Expression and characterization of LTx2, a neurotoxin from Lasiodora sp effecting on calcium channels

Here, we described the expression and characterization of the recombinant toxin LTx2, which was previously isolated from the venomous cDNA library of a Brazilian spider, Lasiodora sp. (Mygalomorphae, Theraphosidae). The recombinant toxin found in the soluble and insoluble fractions was purified by reverse phase high-performance liquid chromatography (HPLC). Ca2+ imaging analysis revealed that the recombinant LTx2 acts on calcium channels of BC3H1 cells, blocking L-type calcium channels. (C) 2008 Elsevier Inc. All rights reserved.

Triiodothyronine Increases mRNA and Protein Leptin Levels in Short Time in 3T3-L1 Adipocytes by PI3K Pathway Activation

The present study aimed to examine the effects of thyroid hormone (TH), more precisely triiodothyronine (T3), on the modulation of leptin mRNA expression and the involvement of the phosphatidyl inositol 3 kinase (PI3K) signaling pathway in adipocytes, 3T3-L1, cell culture. We examined the involvement of this pathway in mediating TH effects by treating 3T3-L1 adipocytes with physiological (P=10nM) or supraphysiological (SI=100 nM) T3 dose during one hour (short time), in the absence or the presence of PI3K inhibitor (LY294002). The absence of any treatment was considered the control group (C). RT-qPCR was used for mRNA expression analyzes. For data analyzes ANOVA complemented with Tukey's test was used at 5% significance. T3 increased leptin mRNA expression in P (2.26 ± 0.36, p< 0.001), SI (1.99 ±0.22, p< 0.01) compared to C group (1± 0.18). This increase was completely abrogated by LY294002 in P (1.31±0.05, p< 0.001) and SI (1.33±0.31, p< 0.05). Western blotting confirmed these results at protein level, indicating the PI3K pathway dependency. To examine whether leptin is directly induced by T3, we used the translation inhibitor cycloheximide (CHX). In P, the presence of CHX maintained the levels mRNA leptin, but was completely abrogated in SI (1.14±0.09, p> 0.001). These results demonstrate that the activation of the PI3K signaling pathway has a role in TH-mediated direct and indirect leptin gene expression in 3T3-L1 adipocytes. © 2013 Oliveira et al.

Ação de diferentes doses de triiodotironina (T3) sobre a expressão gênica dos receptores de hormônios tireoidianos e leptina em cultura celular de adipócitos, 3T3-L1

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Influência do bloqueio de receptores do tipo 1 (AT1) sobre o perfil metabólico, endócrino e cardiovascular de ratos obesos

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Short-term effects of triiodothyronine on thyroid hormone receptor alpha by PI3K pathway in adipocytes, 3T3-L1

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Mechanotransduction pathways in skeletal muscle hypertrophy

In the last decade, molecular biology has contributed to define some of the cellular events that trigger skeletal muscle hypertrophy. Recent evidence shows that insulin like growth factor 1/phosphatidyl inositol 3-kinase/protein kinase B (IGF-1/PI3K/Akt) signaling is not the main pathway towards load-induced skeletal muscle hypertrophy. During load-induced skeletal muscle hypertrophy process, activation of mTORC1 does not require classical growth factor signaling. One potential mechanism that would activate mTORC1 is increased synthesis of phosphatidic acid (PA). Despite the huge progress in this field, it is still early to affirm which molecular event induces hypertrophy in response to mechanical overload. Until now, it seems that mTORC1 is the key regulator of load-induced skeletal muscle hypertrophy. On the other hand, how mTORC1 is activated by PA is unclear, and therefore these mechanisms have to be determined in the following years. The understanding of these molecular events may result in promising therapies for the treatment of muscle-wasting diseases. For now, the best approach is a good regime of resistance exercise training. The objective of this point-of-view paper is to highlight mechanotransduction events, with focus on the mechanisms of mTORC1 and PA activation, and the role of IGF-1 on hypertrophy process.

Circumvention of cellular defense responses to DNA -directed nucleoside analogues by the protein kinase inhibitor, UCN-01

DNA-directed nucleoside analogues, such as ara-C, fludarabine, and gemcitabine, are antimetabolites effective in the treatment of a variety of cancers. However, resistance to nucleoside analogue-based chemotherapy in treatments is still a major problem in therapy. Therefore, it is essential to develop rationales for optimizing the use of nucleoside analogues in combination with other anticancer drugs or modalities such as radiation. The present study focuses on establishing mechanism-based combination strategy to overcome resistance to nucleoside analogues. ^ I hypothesized that the cytostatic concentrations of nucleoside analogues may cause S-phase arrest by activating an S-phase checkpoint that consists of a series of kinases. This may allow cells to repair damaged DNA over time and spare cytotoxicity. Thus, the ability of cells to enact an S-phase arrest in response to incorporation of potentially lethal amounts of nucleoside analogue may serve as a mechanism of resistance to S-phase-specific agents. As a corollary, the addition of a kinase inhibitor, such as UCN-01, may dysregulate the checkpoint response and abrogate the survival of S-phase-arrested cells by suppression of the survival signaling pathways. Using gemcitabine as a model of S-phase-specific nucleoside analogues in human acute myelogenous leukemia ML-1 cells, I demonstrated that cells arrested in S-phase in response to cytostatic conditions. Proliferation continued after washing the cells into drug-free medium, suggesting S-phase arrest served as a resistance mechanism of cancer cells to spare cytotoxicity of nucleoside analogues. However, nontoxic concentrations of UCN-01 rapidly killed S-phase-arrested cells by apoptosis. Furthermore, the molecular mechanism for UCN-01-induced apoptosis in S-phase-arrested cells was through inhibition of survival pathways associated with these cells. In this regard, suppression of the PI 3-kinase-Akt-Bad survival pathway as well as the NF-κB signaling pathway were associated with induction of apoptosis in S-phase-arrested cells by UCN-01, whereas the Ras-Raf-MEK-ERK pathway appeared not involved. This study has provided the rationales and strategies for optimizing the design of effective combination therapies to overcome resistance to nucleoside analogues. In fact, a clinical trial of the combination of ara-C with UCN-01 to treat relapsed or refractory AML patients has been initiated at U.T.M.D. Anderson Cancer Center. ^

The Akt kinase: Molecular determinants of oncogenicity

The serine-threonine kinase Akt is a downstream target of phosphoinositide 3-kinase (PI 3-kinase); it is activated by the phosphoinositide 3-phosphate-dependent kinases PDK1 and PDK2. Certain mutated forms of Akt induce oncogenic transformation in chicken embryo fibroblast cultures and hemangiosarcomas in young chickens. This ability to transform cells depends on localization of Akt at the plasma membrane and on the kinase activity of Akt. A transdominant negative form of Akt interferes with oncogenic transformation induced by the p3k oncogene, which codes for an activated form of PI 3-kinase. Akt is therefore an essential mediator of p3k-induced oncogenicity.

The lipid phosphatase activity of PTEN is critical for its tumor supressor function

Since their discovery, protein tyrosine phosphatases have been speculated to play a role in tumor suppression because of their ability to antagonize the growth-promoting protein tyrosine kinases. Recently, a tumor suppressor from human chromosome 10q23, called PTEN or MMAC1, has been identified that shares homology with the protein tyrosine phosphatase family. Germ-line mutations in PTEN give rise to several related neoplastic disorders, including Cowden disease. A key step in understanding the function of PTEN as a tumor suppressor is to identify its physiological substrates. Here we report that a missense mutation in PTEN, PTEN-G129E, which is observed in two Cowden disease kindreds, specifically ablates the ability of PTEN to recognize inositol phospholipids as a substrate, suggesting that loss of the lipid phosphatase activity is responsible for the etiology of the disease. Furthermore, expression of wild-type or substrate-trapping forms of PTEN in HEK293 cells altered the levels of the phospholipid products of phosphatidylinositol 3-kinase and ectopic expression of the phosphatase in PTEN-deficient tumor cell lines resulted in the inhibition of protein kinase (PK) B/Akt and regulation of cell survival.

Epigallocathechin-3 Gallate Selectively Inhibits the PDGF-BB–induced Intracellular Signaling Transduction Pathway in Vascular Smooth Muscle Cells and Inhibits Transformation of sis-transfected NIH 3T3 Fibroblasts and Human Glioblastoma Cells (A172)

Enhanced activity of receptor tyrosine kinases such as the PDGF β-receptor and EGF receptor has been implicated as a contributing factor in the development of malignant and nonmalignant proliferative diseases such as cancer and atherosclerosis. Several epidemiological studies suggest that green tea may prevent the development of cancer and atherosclerosis. One of the major constituents of green tea is the polyphenol epigallocathechin-3 gallate (EGCG). In an attempt to offer a possible explanation for the anti-cancer and anti-atherosclerotic activity of EGCG, we examined the effect of EGCG on the PDGF-BB–, EGF-, angiotensin II-, and FCS-induced activation of the 44 kDa and 42 kDa mitogen-activated protein (MAP) kinase isoforms (p44mapk/p42mapk) in cultured vascular smooth muscle cells (VSMCs) from rat aorta. VSMCs were treated with EGCG (1–100 μM) for 24 h and stimulated with the above mentioned agonists for different time periods. Stimulation of the p44mapk/p42mapk was detected by the enhanced Western blotting method using phospho-specific MAP kinase antibodies that recognized the Tyr204-phosphorylated (active) isoforms. Treatment of VSMCs with 10 and 50 μM EGCG resulted in an 80% and a complete inhibition of the PDGF-BB–induced activation of MAP kinase isoforms, respectively. In striking contrast, EGCG (1–100 μM) did not influence MAP kinase activation by EGF, angiotensin II, and FCS. Similarly, the maximal effect of PDGF-BB on the c-fos and egr-1 mRNA expression as well as on intracellular free Ca2+ concentration was completely inhibited in EGCG-treated VSMCs, whereas the effect of EGF was not affected. Quantification of the immunoprecipitated tyrosine-phosphorylated PDGF-Rβ, phosphatidylinositol 3′-kinase, and phospholipase C-γ1 by the enhanced Western blotting method revealed that EGCG treatment effectively inhibits tyrosine phosphorylation of these kinases in VSMCs. Furthermore, we show that spheroid formation of human glioblastoma cells (A172) and colony formation of sis-transfected NIH 3T3 cells in semisolid agar are completely inhibited by 20–50 μM EGCG. Our findings demonstrate that EGCG is a selective inhibitor of the tyrosine phosphorylation of PDGF-Rβ and its downstream signaling pathway. The present findings may partly explain the anti-cancer and anti-atherosclerotic activity of green tea.

The Phosphatidylinositol 3-Phosphate Binding Protein Vac1p Interacts with a Rab GTPase and a Sec1p Homologue to Facilitate Vesicle-mediated Vacuolar Protein Sorting

Activated GTP-bound Rab proteins are thought to interact with effectors to elicit vesicle targeting and fusion events. Vesicle-associated v-SNARE and target membrane t-SNARE proteins are also involved in vesicular transport. Little is known about the functional relationship between Rabs and SNARE protein complexes. We have constructed an activated allele of VPS21, a yeast Rab protein involved in vacuolar protein sorting, and demonstrated an allele-specific interaction between Vps21p and Vac1p. Vac1p was found to bind the Sec1p homologue Vps45p. Although no association between Vps21p and Vps45p was seen, a genetic interaction between VPS21 and VPS45 was observed. Vac1p contains a zinc-binding FYVE finger that may bind phosphatidylinositol 3-phosphate [PtdIns(3)P]. In other FYVE domain proteins, this motif and PtdIns(3)P are necessary for membrane association. Vac1 proteins with mutant FYVE fingers still associated with membranes but showed vacuolar protein sorting defects and reduced interactions with Vps45p and activated Vps21p. Vac1p membrane association was not dependent on PtdIns(3)P, Pep12p, Vps21p, Vps45p, or the PtdIns 3-kinase, Vps34p. Vac1p FYVE finger mutant missorting phenotypes were suppressed by a defective allele of VPS34. These data indicate that PtdIns(3)P may perform a regulatory role, possibly involved in mediating Vac1p protein–protein interactions. We propose that activated-Vps21p interacts with its effector, Vac1p, which interacts with Vps45p to regulate the Golgi to endosome SNARE complex.

Requirement of Sequences outside the Conserved Kinase Domain of Fission Yeast Rad3p for Checkpoint Control

The fission yeast Rad3p checkpoint protein is a member of the phosphatidylinositol 3-kinase-related family of protein kinases, which includes human ATMp. Mutation of the ATM gene is responsible for the disease ataxia-telangiectasia. The kinase domain of Rad3p has previously been shown to be essential for function. Here, we show that although this domain is necessary, it is not sufficient, because the isolated kinase domain does not have kinase activity in vitro and cannot complement a rad3 deletion strain. Using dominant negative alleles of rad3, we have identified two sites N-terminal to the conserved kinase domain that are essential for Rad3p function. One of these sites is the putative leucine zipper, which is conserved in other phosphatidylinositol 3-kinase-related family members. The other is a novel motif, which may also mediate Rad3p protein–protein interactions.

Role of Akt and c-Jun N-terminal Kinase 2 in Apoptosis Induced by Interleukin-4 Deprivation

We have shown previously that interleukin-4 (IL-4) protects TS1αβ cells from apoptosis, but very little is known about the mechanism by which IL-4 exerts this effect. We found that Akt activity, which is dependent on phosphatidylinositol 3 kinase, is reduced in IL-4-deprived TS1αβ cells. Overexpression of wild-type Akt or a constitutively active Akt mutant protects cells from IL-4 deprivation-induced apoptosis. Readdition of IL-4 before the commitment point is able to restore Akt activity. We also show expression and c-Jun N-terminal kinase 2 activation after IL-4 deprivation. Overexpression of the constitutively activated Akt mutant in IL-4-deprived cells correlates with inhibition of c-Jun N-terminal kinase 2 activity. Finally, TS1αβ survival is independent of Bcl-2, Bcl-x, or Bax.

Phosphorylation of protein kinase N by phosphoinositide-dependent protein kinase-1 mediates insulin signals to the actin cytoskeleton

Growth factors such as insulin regulate phosphatidylinositol 3-kinase-dependent actin cytoskeleton rearrangement in many types of cells. However, the mechanism by which the insulin signal is transmitted to the actin cytoskeleton remains largely unknown. Yeast two-hybrid screening revealed that the phosphatidylinositol 3-kinase downstream effector phosphoinositide-dependent protein kinase-1 (PDK1) interacted with protein kinase N (PKN), a Rho-binding Ser/Thr protein kinase potentially implicated in a variety of cellular events, including phosphorylation of cytoskeletal components. PDK1 and PKN interacted in vitro and in intact cells, and this interaction was mediated by the kinase domain of PDK1 and the carboxyl terminus of PKN. In addition to a direct interaction, PDK1 also phosphorylated Thr774 in the activation loop and activated PKN. Insulin treatment or ectopic expression of the wild-type PDK1 or PKN, but not protein kinase Cζ, induced actin cytoskeleton reorganization and membrane ruffling in 3T3-L1 fibroblasts and Rat1 cells that stably express the insulin receptor (Rat1-IR). However, the insulin-stimulated actin cytoskeleton reorganization in Rat1-IR cells was prevented by expression of kinase-defective PDK1 or PDK1-phosphorylation site-mutated PKN. Thus, phosphorylation by PDK1 appears to be necessary for PKN to transduce signals from the insulin receptor to the actin cytoskeleton.

The TEL/platelet-derived growth factor β receptor (PDGFβR) fusion in chronic myelomonocytic leukemia is a transforming protein that self-associates and activates PDGFβR kinase-dependent signaling pathways

The TEL/PDGFβR fusion protein is the product of the t(5;12) translocation in patients with chronic myelomonocytic leukemia. The TEL/PDGFβR is an unusual fusion of a putative transcription factor, TEL, to a receptor tyrosine kinase. The translocation fuses the amino terminus of TEL, containing the helix-loop-helix (HLH) domain, to the transmembrane and cytoplasmic domain of the PDGFβR. We hypothesized that TEL/PDGFβR self-association, mediated by the HLH domain of TEL, would lead to constitutive activation of the PDGFβR tyrosine kinase domain and cellular transformation. Analysis of in vitro-translated TEL/PDGFβR confirmed that the protein self-associated and that self-association was abrogated by deletion of 51 aa within the TEL HLH domain. In vivo, TEL/PDGFβR was detected as a 100-kDa protein that was constitutively phosphorylated on tyrosine and transformed the murine hematopoietic cell line Ba/F3 to interleukin 3 growth factor independence. Transformation of Ba/F3 cells required the HLH domain of TEL and the kinase activity of the PDGFβR portion of the fusion protein. Immunoblotting demonstrated that TEL/PDGFβR associated with multiple signaling molecules known to associate with the activated PDGFβR, including phospholipase C γ1, SHP2, and phosphoinositol-3-kinase. TEL/PDGFβR is a novel transforming protein that self-associates and activates PDGFβR-dependent signaling pathways. Oligomerization of TEL/PDGFβR that is dependent on the TEL HLH domain provides further evidence that the HLH domain, highly conserved among ETS family members, is a self-association motif.