[Show abstract][Hide abstract] ABSTRACT: Calcium is a universal messenger that mediates egg activation at fertilization in all sexually reproducing species studied. However, signaling pathways leading to calcium generation and the mechanisms of calcium-induced exit from meiotic arrest vary substantially among species. Here, we review the pathways of calcium signaling and the mechanisms of meiotic exit at fertilization in the eggs of the established developmental model, African clawed frog, Xenopus laevis. We also discuss calcium involvement in the early fertilization-induced events in Xenopus egg, such as membrane depolarization, the increase in intracellular pH, cortical granule exocytosis, cortical contraction, contraction wave, cortical rotation, reformation of the nuclear envelope, sperm chromatin decondensation and sister chromatid segregation.
International Journal of Molecular Sciences 10/2014; 15(10):18659-18676. DOI:10.3390/ijms151018659 · 2.86 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In Xenopus laevis, sperm-egg interaction promotes partial proteolysis and/or tyrosine phosphorylation of uroplakin III (UPIII) and the tyrosine kinase Src, which both localize to the cholesterol-enriched egg membrane microdomains (MDs). Here we show that sperm promote proteolysis and/or tyrosine phosphorylation of UPIII and Src in MDs isolated from ovulated and unfertilized eggs (UF-MDs). An antibody against the extracellular domain of UPIII interferes with these events. Inhibition of fertilization by anti-UPIII antibody is rescued by co-incubation with UF-MDs. This suggests that, like MDs in intact eggs, the isolated UF-MDs are capable of interacting with sperm, an interaction that does not interfere with normal fertilization but rather augments the ability of sperm to fertilize eggs pretreated with anti-UPIII antibody. This unexpected effect of UF-MDs on sperm requires UPIII function in UF-MDs and protein kinase activity in sperm. MDs isolated from progesterone-treated mature oocytes, but not ovarian immature oocytes, are similarly functional as UF-MDs. The anti-UPIII extracellular domain antibody binds more effectively to the surface of mature than immature ovarian oocytes. We propose that the structural and functional competency of the UPIII-Src signaling system in MDs is strictly regulated during oocyte maturation and subsequently in sperm-mediated egg activation and fertilization. The fertilization-related signaling properties seen in UF-MDs can be partially reconstituted in MDs of human embryonic kidney 293 cells (293-MDs) expressing UPIII, Src and uroplakin Ib. However, 293-MDs expressing a proteolysis-resistant mutant of UPIII are less functional, suggesting that the availability of UPIII to protease action is important for MD function.
Development 04/2014; 141(8):1705-14. DOI:10.1242/dev.105510 · 6.46 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Protein structural research in plants lags behind that in animal and bacterial species. This lag concerns both the structural analysis of individual proteins and the proteome-wide characterization of structure-related properties. Until now, no systematic study concerning the relationships between protein disorder and multiple post-translational modifications (PTMs) in plants has been presented.
In this work, we calculated the global degree of intrinsic disorder in the complete proteomes of 8 typical monocotyledonous and dicotyledonous plant species. We further predicted multiple sites for phosphorylation, glycosylation, acetylation and methylation and examined the correlations of protein disorder with the presence of the predicted PTM sites. It was found that phosphorylation, acetylation, and O-glycosylation displayed a clear preference for occurrence in disordered regions of plant proteins. In contrast, methylation tended to avoid disordered sequence, whereas N-glycosylation did not show a universal structural preference in monocotyledonous and dicotyledonous plants. In addition, the analysis performed revealed significant differences between the integral characteristics of monocot and dicot proteomes. They included elevated disorder degree, increased rate of O-glycosylation and R-methylation, decreased rate of N-glycosylation, K-acetylation and K-methylation in monocotyledonous plant species, as compared to dicotyledonous species. Altogether, our study provides the most compelling evidence so far for the connection between protein disorder and multiple PTMs in plants.
email@example.com; firstname.lastname@example.org SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
[Show abstract][Hide abstract] ABSTRACT: Oocytes and eggs of the African clawed frog, Xenopus laevis, are commonly used in gene expression studies. However, monitoring transcript levels in the individual living oocytes remains challenging. To address this challenge, we used a technique based on multiple repeated collections of nanoliter volumes of cytoplasmic material from a single oocyte. Transcript quantification was performed by quantitative RT-PCR. The technique allowed monitoring of heterologous gene expression in a single oocyte without affecting its viability. We also used this approach to profile the expression of endogenous genes in living Xenopus oocytes. Although frog oocytes are traditionally viewed as a homogenous cell population, a significant degree of gene expression variation was observed among the individual oocytes. A lognormal distribution of transcript levels was revealed in the oocyte population. Finally, using this technique, we observed a dramatic decrease in the content of various cytoplasmic mRNAs in aging unfertilized eggs but not in oocytes, suggesting a link between mRNA degradation and egg apoptosis.
[Show abstract][Hide abstract] ABSTRACT: Cell-free protein synthesis offers substantial advantages over cell-based expression, allowing direct access to the protein synthetic reaction and meticulous control over the reaction conditions. Recently, we identified a number of statistically significant correlations between calculated and predicted properties of amino acid sequences and their amenability to heterologous cell-free expression. These correlations can be of practical use for predicting expression success and optimizing cell-free protein synthesis. In this chapter, we describe our approach and demonstrate how computational and predictive bioinformatics can be used to analyze and optimize cell-free protein expression.
[Show abstract][Hide abstract] ABSTRACT: Background
In several species with external fertilization, including frogs, laid unfertilized eggs were found to die by apoptosis outside of the animal body. However, there is no apparent reason for the externally laid eggs to degrade by this process, considering that apoptosis developed as a mechanism to reduce the damaging effect of individual cell death to the whole organism.
Here, we demonstrate that a number of eggs are retained in the genital tract of the African clawed frog Xenopus laevis after gonadotropin-induced ovulation. The majority of these eggs exit meiotic arrest within 24 hours of hormone administration. Subsequently, post-meiotic eggs die in the frog genital tract by a well-defined apoptotic process. The hallmarks of egg degradation include prominent morphological changes, cytochrome c release, caspase activation, increase in ADP/ATP ratio, progressive intracellular acidification, egg swelling and all-out proteolysis of egg proteins. The sustained presence of post-apoptotic eggs in the genital tract of ageing frogs evidenced age-associated worsening of apoptotic clearance.
The direct observation of egg degradation in the Xenopus genital tract provides a clue to the physiological relevance of frog egg apoptosis. It works to eliminate the mature unlaid eggs retained in the animal body after ovulation. Our findings establish egg apoptosis as a major physiological process accompanying ovulation in frogs.
[Show abstract][Hide abstract] ABSTRACT: Our previous study demonstrated that tyrosine phosphorylation of p145(met)/β-subunit of hepatocyte growth factor receptor by epidermal growth factor receptor and Src contributes to the anti-apoptotic growth of human bladder carcinoma cell 5637 under serum-starved conditions. Here, we show that some other cell lines of human bladder carcinoma, but not other types of human cancer cells, also exhibit Src-dependent, anti-apoptotic proliferation under serum-starved conditions, and that low-density, detergent-insoluble membrane microdomains (MD) serve as a structural platform for signaling events involving p145(met), EGFR, and Src. As an MD-associated molecule that may contribute to bladder carcinoma-specific cellular function, we identified uroplakin IIIa (UPIIIa), an urothelium-specific protein. Results obtained so far revealed: 1) UPIIIa undergoes partial proteolysis in serum-starved cells; 2) a specific antibody to the extracellular domain of UPIIIa inhibits the proteolysis of UPIIIa and the activation of Src, and promotes apoptosis in serum-starved cells; and 3) knockdown of UPIIIa by short interfering RNA also promotes apoptosis in serum-starved cells. GM6001, a potent inhibitor of matrix metalloproteinase (MMP), inhibits the proteolysis of UPIIIa and promotes apoptosis in serum-starved cells. Furthermore, serum starvation promotes expression and secretion of the heparin-binding EGF-like growth factor in a manner that depends on the functions of MMP, Src, and UPIIIa. These results highlight a hitherto unknown signaling network involving a subset of MD-associated molecules in the anti-apoptotic mechanisms of human bladder carcinoma cells.
Biology Open 10/2012; 1(10):1024-34. DOI:10.1242/bio.20121115 · 2.42 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Post-translational modifications (PTMs) are required for proper folding of many proteins. The low capacity for PTMs hinders the production of heterologous proteins in the widely used prokaryotic systems of protein synthesis. Until now, a systematic and comprehensive study concerning the specific effects of individual PTMs on heterologous protein synthesis has not been presented. To address this issue, we expressed 1488 human proteins and their domains in a bacterial cell-free system, and we examined the correlation of the expression yields with the presence of multiple PTM sites bioinformatically predicted in these proteins. This approach revealed a number of previously unknown statistically significant correlations. Prediction of some PTMs, such as myristoylation, glycosylation, palmitoylation, and disulfide bond formation, was found to significantly worsen protein amenability to soluble expression. The presence of other PTMs, such as aspartyl hydroxylation, C-terminal amidation, and Tyr sulfation, did not correlate with the yield of heterologous protein expression. Surprisingly, the predicted presence of several PTMs, such as phosphorylation, ubiquitination, SUMOylation, and prenylation, was associated with the increased production of properly folded soluble proteins. The plausible rationales for the existence of the observed correlations are presented. Our findings suggest that identification of potential PTMs in polypeptide sequences can be of practical use for predicting expression success and optimizing heterologous protein synthesis. In sum, this study provides the most compelling evidence so far for the role of multiple PTMs in the stability and solubility of heterologously expressed recombinant proteins.
[Show abstract][Hide abstract] ABSTRACT: The transcription factor signal transducer and activator of transcription 3 (STAT3) has two important phosphorylation sites, Tyr705 and Ser727, for its activation. Ser727 phosphorylation has been considered to be a secondary event after Tyr705 phosphorylation. In this study, the role and regulation of Ser727 phosphorylation in STAT3 in melanocytic cells were examined. STAT3 was phosphorylated on Ser727 in the absence of Tyr705 phosphorylation in melanocytes. 12-O-tetradecanoylphorbol-13-acetate-induced increase in cell survival activity and nuclear translocation of STAT3 was associated with Ser727 phosphorylation. Ser727 was constitutively phosphorylated in all melanoma cell lines examined irrespective of Tyr705 phosphorylation. The possible involvement of Ser727 phosphorylation in STAT3 in cell survival activity and nuclear translocation of STAT3 in melanocytes was demonstrated also in melanoma cells. The constitutive Ser727 phosphorylation in melanoma cells was partially mediated by the B-Raf-MEK-ERK1/2 pathway. Immunohistochemical studies on specimens of primary lesions of acral lentiginous melanoma revealed that Ser727 phosphorylation precedes Tyr705 phosphorylation in the early stages of melanoma progression. Our results indicate that Ser727 phosphorylation on STAT3 is not necessarily a secondary event after Tyr705 phosphorylation and suggest that it has a role in the regulation of cell survival activity and nuclear translocation of STAT3 in melanocytic cells.
[Show abstract][Hide abstract] ABSTRACT: A characteristic feature of frog reproduction is external fertilization accomplished outside the female's body. Mature fertilization-competent frog eggs are arrested at the meiotic metaphase II with high activity of the key meiotic regulators, maturation promoting factor (MPF) and cytostatic factor (CSF), awaiting fertilization. If the eggs are not fertilized within several hours of ovulation, they deteriorate and ultimately die by as yet unknown mechanism.
Here, we report that the vast majority of naturally laid unfertilized eggs of the African clawed frog Xenopus laevis spontaneously exit metaphase arrest under various environmental conditions and degrade by a well-defined apoptotic process within 48 hours after ovulation. The main features of this process include cytochrome c release, caspase activation, ATP depletion, increase of ADP/ATP ratio, apoptotic nuclear morphology, progressive intracellular acidification, and egg swelling. Meiotic exit seems to be a prerequisite for execution of the apoptotic program, since (i) it precedes apoptosis, (ii) apoptotic events cannot be observed in the eggs maintaining high activity of MPF and CSF, and (iii) apoptosis in unfertilized frog eggs is accelerated upon early meiotic exit. The apoptotic features cannot be observed in the immature prophase-arrested oocytes, however, the maturation-inducing hormone progesterone renders oocytes susceptible to apoptosis.
The study reveals that naturally laid intact frog eggs die by apoptosis if they are not fertilized. A maternal apoptotic program is evoked in frog oocytes upon maturation and executed after meiotic exit in unfertilized eggs. The meiotic exit is required for execution of the apoptotic program in eggs. The emerging anti-apoptotic role of meiotic metaphase arrest needs further investigation.
[Show abstract][Hide abstract] ABSTRACT: Fertilization is the fundamental system of biological reproduction in many organisms, including animals, plants, and algae. A growing body of knowledge has emerged to explain how fertilization and activation of development are accomplished. Studies on the molecular mechanisms of fertilization are in progress for a wide variety of multicellular organisms. In this review, we summarize recent findings and debates about the long-standing questions concerning fertilization: how egg and sperm become competent for their interaction with each other, how the binding and fusion of these gamete cells are made possible, and how the fertilized eggs initiate development to a newborn. We will focus on the structure and function of the membrane microdomains (MDs) of egg and sperm that may serve as a platform or signaling center for the aforementioned cellular functions. In particular, we provide evidence that MDs of eggs from the African clawed frog, Xenopus laevis, play a pivotal role in receiving extracellular signals from fertilizing sperm and then transmitting them to the egg cytoplasm, where the tyrosine kinase Src is present and responsible for the subsequent signaling events collectively called egg activation. The presence of a new signaling axis involving uroplakin III, an MD-associated transmembrane protein, and Src in this system will be highlighted and discussed.
Molecular Reproduction and Development 10/2011; 78(10-11):814-30. DOI:10.1002/mrd.21336 · 2.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A tyrosine-phosphorylated protein of 33 kDa is shown to be present in the solubilized yolk fraction of Xenopus laevis oocytes, eggs, and early embryos. The phosphoprotein is lipovitellin 2 as demonstrated by immunoprecipitation and mass spectrometry studies, and is termed pp33/LV2. Sub-cellular fractionation and immunoblotting studies demonstrate that pp33/LV2 is stably present in the Triton X-100-resistant and SDS-soluble yolk fractions during oogenesis, oocyte maturation, and early embryogenesis. From after the swimming tadpole stages (stage 39∼), however, it becomes partly soluble to Triton X-100-containing buffer and all disappear thereafter (stage 48∼49). In vitro enzyme assays with the use of the tyrosine phosphatase LAR and the tyrosine kinase Src demonstrate the reversible nature of the tyrosine phosphorylation of pp33/LV2. Microinjection studies demonstrate that the solubilized yolk fractions, but not those immunodepleted of pp33/LV2 or those pretreated with LAR, inhibit progesterone- or insulin-induced oocyte maturation. A pp33/LV2-like protein seems to present in two Xenopus subspecies, one other frog species, and two fish species, but not in other amphibian species, such as newt and salamander. These results suggest that LV2, in its tyrosine-phosphorylated form, serves in a cellular function in a species-specific manner, but the mechanism is still unknown.
[Show abstract][Hide abstract] ABSTRACT: Coplanar polychlorinated biphenyls included in dioxin-like compounds are bio-accumulated and adversely affect wildlife and human health. Although many researchers have studied the metabolism of PCBs, there have been few reports of the in vitro metabolism of 3,3',4,4',5-pentachlorobiphenyl (PCB126), despite the fact that it has the highest toxicity among PCB congeners. Cytochrome P450 (CYP) 1A1 proteins can metabolize some dioxins and PCBs by hydroxylation, but the activities of human and rat CYP1A1 proteins are very different. The mechanism remains unclear. From our results, rat CYP1A1 metabolized PCB126 into 4-OH-3,3',4',5-tetrachlorobiphenyl and 4-OH-3,3',4',5,5'-pentachlorobiphenyl, but human CYP1A1 did not metabolize. Homology models of the two CYP proteins, and docking studies, showed that differences in the amino acid residues forming their substrate-binding cavities led to differences in the size and shape of the cavities; only the cavity of rat CYP1A1 allowed PCB126 close enough to the haem to be metabolized. Comparison of the amino acid residues of other mammalian CYP1A1 proteins suggested that rats have a unique metabolism of xenobiotics. Our results suggest that it is necessary to be careful in human extrapolation of toxicity data estimated by using the rat as an experimental animal, especially in the case of compounds metabolized by CYP1A1.
Journal of Biochemistry 03/2011; 149(4):487-94. DOI:10.1093/jb/mvr009 · 2.58 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Good donors in breeding for salt tolerance are a prerequisite for food security under changing climatic
conditions. Horkuch, a farmer-popular salt tolerant rice (Oryza sativa L.) variety from the south-west coast of Bangladesh
was characterised up to maturity under NaCl stress, together with a modern variety (BRRI dhan41), a sensitive control (BRRI
dhan29) and Pokkali, the salt-tolerant benchmark for rice. Horkuch had low reduction in shoot biomass, a low Na :K ratio
in !ag leaves, a low percent reduction in yield and good partitioning of Na in the older leaves, and maintained high levels of
Ca and Mg in the !ag leaves. In order to understand the physiology at the molecular level, the expression of salt-responsive
genes was investigated using microarray analysis. Salt-stressed cDNA of Horkuch seedlings were hybridised with cDNA
probes synthesised mainly from database sequences of Arabidopsis thaliana (L.) Heynh. The upregulated genes included
transcription factors, signal transducers, metabolic enzymes, reactive oxygen species (ROS) scavengers, osmoprotectants
and some speci"c salt-induced transcripts. An increase in expression of photosynthesis-related genes as well ROS
scavengers suggested that this could be the reason for the better yield performance of Horkuch. The data therefore
indicate Horkuch as a potential donor alternative to Pokkali in breeding programs for salt tolerance.
[Show abstract][Hide abstract] ABSTRACT: Single-subunit bacteriophage T7 RNA polymerase (T7 RNAP) is universally employed for in vivo and in vitro transcription of genes put under control of the T7 promoter. The enzyme is capable of transcribing a complete gene without additional proteins. In this study, we reveal the presence of a low molecular weight factor, which induces several-fold activation of T7 RNAP in the cytoplasm of oocytes and eggs from Xenopus laevis. Cell-free reconstitution of the T7 RNAP activation allowed us to investigate the molecular properties of the activator, establish its peptide nature and suggest T7 RNAP activation mechanism. In contrast to the previously described nonspecific transcriptional activators, which interact with scattered ionic sites on nucleic acids, the peptide activator associates with T7 RNAP molecule, thus being a bona fide activator of the polymerase. To our knowledge, this is the first report concerning the specific activation of T7 RNAP by a factor of peptide or protein origin. Besides rather obvious merits in gaining more efficient transcription with T7 RNAP, this finding can provide additional insights into regulatory mechanisms of transcription. The study also introduces a novel highly sensitive luminescent assay of T7 RNAP activity.
Genes to Cells 11/2010; 15(11):1136-44. DOI:10.1111/j.1365-2443.2010.01447.x · 2.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Intracellular signaling during egg activation/fertilization has been extensively studied using intact eggs, which can be manipulated by microinjection of different mRNAs, proteins, or chemical drugs. Furthermore, egg extracts, which retain high CSF activity (CSF-arrested extracts), were developed for studying fertilization/activation signal transduction, which have significant advantages as a model system. The addition of calcium to CSF-arrested extracts initiates a plethora of signaling events that take place during egg activation. Hence, the signaling downstream of calcium mobilization has been successfully studied in the egg extracts. Moreover, despite disruption of membrane-associated signaling compartments and ordered compartmentalization during extract preparation, CSF-arrested extracts can be successfully used to study early signaling events, which occur upstream of calcium release during egg activation/fertilization. In combination with the CSF-arrested extracts, activated egg rafts can reproduce some events of egg activation, including PLCgamma activation, IP3 production, transient calcium release, MAPK inactivation, and meiotic exit. This becomes possible due to complementation of the sperm-induced egg activation signaling machinery present in the rafts with the components of signal transduction system localized in the extracts. Herein, we describe protocols for studying molecular mechanisms of egg fertilization/activation using cell-free extracts and membrane rafts prepared from metaphase-arrested Xenopus eggs.
[Show abstract][Hide abstract] ABSTRACT: Studies have examined the function of PI 3-kinase in the early developmental processes that operate in oocytes or early embryos of various species. However, the roles of egg-associated PI 3-kinase and Akt, especially in signal transduction at fertilization, are not well understood.
Here we show that in Xenopus eggs, a potent inhibitor of phosphatidylinositol 3-kinase (PI 3-kinase), LY294002 inhibits sperm-induced activation of the tyrosine kinase Src and a transient increase in the intracellular concentration of Ca2+ at fertilization. LY294002 also inhibits sperm-induced dephosphorylation of mitogen-activated protein kinase, breakdown of cyclin B2 and Mos, and first embryonic cleavage, all of which are events of Ca2+-dependent egg activation. In fertilized eggs, an 85-kDa subunit of PI 3-kinase (p85) undergoes a transient translocation to the low-density, detergent-insoluble membranes (membrane microdomains) where Src tyrosine kinase signaling is operating. However, the tyrosine phosphorylation of p85 in fertilized eggs is not as evident as that in H2O2-activated eggs, arguing against the possibility that PI 3-kinase is activated by Src phosphorylation. Nevertheless, sperm-induced activation of PI 3-kinase has been demonstrated by the finding that Akt, a serine/threonine-specific protein kinase, is phosphorylated at threonine-308. The threonine-phosphorylated Akt also localizes to the membrane microdomains of fertilized eggs. Application of bp(V), an inhibitor of PTEN that dephosphorylates PIP3, the enzymatic product of PI 3-kinase, promotes parthenogenetic activation of Xenopus eggs. In vitro kinase assays demonstrate that PIP3 activates Src in a dose-dependent manner.
These results suggest that PI 3-kinase is involved in sperm-induced egg activation via production of PIP3 that would act as a positive regulator of the Src signaling pathway in Xenopus fertilization.
[Show abstract][Hide abstract] ABSTRACT: High-throughput cell-free protein synthesis is being used increasingly in structural/functional genomics projects. However, the factors determining expression success are poorly understood. Here, we evaluated the expression of 3066 human proteins and their domains in a bacterial cell-free system and analyzed the correlation of protein expression with 39 physicochemical and structural properties of proteins. As a result of the bioinformatics analysis performed, we determined the 18 most influential features that affect protein amenability to cell-free expression. They include protein length; hydrophobicity; pI; content of charged, nonpolar, and aromatic residues;, cysteine content; solvent accessibility; presence of coiled coil; content of intrinsically disordered and structured (alpha-helix and beta-sheet) sequence; number of disulfide bonds and functional domains; presence of transmembrane regions; PEST motifs; and signaling sequences. This study represents the first comprehensive bioinformatics analysis of heterologous protein synthesis in a cell-free system. The rules and correlations revealed here provide a plethora of important insights into rationalization of cell-free protein production and can be of practical use for protein engineering with the aim of increasing expression success.-Kurotani, A., Takagi, T., Toyama, M., Shirouzu, M., Yokoyama, S., Fukami, Y., Tokmakov, A. A. Comprehensive bioinformatics analysis of cell-free protein synthesis: identification of multiple protein properties that correlate with successful expression.
The FASEB Journal 11/2009; 24(4):1095-104. DOI:10.1096/fj.09-139527 · 5.04 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The growth of most melanoma cells in vitro is inhibited by the tumor-promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). In this study, the involvement of the signal transducer and activator of transcription 3 (STAT3) in the TPA-induced growth inhibition of melanoma cells was examined. The in vitro growth and DNA synthesis of five melanoma cell lines, whose STAT3 was activated (phosphorylated), was inhibited by TPA, whereas that of WM35 and WM39 cells, whose STAT3 activity was at negligible levels, was considerably slow and not affected by TPA. Blockade of STAT3 activity by small interfering RNAs suppressed the growth of WM1205Lu cells containing constitutively activated STAT3. Treatment of WM1205Lu cells with TPA decreased both the phosphorylated STAT3 and the DNA-binding activity of STAT3. Pretreatment of WM1205Lu cells with either a protein-tyrosine phosphatase inhibitor or a protein kinase C (PKC) inhibitor prevented the inhibitory effects of TPA on the level of phosphorylated STAT3. The five melanoma cell lines containing phosphorylated STAT3 commonly expressed PKCalpha, PKCdelta, and PKCepsilon. Introduction of the dominant negative mutant of one of these PKC isoforms into WM1205Lu cells inhibited the TPA-induced dephosphorylation of STAT3. A Src inhibitor attenuated the STAT3 phosphorylation in WM1205Lu cells. These results indicate that constitutively activated STAT3 is positively regulated by c-Src and negatively regulated by a PKC-activated tyrosine phosphatase(s) in melanoma cells. Because TPA did not affect c-Src activity, we conclude that the growth inhibitory effect of TPA on melanoma cells is mediated through inactivation of STAT3 by a PKC-activated tyrosine phosphatase(s).
[Show abstract][Hide abstract] ABSTRACT: The complete family of expressed pyruvate dehydrogenase kinase (PDK) genes in the tissues of the African clawed frog, Xenopus laevis, consists of four members. Our previous study [Terazawa, Y., Tokmakov, A., Shirouzu, M., Yokoyama, S., 2005. Molecular cloning and expression analysis of PDK family genes in Xenopus laevis reveal oocyte-specific PDK isoform. Biochem. Biophys. Res. Commun. 338, 1798-1804] revealed that expression patterns of PDK genes differ greatly in the oocytes and somatic tissues of the adult frog. In the present work, using quantitative reverse-transcriptase PCR analysis, we demonstrate that the major transition from the oocyte-specific to somatic tissue-specific xPDK expression pattern occurs at the late stages of Xenopus embryogenesis after mid-blastula transition (MBT). Also, we show that the content of mRNA for xPDKo3, which is the predominant PDK isoform in oocytes and eggs, increases by about 3-fold during maturation. Other PDK family genes are down-regulated during oogenesis, thus being at their lowest expression levels in the grown-up oocytes, matured eggs, and early embryos. The expression of all PDK genes increases several-fold in the embryogenesis following MBT. Analysis of protein expression using an antibody raised against C-terminal of xPDKo3 confirmed isoform-specific up-regulation of xPDKo3 late in maturation and revealed cytoplasmic and mitochondrial localization of this protein. Bioinformatics and mass-spectrometric analyses allowed identification of an N-terminal mitochondrial targeting signal and a peptide cleavage site in xPDKo3 molecule.