Exploring the function of Tyr83 in bacteriorhodopsin: features of the Y83F and Y83N mutants.

Tyrosine-83, a residue which is conserved in all halobacterial retinal proteins, is located at the extracellular side in helix C of bacteriorhodopsin. Structural studies indicate that its hydroxyl group is hydrogen bonded to Trp189 and possibly to Glu194, a residue which is part of the proton release complex (PRC) in bacteriorhodopsin. To elucidate the role of Tyr83 in proton transport, we studied the Y83F and Y83N mutants. The Y83F mutation causes an 11 nm blue shift of the absorption spectrum and decreases the size of the absorption changes seen upon dark adaptation. The light-induced fast proton release, which accompanies formation of the M intermediate, is observed only at pH above 7 in Y83F. The pK(a) of the PRC in M is elevated in Y83F to about 7.3 (compared to 5.8 in WT). The rate of the recovery of the initial state (the rate of the O --> BR transition) and light-induced proton release at pH below 7 is very slow in Y83F (ca. 30 ms at pH 6). The amount of the O intermediate is decreased in Y83F despite the longer lifetime of O. The Y83N mutant shows a similar phenotype in respect to proton release. As in Y83F, the recovery of the initial state is slowed several fold in Y83N. The O intermediate is not seen in this mutant. The data indicate that the PRC is functional in Y83F and Y83N but its pK(a) in M is increased by about 1.5 pK units compared to the WT. This suggests that Tyr83 is not the main source for the proton released upon M formation in the WT; however, Tyr83 is involved in the proton release affecting the pK(a) of the PRC in M and the rate of proton transport from Asp85 to PRC during the O --> bR transition. Both the Y83F and the Y83N mutations lead to a greatly decreased functionality of the pigment at high pH because most of the pigment is converted into the inactive P480 species, with a pK(a) 8-9.     

温度和铜绿微囊藻毒性对萼花臂尾轮虫生活史参数的影响

文章开展了25℃下两个品系铜绿微囊藻(有毒与无毒)不同浓度对萼花臂尾轮虫生活史的影响研究, 及在5个温度下不同浓度有毒铜绿微囊藻对萼花臂尾轮虫生活史影响的研究。结果表明铜绿微囊藻毒性、浓度及二者交互作用对轮虫生活史参数净生殖率(R₀; F=31.83, P<0.01; F=30.36, P<0.01; F=13.51, P<0.01)、内禀增长率(r_m; F=34.67, P<0.01; F=18.73, P<0.01; F=12.99, P<0.01)均有显著影响; 温度、铜绿微囊藻浓度及二者交互作用对轮虫生活史参数净生殖率、内禀增长率也均有显著影响。无毒铜绿微藻在低浓度(1×104 cells/mL)下对轮虫种群有促进作用, 可作为轮虫食物来源, 但缺乏脂肪酸等营养物质, 食物质量比蛋白核小球藻低; 在高浓度(1×105和5×105 cells/mL)下轮虫摄食无毒铜绿微囊藻机率变大, 整体食物品质下降, 对轮虫有抑制作用。有毒铜绿微囊藻对轮虫种群的抑制作用更加明显, 微囊藻浓度升高, 净生殖率和内禀增长率显著下降。研究结果还表明30℃和35℃高温下轮虫生长繁殖变快, 世代时间缩短。在高温(30℃和35℃)环境下, 铜绿微囊藻浓度升高对轮虫抑制作用更加明显。     

Intracellular signaling by a mutant human insulin receptor lacking the carboxyl-terminal tyrosine autophosphorylation sites.

We have recently characterized a mutant insulin receptor (Y/F2) in which the two tyrosines in the carboxyl terminus (Tyr1316, Tyr1322) were mutated to phenylalanine. Compared with wild type receptors, the Y/F2 receptor exhibited markedly enhanced sensitivity to insulin-stimulated DNA synthesis with normal insulin-stimulated glucose uptake (Takata, Y., Webster, N. J. G., and Olefsky, J. M. (1991) J. Biol. Chem. 266, 9135-9139). In this paper, we present further evidence for the divergence of the metabolic and mitogenic signaling pathways utilized by the insulin receptor. The mutant receptor showed normal sensitivity and responsiveness for insulin-stimulated glucose incorporation into glycogen. The insulin sensitivity for phosphorylation of two substrates (pp180 and pp220) was the same in both Y/F2 cells and HIRc cells. Phosphotyrosine content, however, was greater in Y/F2 cells than in HIRc cells, especially in the basal state. Insulin stimulated S6 kinase activity 2-6-fold, with an ED50 of -10 nM in Rat 1 cells and 0.5 nM in HIRc cells. The sensitivity to insulin was enhanced in Y/F2 cells with an ED50 of 0.1 nM. These effects were insulin-specific, since insulin-like growth factor (IGF)-I-stimulated mitogenesis was normal. In summary: 1) Y/F2 receptors exhibit normal metabolic and enhanced mitogenic signaling; 2) the enhanced mitogenic signaling is specific for the insulin receptor in the Y/F2 cells, since IGF-I-stimulated mitogenesis is normal; 3) Y/F2 cells display increased endogenous substrate phosphorylation and augmented insulin-stimulated S6 kinase activity placing these responses among insulin's mitogenic effects; and 4) these results are consistent with the concept that the COOH-terminal tyrosine residues of the insulin receptor are normally inhibitory to mitogenic signaling.     

Macrophage activation and histopathological findings in Calomys callosus and Swiss mice infected with several strains of Trypanosoma cruzi.

Peritoneal macrophage activation as measured by H2O2 release and histopathology was compared between Swiss mice and Calomys callosus, a wild rodent, reservoir of Trypanosoma cruzi, during the course of infection with four strains of this parasite. In mice F and Y strain infections result in high parasitemia and mortality while with silvatic strains Costalimai and M226 parasitemia is sub-patent, with very low mortality. H2O2 release peaked at 33.6 and 59 nM/2 x 10(6) cells for strains Y and F, respectively, 48 and 50 nM/2 x 10(6) for strains Costalimai and M226, at different days after infection. Histopathological findings of myositis, myocarditis, necrotizing arteritis and absence of macrophage parasitism were found for strains F and Costalimai. Y strain infection presented moderate myocarditis and myositis, with parasites multiplying within macrophages. In C. callosus all four strains resulted in patent parasitemia which was eventually overcome, with scarce mortality. H2O2 release for strains Y and F was comparable to that of mice-peaks of 27 and 53 nM/2 x 10(6) cells, with lower values for strains Costalimai and M226-16.5 and 4.6 nM/2 x 10(6) cells, respectively. Histopathological lesions with Y and F strain injected animals were comparable to those of mice at the onset of infections; they subsided completely at the later stages with Y strain and partially with F strain infected C. callosus. In Costalimai infected C. callosus practically no histopathological alterations were observed.     

Tyrosine phosphorylation sites at amino acids 239 and 240 of Shc are involved in epidermal growth factor-induced mitogenic signaling that is distinct from Ras/mitogen-activated protein kinase activation.

Epidermal growth factor (EGF) induces tyrosine phosphorylation of the Shc adapter protein, which plays an important role in EGF-stimulated mitogenesis. Shc stimulates Ras/mitogen-activated protein kinase (MAPK) through forming a complex with Grb2 at the phosphorylated tyrosine (Y) residue 317. In this study, we identified novel phosphorylation sites of Shc, at Y239 and Y240. To define the Shc pathway further, we used NIH 3T3 cells expressing the previously characterized mutant EGF receptor (EGF-R) which lacks all known autophosphorylation sites but retains EGF-stimulated mitogenesis with selective phosphorylation of Shc. We constructed wild-type (WT) or mutant Shc cDNAs in which Y317 or/and Y239 and Y240 are replaced with phenylalanine (F) and introduced them into NIH 3T3 cells expressing WT or mutant EGF-R. In the WT EGF-R-expressing cells, the Y239/240/317F Shc, but not Y317F or Y239/240F Shc, decreased EGF-stimulated cell growth. In the mutant EGF-R-expressing cells, Y317F Shc or Y239/240F Shc decreased EGF-stimulated cell growth significantly, though Y317F was a little more potent than Y239/240F. Although cells expressing the Y317F Shc hardly activated MAPK in response to EGF, cells expressing the Y239/240F Shc fully activated MAPK. In contrast, Y239/240F Shc, but not Y317F Shc, reduced the EGF-induced c-myc message. These results suggest that Shc activates two distinct signaling pathways, Y317 to Ras/MAPK and Y239 and Y240 to another pathway including Myc, and that both are involved in EGF-induced mitogenic signaling.     

Selective Cytotoxicity of 1,3,4-Thiadiazolium Mesoionic Derivatives on Hepatocarcinoma Cells (HepG2)

In this work, we evaluated the cytotoxicity of mesoionic 4-phenyl-5-(2-Y, 4-X or 4-X-cinnamoyl)-1,3,4-thiadiazolium-2-phenylamine chloride derivatives (MI-J: X=OH, Y=H; MI-D: X=NO₂, Y=H; MI-4F: X=F, Y=H; MI-2,4diF: X=Y=F) on human hepatocellular carcinoma (HepG2), and non-tumor cells (rat hepatocytes) for comparison. MI-J, M-4F and MI-2,4diF reduced HepG2 viability by ~ 50% at 25 μM after 24-h treatment, whereas MI-D required a 50 μM concentration, as shown by 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyltetrazolium bromide assays. The cytotoxicity was confirmed with lactate dehydrogenase assay, of which activity was increased by 55, 24 and 16% for MI-J, MI-4F and MI-2,4diF respectively (at 25 μM after 24 h). To identify the death pathway related to cytotoxicity, the HepG2 cells treated by mesoionic compounds were labeled with both annexin V and PI, and analyzed by flow cytometry. All compounds increased the number of doubly-stained cells at 25 μM after 24 h: by 76% for MI-J, 25% for MI-4F and MI-2,4diF, and 11% for MI-D. It was also verified that increased DNA fragmentation occurred upon MI-J, MI-4F and MI-2,4diF treatments (by 12%, 9% and 8%, respectively, at 25 μM after 24 h). These compounds were only weakly, or not at all, transported by the main multidrug transporters, P-glycoprotein, ABCG2 and MRP1, and were able to slightly inhibit their drug-transport activity. It may be concluded that 1,3,4-thiadiazolium compounds, especially the hydroxy derivative MI-J, constitute promising candidates for future investigations on in-vivo treatment of hepatocellular carcinoma.     

RACK1-mediated integration of adhesion and insulin-like growth factor I (IGF-I) signaling and cell migration are defective in cells expressing an IGF-I receptor mutated at tyrosines 1250 and 1251

The scaffolding protein receptor for activated C kinase (RACK1) has been proposed to mediate the integration of insulin-like growth factor I receptor (IGF-IR) and adhesion signaling. Here we investigated the mechanism of this integration of signaling, by using an IGF-IR mutant (Y1250F/Y1251F) that is deficient in anti-apoptotic and transforming function. RACK1 was found to associate with the IGF-IR only in adherent cells and did not associate with the IGF-IR in nonadherent cells, lymphocytic cells, or cells expressing the Y1250F/Y1251F mutant. In R- cells transiently expressing the Y1250F/Y1251F mutant RACK1 became constitutively associated with beta1 integrin and did not associate with Shc, Src, or Shp2. This was accompanied by the loss of formation of a complex containing the IGF-IR, RACK1, and beta1 integrin; loss of migratory capacity; enhanced Src and FAK activity; enhanced Akt phosphorylation; and decreased p38 mitogen-activated protein kinase activity. Shc was not phosphorylated in response to IGF-I in cells expressing the Y1250F/Y1251F mutant and remained associated with protein phosphatase 2A. Similar alterations in signaling were observed in cells that were stimulated with IGF-I in nonadherent cultures. Our data suggest that disruption of RACK1 scaffolding function in cells expressing the Y1250F/Y1251F mutant results in the loss of adhesion signals that are necessary to regulate Akt activity and to promote turnover of focal adhesions and cell migration.     

Expression of a Y559F Mutant CSF-1 Receptor in M1 Myeloid Cells: A Role for Src Kinases in CSF-1 Receptor-Mediated Differentiation

We have established two M1 myeloid cell lines, M1/WT cells overexpressing the wild-type CSF-1 receptor and M1/Y559F cells expressing a specific tyrosine mutant. M1/WT cells differentiated in response to CSF-1, with a reduction in their proliferative capacity. CSF-1-mediated differentiation was partially abrogated in the M1/Y559F cells, with a less marked reduction in proliferative capacity. The Src tyrosine kinases c-Src, c-Yes, c-Fyn, and c-Hck were tyrosine phosphorylated in the M1/WT cells in response to CSF-1 and bound to the WT CSF-1R through their SH2 domains. Binding of the Src kinases to the CSF-1 receptor was greatly reduced in the M1/Y559F cells. CSF-1-mediated activation of STAT3 was also abrogated in the M1/Y559F cell line. Treatment of M1/WT cells with the Src family inhibitor PP2 resulted in an inhibition of CSF-1-mediated differentiation, equivalent to that observed in the M1/Y559F cells. These data suggest that the reduced Src binding observed in the M1/Y559F cells may contribute to their reduced ability to differentiate.     

YEAST two-hybrid and itc studies of alpha and beta spectrin interaction at the tetramerization site

Yeast two-hybrid (Y2H) and isothermal titration calorimetry (ITC) methods were used to further study the mutational effect of non-erythroid alpha spectrin (αII) at position 22 in tetramer formation with beta spectrin (βII). Four mutants, αII-V22D, V22F, V22M and V22W, were studied. For the Y2H system, we used plasmids pGBKT7, consisting of the cDNA of the first 359 residues at the N-terminal region of αII, and pGADT7, consisting of the cDNA of residues 1697–2145 at the C-terminal region of βII. Strain AH109 yeast cells were used for colony growth assays and strain Y187 was used for β-galactosidase activity assays. Y2H results showed that the C-terminal region of βII interacts with the N-terminal region of αII, either the wild type, or those with V22F, V22M or V22W mutations. The V22D mutant did not interact with βII. For ITC studies, we used recombinant proteins of the αII N-terminal fragment and of the erythroid beta spectrin (βI) C-terminal fragment; results showed that the K_d values for V22F were similar to those for the wild-type (about 7 nM), whereas the K_d values were about 35 nM for V22M and about 90 nM for V22W. We were not able to detect any binding for V22D with ITC methods. This study clearly demonstrates that the single mutation at position 22 of αII, a region critical to the function of nonerythroid α spectrin, may lead to a reduced level of spectrin tetramers and abnormal spectrin-based membrane skeleton. These abnormalities could cause abnormal neural activities in cells.     

Harvesting in seasonal environments

Most harvest theory is based on an assumption of a constant or stochastic environment, yet most populations experience some form of environmental seasonality. Assuming that a population follows logistic growth we investigate harvesting in seasonal environments, focusing on maximum annual yield (M.A.Y.) and population persistence under five commonly used harvest strategies. We show that the optimal strategy depends dramatically on the intrinsic growth rate of population and the magnitude of seasonality. The ordered effectiveness of these alternative harvest strategies is given for different combinations of intrinsic growth rate and seasonality. Also, for piecewise continuous-time harvest strategies (i.e., open / closed harvest, and pulse harvest) harvest timing is of crucial importance to annual yield. Optimal timing for harvests coincides with maximal rate of decline in the seasonally fluctuating carrying capacity. For large intrinsic growth rate and small environmental variability several strategies (i.e., constant exploitation rate, linear exploitation rate, and time-dependent harvest) are so effective that M.A.Y. is very close to maximum sustainable yield (M.S.Y.). M.A.Y. of pulse harvest can be even larger than M.S.Y. because in seasonal environments population size varies substantially during the course of the year and how it varies relative to the carrying capacity is what determines the value relative to optimal harvest rate. However, for populations with small intrinsic growth rate but subject to large seasonality none of these strategies is particularly effective with M.A.Y. much lower than M.S.Y. Finding an optimal harvest strategy for this case and to explore harvesting in populations that follow other growth models (e.g., involving predation or age structure) will be an interesting but challenging problem.     

Complementation of growth factor receptor-dependent mitogenic signaling by a truncated type I phosphatidylinositol 4-phosphate 5-kinase.

Substitution of phenylalanine for tyrosine at codon 809 (Y809F) of the human colony-stimulating factor 1 (CSF-1) receptor (CSF-1R) impairs ligand-stimulated tyrosine kinase activity, prevents induction of c-MYC and cyclin D1 genes, and blocks CSF-1-dependent progression through the G1 phase of the cell cycle. We devised an unbiased genetic screen to isolate genes that restore the ability of CSF-1 to stimulate growth in cells that express mutant CSF-1R (Y809F). This screen led us to identify a truncated form of the murine type Ibeta phosphatidylinositol 4-phosphate 5-kinase (mPIP5K-Ibeta). This truncated protein lacks residues 1 to 238 of mPIP5K-Ibeta and is catalytically inactive. When we transfected cells expressing CSF-1R (Y809F) with mPIP5K-Ibeta (delta1-238), CSF-1-dependent induction of c-MYC and cyclin D1 was restored and ligand-dependent cell proliferation was sustained. CSF-1 normally triggers the rapid disappearance of CSF-1R (Y809F) from the cell surface; however, transfection of cells with mPIP5K-Ibeta (delta1-238) stabilized CSF-1R (Y809F) expression on the cell surface, resulting in elevated levels of ligand-activated CSF-1R (Y809F). These results suggest a role for PIP5K-Ibeta in receptor endocytosis and that the truncated enzyme compensated for a mitogenically defective CSF-1R by interfering with this process.     

Conserved tyrosine-369 in the active site of Escherichia coli copper amine oxidase is not essential.

Copper amine oxidases are homodimeric enzymes that catalyze two reactions: first, a self-processing reaction to generate the 2,4,5-trihydroxyphenylalanine (TPQ) cofactor from an active site tyrosine by a single turnover mechanism; second, the oxidative deamination of primary amine substrates with the production of aldehyde, hydrogen peroxide, and ammonia catalyzed by the mature enzyme. The importance of active site residues in both of these processes has been investigated by structural studies and site-directed mutagenesis in enzymes from various organisms. One conserved residue is a tyrosine, Tyr369 in the Escherichia coli enzyme, whose hydroxyl is hydrogen bonded to the O4 of TPQ. To explore the importance of this site, we have studied a mutant enzyme in which Tyr369 has been mutated to a phenylalanine. We have determined the X-ray crystal structure of this variant enzyme to 2.1 A resolution, which reveals that TPQ adopts a predominant nonproductive conformation in the resting enzyme. Reaction of the enzyme with the irreversible inhibitor 2-hydrazinopyridine (2-HP) reveals differences in the reactivity of Y369F compared with wild type with more efficient formation of an adduct (lambda(max) = 525 nm) perhaps reflecting increased mobility of the TPQ adduct within the active site of Y369F. Titration with 2-HP also reveals that both wild type and Y369F contain one TPQ per monomer, indicating that Tyr369 is not essential for TPQ formation, although we have not measured the rate of TPQ biogenesis. The UV-vis spectrum of the Y369F protein shows a broader peak and red-shifted lambda(max) at 496 nm compared with wild type (480 nm), consistent with an altered electronic structure of TPQ. Steady-state kinetic measurements reveal that Y369F has decreased catalytic activity particularly below pH 6.5 while the K(M) for substrate beta-phenethylamine increases significantly, apparently due to an elevated pK(a) (5.75-6.5) for the catalytic base, Asp383, that should be deprotonated for efficient binding of protonated substrate. At pH 7.0, the K(M) for wild type and Y369F are similar at 1.2 and 1.5 microM, respectively, while k(cat) is decreased from 15 s(-1) in wild type to 0.38 s(-1), resulting in a 50-fold decrease in k(cat)/K(M) for Y369F. Transient kinetics experiments indicate that while the initial stages of enzyme reduction are slower in the variant, these do not represent the rate-limiting step. Previous structural and solution studies have implicated Tyr369 as a component of a proton shuttle from TPQ to dioxygen. The moderate changes in kinetic parameters observed for the Y369F variant indicate that if this is the case, then the absence of the Tyr369 hydroxyl can be compensated for efficiently within the active site.     

Multiple autophosphorylation site mutations of the epidermal growth factor receptor. Analysis of kinase activity and endocytosis

We have utilized site-directed mutants to study the role of autophosphorylation of the epidermal growth factor (EGF) receptor in the regulation of receptor kinase activity and ligand-induced endocytosis. A single mutation of the major autophosphorylation site, Y1173, and a double mutation of two autophosphorylation sites, Y1173 and Y1148, did not inhibit kinase activity in vivo, using PLC gamma 1 as a specific substrate for the EGF receptor kinase. The simultaneous mutation of three major autophosphorylation sites (Y1173, Y1148, Y1068), however, caused more than a 50% decrease in EGF-induced tyrosine phosphorylation of PLC gamma 1. The triple mutation also resulted in a substantial inhibition of the EGF-receptor endocytic system. We have used three types of experiments to analyze internalization, recycling, and degradation of EGF in cells with these mutants or the wild-type receptor. Using a simple mathematical model we have shown that the internalization rate constant is 2-fold lower in cells expressing the triple mutation receptor (F3 cells) than in cells expressing wild-type EGF receptor (wild-type cells). However, the rate constant for recycling was similar in both cell types. The EGF degradation rate constant was also lower in F3 cells. EGF-induced EGF receptor degradation was slower in F3 cells (t1/2 = 4 h) than in wild-type cells (t1/2 = 1 h). Therefore, our results suggest that multiple autophosphorylations of the carboxyl terminus of the EGF receptor are required for EGF receptor kinase activation, and for the internalization and intracellular processing of the EGF.receptor complex.     

Immunochemical characterization of Fraction I of Yersinia pestis strains by CAF1M gene

The role of caf1M gene in biogenesis of Yersinia pestis capsule was studied in natural strains of the agent with Fra+/- phenotypes and recombinant variants with ycaA (caf1+;caf1M;caf1A+;caf1R+) locus defect. These bacteria did not form a clearly discernible capsule stained by classical methods but synthesized Cafl, whose content in the cells was many times higher than in lysates, in external cell wall, and in the medium with reference Y. pestis EV NIIEG culture (caf1+;caf1M;caf1A+;caf1R+). However Caf1 was not detected on the surface or culture fluid of natural and mutant Y. pestis cells. Exclusive role of Caf1M in Caf1 delivery to Y. pestis cell surface, but not in F1 monomer folding, was proven. Retention of lipopolysaccharide (LPS), a typical SR-LPS configuration and epitope specificity of its components was demonstrated, ensuring similar reactivity in solid-phase enzyme immunoassay with a panel of monoclonal antibodies to Y. pestis LPS. Study of immunochemical properties of antigenic substances derived from caf1M-defective Y. pestis cells by isolation of F1 showed that these substances represent an envelope protein involved in the caf1+ strains (together with Caf1) in assembly of "mature" F1 molecule as a result of posttranslation modification of various genes products. Variants of identification of Y. pestis with Fra+ phenotype by means of monoclonal antibodies to F1, fibrinolysis/coagulase, or LPS in solid-phase enzyme immunoassay are discussed.     

Tyrosyl phosphorylation of Shp2 is required for normal ERK activation in response to some,but not all,growth factors

The protein-tyrosine phosphatase Shp2 is required for normal activation of the ERK mitogen-activated protein kinase in multiple receptor tyrosine kinase signaling pathways. In fibroblasts, Shp2 undergoes phosphorylation at two C-terminal tyrosyl residues in response to some (fibroblast growth factor and platelet-derived growth factor (PDGF)) but not all (epidermal growth factor and insulin-like growth factor) growth factors. Whereas the catalytic activity of Shp2 is required for all Shp2 actions, the effect of tyrosyl phosphorylation on Shp2 function has been controversial. To clarify the role of Shp2 tyrosyl phosphorylation, we infected Shp2-mutant fibroblasts with retroviruses expressing wild type Shp2 or mutants of either (Y542F or Y580F) or both (Y542F,Y580F) C-terminal tyrosines. Compared with wild type cells, ERK activation was decreased in Y542F- or Y580F-infected cells in response to fibroblast growth factor and PDGF but not the epidermal growth factor. Mutation of both phosphorylation sites resulted in a further decrease in growth factor-evoked ERK activation, although not to the level of the vector control. Immunoblot analyses confirm that Tyr-542 and Tyr-580 are the major sites of Shp2 tyrosyl phosphorylation and that Tyr-542 is the major Grb2 binding site. However, studies with antibodies specific for individual Shp2 phosphorylation sites reveal unexpected complexity in the mechanism of Shp2 tyrosyl phosphorylation by different receptor tyrosine kinases. Moreover, because Y580F mutants retain nearly wild type Grb2-binding ability, yet exhibit defective PDGF-evoked ERK activation, our results show that the association of Grb2 with Shp2 is not sufficient for promoting full ERK activation in response to these growth factors, thereby arguing strongly against the "Grb2-adapter" model of Shp2 action.     

Tyrosine phosphorylation of lactate dehydrogenase A is important for NADH/NAD(+) redox homeostasis in cancer cells

The Warburg effect describes an increase in aerobic glycolysis and enhanced lactate production in cancer cells. Lactate dehydrogenase A (LDH-A) regulates the last step of glycolysis that generates lactate and permits the regeneration of NAD(+). LDH-A gene expression is believed to be upregulated by both HIF and Myc in cancer cells to achieve increased lactate production. However, how oncogenic signals activate LDH-A to regulate cancer cell metabolism remains unclear. We found that the oncogenic receptor tyrosine kinase FGFR1 directly phosphorylates LDH-A. Phosphorylation at Y10 and Y83 enhances LDH-A activity by enhancing the formation of active, tetrameric LDH-A and the binding of LDH-A substrate NADH, respectively. Moreover, Y10 phosphorylation of LDH-A is common in diverse human cancer cells, which correlates with activation of multiple oncogenic tyrosine kinases. Interestingly, cancer cells with stable knockdown of endogenous LDH-A and rescue expression of a catalytic hypomorph LDH-A mutant, Y10F, demonstrate increased respiration through mitochondrial complex I to sustain glycolysis by providing NAD(+). However, such a compensatory increase in mitochondrial respiration in Y10F cells is insufficient to fully sustain glycolysis. Y10 rescue cells show decreased cell proliferation and ATP levels under hypoxia and reduced tumor growth in xenograft nude mice. Our findings suggest that tyrosine phosphorylation enhances LDH-A enzyme activity to promote the Warburg effect and tumor growth by regulating the NADH/NAD(+) redox homeostasis, representing an acute molecular mechanism underlying the enhanced lactate production in cancer cells.     

Functions of the major tyrosine phosphorylation site of the PDGF receptor beta subunit.

Two tyrosine phosphorylation sites in the human platelet-derived growth factor receptor (PDGFR) beta subunit have been mapped previously to tyrosine (Y)751, in the kinase insert, and Y857, in the kinase domain. Y857 is the major site of tyrosine phosphorylation in PDGF-stimulated cells. To evaluate the importance of these phosphorylations, we have characterized the wild-type (WT) and mutant human PDGF receptor beta subunits in dog kidney epithelial cells. Replacement of either Y751 or Y857 with phenylalanine (F) reduced PDGF-stimulated DNA synthesis to approximately 50% of the WT level. A mutant receptor with both tyrosines mutated was unable to initiate DNA synthesis, as was a kinase-inactive mutant receptor. Transmodulation of the epidermal growth factor receptor required Y857 but not Y751. We also tested the effects of phosphorylation site mutations on PDGF-stimulated receptor kinase activity. PDGF-induced tyrosine phosphorylation of two cellular proteins, phospholipase C gamma 1 (PLC gamma 1) and the GTPase activating protein of Ras (GAP), was assayed in epithelial cells expressing each of the mutant receptors. Tyrosine phosphorylation of GAP and PLC gamma 1 was reduced markedly by the F857 mutation but not significantly by the F751 mutation. Reduced kinase activity of F857 receptors was also evident in vitro. Immunoprecipitated WT receptors showed a two- to fourfold increase in specific kinase activity if immunoprecipitated from PDGF-stimulated cells. The F751 receptors showed a similar increase in activity, but F857 receptors did not. Our data suggest that phosphorylation of Y857 may be important for stimulation of kinase activity of the receptors and for downstream actions such as epidermal growth factor receptor transmodulation and mitogenesis.     

Suppression of urokinase expression and invasion by a soybean Kunitz trypsin inhibitor are mediated through inhibition of Src-dependent signaling pathways

A soybean Kunitz trypsin inhibitor (KTI) interacts with cells as a negative modulator of the invasive cells. Using complementary pharmacological and genetic approaches, we provide novel findings regarding mechanisms by which KTI inhibits signaling pathways in ovarian cancer cells leading to invasion. Transforming growth factor-beta1 (TGF-beta1) directly activates Src kinase, which in turn activates ERK-phosphatidylinositol 3-kinase/Akt, the downstream targets of Src, for urokinase-type plasminogen activator (uPA) up-regulation in human ovarian cancer HRA cells. Preincubation of the HRA cells with KTI reduced the ability of TGF-beta1 to trigger the uPA expression at the gene level and at the protein level. To further elucidate the mechanism of the KTI-dependent suppressive effect of TGF-beta1-induced uPA expression and invasion, we investigated which signaling pathway transduced by KTI is responsible for this inhibitory effect. Here, we show that 1) KTI suppressed TGF-beta1-induced phosphorylation of Src, ERK1/2, and Akt by 40-60%; 2) KTI was insensitive to suppress the phosphorylation of ERK1/2 and Akt in the constitutively active (CA)-c-Src (Y529F) cells; 3) uPA expression was up-regulated in TGF-beta1-stimulated HRA cells and in unstimulated Y529F cells; 4) the addition of KTI reduced the TGF-beta1-induced increase of uPA gene and protein expression in the wild-type c-Src-transfected cells (in contrast, KTI could not inhibit uPA expression in the Y529F cells); and 5) CA-c-Src transfection resulted in a 2-fold increase in invasiveness, whereas KTI did not reduce invasion of the Y529F cells. Using additional complementary genetic approaches (CA-MEK1, CA-Akt, or kinase-dead-Akt), we conclude that KTI may suppress uPA expression and promotion of invasion possibly through one or more upstream targets of Src.