate binding to urease and of sulfate binding to A protein phosphatase. The structure and kinetics support the hypothesis that the bridging oxygen atom initiates hydrolysis.

Role of the dsRNA-dependent protein kinase (PKR) in the attenuation of protein loss from muscle by insulin and insulin-like growth factor-I (IGF-I)

Proteolysis-inducing factor (PIF), a tumour-produced cachectic factor, induced a dose-dependent decrease in protein synthesis in murine myotubes, together with an increase in phosphorylation of eucaryotic initiation factor 2 (eIF2) on the alpha-subunit. Both insulin (1 nM) and insulin-like growth factor I (IGF-I) (13.2 nM) attenuated the depression of protein synthesis by PIF and the increased phosphorylation of eIF2alpha, by inhibiting the activation (autophosphorylation) of the dsRNA-dependent protein kinase (PKR) by induction of protein phosphatase 1. A low-molecular weight inhibitor of PKR also reversed the depression of protein synthesis by PIF to the same extent, as did insulin and IGF-I. Both insulin and IGF-I-stimulated protein synthesis in the presence of PIF, and this was attenuated by Salubrinal, an inhibitor of phospho eIF2alpha phosphatase, suggesting that at least part of this action was due to their ability to inhibit phosphorylation of eIF2alpha. Both insulin and IGF-I also attenuated the induction of protein degradation in myotubes induced by PIF, this effect was also attenuated by Salubrinal. These results suggest an alternative mechanism involving PKR to explain the effect of insulin and IGF-I on protein synthesis and degradation in skeletal muscle in the presence of catabolic factors.

Mechanism of attenuation of angiotensin-II-induced protein degradation by insulin-like growth factor-I (IGF-I)

Insulin-like growth factor-I (IGF-I) has been shown to attenuate protein degradation in murine myotubes induced by angiotensin II through downregulation of the ubiquitin-proteasome pathway, although the mechanism is not known. Angiotensin II is known to upregulate this pathway through a cellular signalling mechanism involving release of arachidonic acid, activation of protein kinase Cα (PKCα), degradation of inhibitor-κB (I-κB) and nuclear migration of nuclear factor-κB (NF-κB), and all of these events were attenuated by IGF-I (13.2 nM). Induction of the ubiquitin-proteasome pathway has been linked to activation of the RNA-activated protein kinase (PKR), since an inhibitor of PKR attenuated proteasome expression and activity in response to angiotensin II and prevented the decrease in the myofibrillar protein myosin. Angiotensin II induced phosphorylation of PKR and of the eukaryotic initiation factor-2 (eIF2) on the α-subunit, and this was attenuated by IGF-I, by induction of the expression of protein phosphatase 1, which dephosphorylates PKR. Release of arachidonic acid and activation of PKCα by angiotensin II were attenuated by an inhibitor of PKR and IGF-I, and the effect was reversed by Salubrinal (15 μM), an inhibitor of eIF2α dephosphorylation, as was activation of PKCα. In addition myotubes transfected with a dominant-negative PKR (PKRΔ6) showed no release of arachidonate in response to Ang II, and no activation of PKCα. These results suggest that phosphorylation of PKR by angiotensin II was responsible for the activation of the PLA2/PKC pathway leading to activation of NF-κB and that IGF-I attenuates protein degradation due to an inhibitory effect on activation of PKR. © 2007 Elsevier Inc. All rights reserved.

Uncovering changes in the redox protein interactome of PTEN

Phosphatase and tensin homolog (PTEN) is a redox-sensitive, dual-specificity protein phosphatase involved in regulating a number of cellular processes including metabolism, apoptosis, cell proliferation and survival. It acts as a tumor suppressor by negatively regulating the PI3K/Akt pathway. While direct evidence of a redox regulation of PTEN downstream signaling has been reported, the effect of cellular oxidative stress or direct PTEN oxidation on the PTEN interactome is still poorly defined. To investigate this, PTEN-GST fusion protein was prepared in its reduced form and an H2O2-oxidized form that was reversible by DTT treatment, and these were immobilized on a glutathione-sepharose-based support. The immobilized protein was incubated with cell lysate to capture interacting proteins. Captured proteins were eluted from the beads, analyzed by LC-MSMS and comparatively quantified using label-free methods. After subtraction of interactors that were also present in the resin and GST controls, 97 individual protein interactors were identified, including several that are novel. Fourteen interactors that varied significantly with the redox status of PTEN were identified, including thioredoxin and peroxiredoxin-1. Except for one interactor, their binding was higher for oxidized PTEN. Using western blotting, altered binding to PTEN was confirmed for 3 selected interactors (Prdx1, Trx, and Anxa2) and DDB1 was validated as a novel interactor with unaltered binding. Our results suggest that the redox status of PTEN causes a functional variation in the PTEN interactome which is important for the cellular function of PTEN. The resin capture method developed had distinct advantages in that the redox status of PTEN could be directly controlled and measured.

Reversible oxidation of phosphatase and tensin homolog (PTEN) alters its interactions with signaling and regulatory proteins

Phosphatase and tensin homolog (PTEN) is involved in a number of different cellular processes including metabolism, apoptosis, cell proliferation and survival. It is a redox-sensitive dual-specificity protein phosphatase that acts as a tumor suppressor by negatively regulating the PI3K/Akt pathway. While direct evidence of redox regulation of PTEN downstream signaling has been reported, the effect of PTEN redox status on its protein-protein interactions is poorly understood. PTEN-GST in its reduced and a DTT-reversible H2O2-oxidized form was immobilized on a glutathione-sepharose support and incubated with cell lysate to capture interacting proteins. Captured proteins were analyzed by LC-MSMS and comparatively quantified using label-free methods. 97 Potential protein interactors were identified, including a significant number that are novel. The abundance of fourteen interactors was found to vary significantly with the redox status of PTEN. Altered binding to PTEN was confirmed by affinity pull-down and Western blotting for Prdx1, Trx, and Anxa2, while DDB1 was validated as a novel interactor with unaltered binding. These results suggest that the redox status of PTEN causes a functional variation in the PTEN interactome. The resin capture method developed had distinct advantages in that the redox status of PTEN could be directly controlled and measured.

Antitumor Activity Of Irradiated Riboflavin On Human Renal Carcinoma Cell Line 786-o.

Riboflavin (vitamin B2) is a precursor for coenzymes involved in energy production, biosynthesis, detoxification, and electron scavenging. Previously, we demonstrated that irradiated riboflavin (IR) has potential antitumoral effects against human leukemia cells (HL60), human prostate cancer cells (PC3), and mouse melanoma cells (B16F10) through a common mechanism that leads to apoptosis. Hence, we here investigated the effect of IR on 786-O cells, a known model cell line for clear cell renal cell carcinoma (CCRCC), which is characterized by high-risk metastasis and chemotherapy resistance. IR also induced cell death in 786-O cells by apoptosis, which was not prevented by antioxidant agents. IR treatment was characterized by downregulation of Fas ligand (TNF superfamily, member 6)/Fas (TNF receptor superfamily member 6) (FasL/Fas) and tumor necrosis factor receptor superfamily, member 1a (TNFR1)/TNFRSF1A-associated via death domain (TRADD)/TNF receptor-associated factor 2 (TRAF) signaling pathways (the extrinsic apoptosis pathway), while the intrinsic apoptotic pathway was upregulated, as observed by an elevated Bcl-2 associated x protein/B-cell CLL/lymphoma 2 (Bax/Bcl-2) ratio, reduced cellular inhibitor of apoptosis 1 (c-IAP1) expression, and increased expression of apoptosis-inducing factor (AIF). The observed cell death was caspase-dependent as proven by caspase 3 activation and poly(ADP-ribose) polymerase-1 (PARP) cleavage. IR-induced cell death was also associated with downregulation of v-src sarcoma (Schmidt-Ruppin A-2) viral oncogene homologue (avian)/protein serine/threonine kinase B/extracellular signal-regulated protein kinase 1/2 (Src/AKT/ERK1/2) pathway and activation of p38 MAP kinase (p38) and Jun-amino-terminal kinase (JNK). Interestingly, IR treatment leads to inhibition of matrix metalloproteinase-2 (MMP-2) activity and reduced expression of renal cancer aggressiveness markers caveolin-1, low molecular weight phosphotyrosine protein phosphatase (LMWPTP), and kinase insert domain receptor (a type III receptor tyrosine kinase) (VEGFR-2). Together, these results show the potential of IR for treating cancer.