Prekeratin expression of simple epithelia in the cells of long-term cultured epithelial lines of the rat liver

Prekeratin of simple epithelia with m.m. 55 kD (PK55) was found in all the studied tumorigenic and non-tumorigenic liver epithelial cell lines of the IAR series by means of indirect immunofluorescent methods in combination with corresponding monoclonal antibodies. The most prominent expression was observed in some tumorigenic cell lines. Expression of PK55 was reversible--the cells lost prekeratin in low density cultures. It has been found that the synthesis of prekeratins with m.m. 49 kD (PK49) and 40 kD (PK40) began on reaching higher cell densities than those needed for PK55 synthesis in IAR6-7 line. The PK40 appeared in cells spread on the substratum, while the PK49 was observed in upper poorly spread cells of ridges in multilayered dense cultures. Thus, the synthesis of prekeratins is not constitutive at least for some types of epithelial cells. Specific cell-to-cell interactions are presumably needed for each particular prekeratin synthesis induction.     

Changes in the expression of prekeratins with molecular weight of 55 and 40 kD in monolayer epithelium during skin morphogenesis in rats

It has been shown by indirect immunofluorescence using monoclonal antibodies against adult rat simple epithelial prekeratins with the molecular weight of 55 kD (PK55) and 40 kD (PK40) that PK55 was expressed in the covering ectoderm until the 11th day of the antenatal period. PK55 expression markedly increased in ectodermal cells lining the heart region, with PK40 appearing in the same cells on day 11. Beginning from the 16th day gradual loss of both prekeratins started with the parallel formation of squamous differentiated epidermis. These proteins were retained in the outer layers of peridermal cells and in some cells of the basal layer (probably Merkel cells). PK55 was re-expressed on the 18th day in cells migrating into dermal layer during hair folliculi formation. PK55 disappeared again in mature folliculi. Thus, a correlation between distinct morphogenetic events and PK55 and PK40 expression has been found.     

The RNA pseudoknots in foot-and-mouth disease virus are dispensable for genome replication,but essential for the production of infectious virus

Non-coding regions of viral RNA (vRNA) genomes are critically important in the regulation of gene expression. In particular, pseudoknot (PK) structures, which are present in a wide range of RNA molecules, have a variety of roles. The 5′ untranslated region (5′ UTR) of foot-and-mouth disease virus (FMDV) vRNA is considerably longer than in other viruses from the picornavirus family and consists of a number of distinctive structural motifs that includes multiple (2, 3 or 4 depending on the virus strain) putative PKs linked in tandem. The role(s) of the PKs in the FMDV infection are not fully understood. Here, using bioinformatics, sub-genomic replicons and recombinant viruses we have investigated the structural conservation and importance of the PKs in the FMDV lifecycle. Our results show that despite the conservation of two or more PKs across all FMDVs, a replicon lacking PKs was replication competent, albeit at reduced levels. Furthermore, in competition experiments, GFP FMDV replicons with less than two (0 or 1) PK structures were outcompeted by a mCherry FMDV wt replicon that had 4 PKs, whereas GFP replicons with 2 or 4 PKs were not. This apparent replicative advantage offered by the additional PKs correlates with the maintenance of at least two PKs in the genomes of FMDV field isolates. Despite a replicon lacking any PKs retaining the ability to replicate, viruses completely lacking PK were not viable and at least one PK was essential for recovery of infections virus, suggesting a role for the PKs in virion assembly. Thus, our study points to roles for the PKs in both vRNA replication and virion assembly, thereby improving understanding the molecular biology of FMDV replication and the wider roles of PK in RNA functions.     

Comparative analysis of pyruvate kinases from the hyperthermophilic archaea Archaeoglobus fulgidus,Aeropyrum pernix,and Pyrobaculum aerophilum and the hyperthermophilic bacterium Thermotoga maritima: unusual regulatory properties in hyperthermophilic archaea

Pyruvate kinases (PK, EC 2.7.1.40) from three hyperthermophilic archaea (Archaeoglobus fulgidus strain 7324, Aeropyrum pernix, and Pyrobaculum aerophilum) and from the hyperthermophilic bacterium Thermotoga maritima were compared with respect to their thermophilic, kinetic, and regulatory properties. PKs from the archaea are 200-kDa homotetramers composed of 50-kDa subunits. The enzymes required divalent cations, Mg2+ and Mn2+ being most effective, but were independent of K+. Temperature optima for activity were 85 degrees C (A. fulgidus) and above 98 degrees C (A. pernix and P. aerophilum). The PKs were highly thermostable up to 110 degrees C (A. pernix) and showed melting temperatures for thermal unfolding at 93 degrees C (A. fulgidus) or above 98 degrees C (A. pernix and P. aerophilum). All archaeal PKs exhibited sigmoidal saturation kinetics with phosphoenolpyruvate (PEP) and ADP indicating positive homotropic cooperative response with both substrates. Classic heterotropic allosteric regulators of PKs from eukarya and bacteria, e.g. fructose 1,6-bisphosphate or AMP, did not affect PK activity of hyperthermophilic archaea, suggesting the absence of heterotropic allosteric regulation. PK from the bacterium T. maritima is also a homotetramer of 50-kDa subunits. The enzyme was independent of K+ ions, had a temperature optimum of 80 degrees C, was highly thermostable up to 90 degrees C, and had a melting temperature above 98 degrees C. The enzyme showed cooperative response to PEP and ADP. In contrast to its archaeal counterparts, the T. maritima enzyme exhibited the classic allosteric response to the activator AMP and to the inhibitor ATP. Sequences of hyperthermophilic PKs showed significant similarity to characterized PKs from bacteria and eukarya. Phylogenetic analysis of PK sequences of all three domains indicates a distinct archaeal cluster that includes the PK from the hyperthermophilic bacterium T. maritima.     

PKs的生物学功能

PKs是最近发现的多功能分泌蛋白,由PK1和PK2组成.在不同的系统中,它们通过两个高度同源G-蛋白偶联受体发挥各种生物学功能.它们与神经和血管的形成以及免疫应答的调节有关,并且对生殖系统的正常生理和促性腺激素释放激素系统的发育都有很大的影响.     

Differential expression of prokineticin receptors by endothelial cells derived from different vascular beds: a physiological basis for distinct endothelial function.

Prokineticins (PKs), multifunctional secreted proteins, activate two endogenous G protein-coupled receptors (R) termed PK-R1 and PK-R2. It was suggested that PK1 acts selectively on the endothelium of endocrine glands, yet PK-Rs were also found in endothelial cells (EC) derived from other tissues. Therefore we examined here the characteristics of PK - system in EC derived from different vascular beds. Corpus luteum (CL)-derived EC (LEC) expressed both PK-R1 and PK-R2. In contrast, EC from the aorta (BAEC) only expressed PK-R1. Interestingly, also EC from brain capillaries (BCEC) expressed only PK-R1. The distinct pattern of PK-R expression may define EC phenotypic heterogeneity. Regulation of receptor expression also differed in BAEC and LEC: TNFalpha markedly reduced PK-R1 only in BAEC, but serum removal decreased PK-R1 in both cell types. Therefore, if cells were initially serum-starved, the anti-apoptotic effect of PKs was retained only in LEC. Yet, addition of PKs concomitant with serum removal enhanced the proliferation and survival of both BAEC and LEC. Immunohistochemical staining showed that in CL and aorta PK1 was expressed in smooth muscle cells in vessel walls, suggesting a paracrine mode of action. PK1 enhanced the net paracellular transport (measured by electrical resistance and Mannitol transport) in LEC but not in BAEC or BCEC. Collectively, these findings indicate that PKs serve as mitogens and survival factors for microvascular (LEC) and macrovascular (BAEC) EC. However, the distinct expression and function of PK receptors suggest different physiological roles for these receptors in various EC types.     

Effects of protein kinase and phosphatidylinositol-3 kinase inhibitors on growth and ultrastructure of Trypanosoma cruzi

An increasing number of protein kinases (PKs) of parasitic protozoa are being evaluated as drug targets. Some PK inhibitors display antiproliferative effects on protozoa. We tested three PK inhibitors on the growth and ultrastructure of epimastigotes of Trypanosoma cruzi and the effect of these drugs on intracellular amastigotes. They were staurosporine (serine/threonine kinase inhibitor), genistein (tyrosine kinase inhibitor), and wortmannin (phosphatidylinositol 3' (PI3) kinase inhibitor). All drugs inhibited epimastigote growth at the concentrations tested. Wortmannin inhibited parasite growth at the lowest concentrations. However, staurosporine was the most effective after 24 h treatment and genistein caused the stronger inhibition during the whole treatment (60-70% inhibition). The IC50 were: staurosporine: 6.43+/-1.28 microM; genistein: 6.54+/-1.86 microM; and wortmannin: 0.056+/-0.014 microM. These PK inhibitors had strong ultrastructural effects on the epimastigotes: abnormal chromatin condensation of the nucleus; loose flagellar membrane with the formation of blebs; incomplete cell division; autophagosomes and myelin-like figures. These drugs did not interfere with the division of intracellular amastigotes or with its differentiation to trypomastigotes. However, as trypanosomes have kinomes that contain a large set of protein kinases and phosphatases, PKs should not be disregarded as an important target for chemotherapy of Chagas disease.     

Cloning and expression of the Zymomonas mobilis pyruvate kinase gene in Escherichia coli

The homotetrameric pyruvate kinases (PK) constitute a fine example of allosteric enzymes subjected to sophisticated regulatory mechanisms. We have cloned and sequenced the Zymomonas mobilis structural gene for the first prokaryotic dimeric PK, as an initial step toward understanding the peculiar properties of this enzyme. The deduced amino acid sequence of the pyk gene consists of 475 residues with a calculated molecular mass of 51.4 kDa and exhibits up to 50% sequence identity with other PKs. Heterologous expression in Escherichia coli was not obtained from the native promoter, but only when the pyk gene was under the control of a strong inducible promoter when a ribosome-binding site was present upstream of the putative TTG start codon of the pyk gene. Kinetic characterization of PK in concentrated crude cell extracts showed that the enzyme is not activated by sugar phosphates or AMP but is slightly inhibited by ATP. Thus, PK of Z. mobilis is unique among the characterized prokaryotic PKs due to its high activity in the absence of any allosteric activator. Amino acid sequence alignments revealed that glutamate 381 may play a role in ineffective binding of the usual PK activator, fructose-1,6-bisphosphate.     

Site-specific phosphorylation profiling of Arabidopsis proteins by mass spectrometry and peptide chip analysis

An estimated one-third of all proteins in higher eukaryotes are regulated by phosphorylation by protein kinases (PKs). Although plant genomes encode more than 1000 PKs, the substrates of only a small fraction of these kinases are known. By mass spectrometry of peptides from cytoplasmic- and nuclear-enriched fractions, we determined 303 in vivo phosphorylation sites in Arabidopsis proteins. Among 21 different PKs, 12 were phosphorylated in their activation loops, suggesting that they were in their active state. Immunoblotting and mutational analysis confirmed a tyrosine phosphorylation site in the activation loop of a GSK3/shaggy-like kinase. Analysis of phosphorylation motifs in the substrates suggested links between several of these PKs and many target sites. To perform quantitative phosphorylation analysis, peptide arrays were generated with peptides corresponding to in vivo phosphorylation sites. These peptide chips were used for kinome profiling of subcellular fractions as well as H 2O 2-treated Arabidopsis cells. Different peptide phosphorylation profiles indicated the presence of overlapping but distinct PK activities in cytosolic and nuclear compartments. Among different H 2O 2-induced PK targets, a peptide of the serine/arginine-rich (SR) splicing factor SCL30 was most strongly affected. SRPK4 (SR protein-specific kinase 4) and MAPKs (mitogen-activated PKs) were found to phosphorylate this peptide, as well as full-length SCL30. However, whereas SRPK4 was constitutively active, MAPKs were activated by H 2O 2. These results suggest that SCL30 is targeted by different PKs. Together, our data demonstrate that a combination of mass spectrometry with peptide chip phosphorylation profiling has a great potential to unravel phosphoproteome dynamics and to identify PK substrates.     

Dependence of aminoglycoside 3'-phosphotransferase VIII activity on protein serine/threonine kinases in Streptomyces rimosus

In Streptomyces rimosus, selection with aminoglycoside kanamycin triggers "silent" aminoglycoside 3'-phosphotransferase (aph) VIII gene. Expression of aphVIII was accompanied by amplification of a chromosomal DNA fragment, which contained aphVIII. Earlier, S. rimosus aphVIII gene was isolated, sequenced, and deduced APHVIII protein sequence was reported. Using in vitro labeling and immunoprecipitation with anti-APHVIII antibody, we demonstrate that one of the abundant proteins phosphorylated by endogenous protein kinases (PKs) in extracts of S. rimosus strain S683 is APHVIII. Phosphoamino acid assay has shown phosphorylation of two seryl residues in APH molecule. The amount of phosphate incorporated into APHVIII in the presence of Ca2+ was 1.84-fold as much as that detected without Ca2+. As shown by in the gel self-phosphorylation and in the substrate-containing gel phosphorylation analyses, two serine PKs with molecular masses of 74 kDa and 55 kDa were active against APHVIII. The 55-kDa PK showed a clear Ca2+ and calmodulin dependency in activity. The specific kanamycin phosphotransferase activity of exhaustedly phosphorylated APHVIII was 3.72-fold as much as that detected in the preparation of nonphosphorylated enzyme. These results suggest involvement of PKs under study in the modulation of APHVIII aminoglycoside phosphorylating activity and in the generation of kanamycin resistance in S. rimosus.     

Intermediates in the conversion of prekeratin into keratin molecules in human epidermis

In order to elucidate the relationship between prekeratin and keratin, we performed pulse-chase experiments using [35S]methionine (35S-Met) in vitro. Of 6 prekeratin molecules (49, 52, 55, 62, 69 and 71 kDa) that incorporated 35S-Met, the 55-kDa prekeratin incorporated the most 35S-Met. In 3 molecules (52, 55 and 62 kDa) incorporation was decreased at 30 min after being chased; however, incorporation of only two molecules (55 and 62 kDa) of the 6 prekeratins was increased at 60 min. From these results and our previous data, we conclude that the initial stage of processing is as follows: 3 prekeratin molecules (52, 55 and 62 kDa) are first cleaved in the N-terminal region, then two prekeratin molecules (55 and 69 kDa) are processed to intermediates (52 and 62 kDa) by some proteolytic enzyme(s).     

Piggy-backing the concept of cancer drugs for schistosomiasis treatment: a tangible perspective?

The fear that schistosomes will become resistant to praziquantel (PZQ) motivates the search for alternatives to treat schistosomiasis. Recent studies of signaling proteins in schistosomes uncovered a way of achieving this goal relatively quickly. It was shown that protein kinases (PKs) control important biological processes in schistosomes. Concurrently, the involvement of mutant forms of PKs was demonstrated in the etiology of cancer. Therefore, different anticancer drugs have been developed to inhibit deregulated PKs. These can also inhibit schistosome PKs, thus blocking parasite development. Recent studies characterizing schistosome PKs are summarized and we discuss the concept of PK inhibitors, including approved cancer drugs, as novel candidate anti-schistosome agents. This is also likely to be of significance for other worm infections.     

Identification and expression profile analysis of the protein kinase gene superfamily in maize development

Eukaryotic protein kinases (ePKs) evolved as a family of highly dynamic molecular switches that serve to orchestrate the activity of almost all cellular processes. Some of the functionally characterized ePKs from plants have been found to be components of signaling networks, such as those for the perception of biotic agents, light quality and quantity, plant hormones, and various adverse environmental conditions. To date, only a tiny fraction of plant ePKs have been functionally identified, and even fewer have been identified in maize [Zea mays (Zm)]. In this study, we have identified 1,241 PK-encoding genes in the maize genome. Phylogenetic analyses identified eight gene groups with considerable conservation among groups, and each group could be further divided into multiple families and/or subfamilies. Similar intron/exon structural patterns were observed in the same families/subfamilies, strongly supporting their close evolutionary relationship. Chromosome distribution and genetic analysis revealed that tandem duplications and segmental/whole-genome duplications might represent two of the major mechanisms contributing to the expansion of the PK superfamily in maize. The dynamic expression patterns of ZmPK genes across the 60 different developmental stages of 11 organs showed that some members of this superfamily exhibit tissue-specific expression, whereas others are more ubiquitously expressed, indicative of their important roles in performing diverse developmental and physiological functions during the maize life cycle. Furthermore, RNA-sequence-based gene expression profiling of PKs along a leaf developmental gradient and in mature bundle sheath and mesophyll cells indicated that ZmPK genes are involved in various physiological processes, such as cell-fate decisions, photosynthetic differentiation, and regulation of stomatal development. Our results provide new insights into the function and evolution of maize PKs and will be useful in studies aimed at revealing the global regulatory network of maize development, thereby contributing to the maize molecular breeding with enhanced quality traits.     

Fluorescent nanoparticles consisting of lipopeptides and fluorescein-modified polyanions for monitoring of protein kinase activity

Protein kinase (PK)-responsive nanoparticles (NPs) comprising a hydrophobically modified peptide substrate for PKs and a fluorescein-labeled polyanion (pA-F) were reported for monitoring PK activity via fluorescence intensity measurements. In this system, the formation of NPs by mixing lipopeptides and pA-Fs results in fluorescence quenching, while the quenched fluorescence recovered following dissociation of the NPs owing to the phosphorylation reaction of PKs. Eleven lipopeptides with different hydrophobic moieties (hydrocarbon and lithocholic acid) and four pA-Fs having main chains with differing flexibilities and fluorescein contents were synthesized and used to fabricate a series of twenty-four PK-responsive NP probes. The responses of the PK-responsive NP probes to PKs were evaluated to screen the most suitable NP probes. The assay system was then used to determine the IC(50) values for five inhibitors, the results of which were very similar to those previously reported. Thus, PK-responsive NPs are useful tools for high-throughput screening (HTS) of PK inhibitors.