Yasuo Fukami

Role and Regulation of STAT3 Phosphorylation at Ser727 in Melanocytes and Melanoma Cells

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.

Characterization of Xenopus egg membrane microdomains containing uroplakin Ib/III complex: roles of their molecular interactions for subcellular localization and signal transduction

A single‐transmembrane protein uroplakin III (UPIII) and its tetraspanin binding‐partner uroplakin Ib (UPIb) are members of the UP proteins that were originally identified in mammalian urothelium. In Xenopus laevis eggs, these proteins: xUPIII and xUPIb, are components of the cholesterol‐enriched membrane microdomains or “rafts” and involved in the sperm–egg membrane interaction and subsequent egg activation signaling via Src tyrosine kinase at fertilization. Here, we investigate whether the xUPIII‐xUPIb complex is in close proximity to CD9, a tetraspanin that has been implicated in the sperm–egg fusion in the mouse and GM1, a ganglioside typically enriched in egg rafts. Preparation of the egg membrane microdomains using different non‐ionic detergents (Brij 98 and Triton X‐100), chemical cross‐linking, co‐immunoprecipitation, in vitro kinase assay and in vitro fertilization experiments demonstrated that GM1, but not CD9, is in association with the xUPIII‐xUPIb complex and contributes to the sperm‐dependent egg activation. Transfection experiments using HEK293 cells demonstrated that xUPIII and xUPIb localized efficiently to the cholesterol‐dependent membrane microdomains when they were co‐expressed, whereas co‐expression of xUPIII and CD9, instead of xUPIb, did not show this effect. Furthermore, xUPIII and xUPIb were shown to suppress kinase activity of the wild type, but not a constitutively active form of, Xenopus Src protein co‐expressed in HEK293 cells. These results provide novel insight into the molecular architecture of the egg membrane microdomains containing xUPIII, xUPIb and Src, which may contribute to the understanding of sperm–egg interaction and signaling during Xenopus fertilization.

Structural basis of species differences between human and experimental animal CYP1A1s in metabolism of 3,3′,4,4′,5-pentachlorobiphenyl

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.

Expression, phosphorylation, and mRNA-binding of heterogeneous nuclear ribonucleoprotein K in Xenopus oocytes, eggs, and early embryos

Here we show that heterogeneous nuclear ribonucleoprotein K (hnRNP K), a member of the K homology domain‐containing proteins, is expressed in Xenopus immature oocytes, unfertilized eggs, and early embryos. Fertilization or egg activation treatment involving upregulation of the egg tyrosine kinase Src promotes a rapid and transient tyrosine phosphorylation of hnRNP K. HnRNP K is also phosphorylated on serine/threonine residues in unfertilized eggs, dephosphorylated after fertilization, and re‐phosphorylated during the premitotic phase of early embryogenesis. In vitro, Src and mitogen‐activated protein kinase (MAPK) were capable of phosphorylating hnRNP K on tyrosine and serine/threonine residues, respectively. In support of this, pretreatment of oocytes, eggs, or embryos with inhibitors for Src (PP2) and MAPK (U0126) blocked effectively the phosphorylation of hnRNP K. We also identify some maternal mRNAs that coimmunoprecipitate with hnRNP K in unfertilized eggs. Specific binding of these mRNAs to hnRNP K was verified by reverse transcriptase–polymerase chain reaction (RT–PCR). In addition, real‐time PCR analyses revealed a subset of the mRNAs whose binding to hnRNP K might be up or downregulated in activated eggs. In vitro binding assay with the use of poly U monopolymeric RNA‐coupled beads demonstrated that the RNA‐binding property of hnRNP K is negatively regulated by tyrosine phosphorylation and positively or neutrally regulated by serine/threonine phosphorylation. Taken together, it is attractive to suggest that hnRNP K is in association with certain pools of maternal mRNAs whose translational activation are modulated by the Src/MAPK phosphorylation of hnRNP K during oocyte‐egg‐embryo transition.

Gamete membrane microdomains and their associated molecules in fertilization signaling

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. Mol. Reprod. Dev. 78:814–830, 2011.

Evidence that phosphatidylinositol 3-kinase is involved in sperm-induced tyrosine kinase signaling in Xenopus egg fertilization

BackgroundStudies 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.ResultsHere 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.ConclusionsThese 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.

Multiple Post-translational Modifications Affect Heterologous Protein Synthesis

Background: Post-translational modifications (PTMs) affect protein folding. Results: Statistically significant correlations are revealed between the yield of heterologous protein expression and the presence of multiple PTM sites bioinformatically predicted in the expressed sequences. Conclusion: Predicting potential PTMs in polypeptide sequences can help optimize heterologous protein synthesis. Significance: Correlations revealed provide insights into the role of specific PTMs in protein stability and solubility. 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.

Physiology and gene expression of the rice landrace Horkuch under salt stress

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 flag 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 flag 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 specific 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.

Comprehensive bioinformatics analysis of cell-free protein synthesis: identification of multiple protein properties that correlate with successful expression

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 (α‐helix and β‐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. FASEB J. 24, 1095–1104 (2010). www.fasebj.org

12-O-Tetradecanoylphorbol-13-acetate Inhibits Melanoma Growth by Inactivation of STAT3 through Protein Kinase C-activated Tyrosine Phosphatase(s)

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 PKCα, PKCδ, and PKCϵ. 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).