Phosphoinositide-dependent protein kinase-1 (PDK1)-independent activation of the protein kinase C substrate, protein kinase D

Phosphoinoisitide dependent kinase l (PDK1) is proposed to phosphorylate a key threonine residue within the catalytic domain of the protein kinase C (PKC) superfamily that controls the stability and catalytic competence of these kinases. Hence, in PDK1-null embryonic stem cells intracellular levels of PKCalpha, PKCbeta1, PKCgamma, and PKCepsilon are strikingly reduced. Although PDK1-null cells have reduced endogenous PKC levels they are not completely devoid of PKCs and the integrity of downstream PKC effector pathways in the absence of PDK1 has not been determined. In the present report, the PDK1 requirement for controlling the phosphorylation and activity of a well characterised substrate for PKCs, the serine kinase protein kinase D, has been examined. The data show that in embryonic stem cells and thymocytes loss of PDK1 does not prevent PKC-mediated phosphorylation and activation of protein kinase D. These results reveal that loss of PDK1 does not functionally inactivate all PKC-mediated signal transduction.     

Depletion of IK causes mitotic arrest through aberrant regulation of mitotic kinases and phosphatases

IK is known to inhibit the expression of major histocompatibility complex (MHC) class II antigen, but other cellular functions of IK remain to be uncovered. In this study, IK depletion caused misalignment of chromosomes through an increase in Aurora A and PLK1 phosphorylation, which was mediated by a decrease in PP1 and PP2A activities. On the other hand, the treatment of a dual inhibitor against CDK and Aurora kinases overrode IK depletion-induced mitotic arrest through the activation of phosphatase activity. These findings imply that IK is an essential protein for achieving correct mitotic progress through the regulation of mitotic kinases and phosphatases.     

TPCK inhibits AGC kinases by direct activation loop adduction at phenylalanine-directed cysteine residues

N-alpha-tosyl-l-phenylalanyl chloromethyl ketone (TPCK) has anti-tumorigenic properties, but its direct cellular targets are unknown. Previously, we showed TPCK inhibited the PDKl-dependent AGC kinases RSK, Akt and S6K1 without inhibiting PKA, ERK1/2, PI3K, and PDK1 itself. Here we show TPCK-inhibition of the RSK-related kinases MSK1 and 2, which can be activated independently of PDK1. Mass spectrometry analysis of RSK1, Aktl, S6K1 and MSK1 immunopurified from TPCK-treated cells identified TPCK adducts on cysteines located in conserved activation loop Phenylalanine-Cysteine (Phe-Cys) motifs. Mutational analysis of the Phe-Cys residues conferred partial TPCK resistance. These studies elucidate a primary mechanism by which TPCK inhibits several AGC kinases, inviting consideration of TPCK-like compounds in chemotherapy given their potential for broad control of cellular growth, proliferation and survival.     

Identification of S6K2 as a centrosome-located kinase

Ribosomal S6 kinase 2 (S6K2) acts downstream of the mammalian target of rapamycin (mTOR). Here, we show that some S6K2 localize at the centrosome throughout the cell cycle. S6K2 is found in the pericentriolar area of the centrosome. S6K2 centrosomal localization is unaffected by serum withdrawal or treatment with rapamycin, wortmannin, U0126, or phorbol-12-myristate-13-acetate (PMA). Unlike S6K2, S6 kinase 1 (S6K1) does not localize at the centrosome, suggesting the two kinases may also have nonoverlapping functions. Our data suggest that centrosomal S6K2 may have a role in the phosphoinositide-3-kinase (PI3K)/Akt/mTOR signaling pathway that has also been detected in the centrosome.     

Isoforms of protein kinase C involved in phorbol ester-induced sphingosine 1-phosphate receptor 1 phosphorylation and desensitization

The role of protein kinase C (PKC) isozymes in phorbol myristate acetate (PMA)-induced sphingosine 1-phosphate (S1P) receptor 1 (S1P₁) phosphorylation was studied. Activation of S1P₁ receptors induced an immediate increase in intracellular calcium, which was blocked by preincubation with PMA. Both S1P and PMA were able to increase S1P₁ phosphorylation in a concentration- and time-dependent fashion. Down-regulation of PKC (overnight incubation with PMA) blocked the subsequent effect of the phorbol ester on S1P₁ phosphorylation, without decreasing that of the natural agonist. Pharmacological inhibition of PKC α prevented the effects of PMA on S1P-triggered intracellular calcium increase and on S1P₁ phosphorylation; no such effect was observed on the effects of the sphingolipid agonist. The presence of PKC α and β isoforms in S1P₁ immunoprecipitates was evidenced by Western blotting. Additionally, expression of dominant-negative mutants of PKC α or β and knockdown of these isozymes using short hairpin RNA, markedly attenuated PMA-induced S1P₁ phosphorylation. Our results indicate that the classical isoforms, mainly PKC α, mediate PMA-induced phosphorylation and desensitization of S1P₁.     

PTEN, a negative regulator of PI3 kinase signalling, alters tau phosphorylation in cells by mechanisms independent of GSK-3

Deregulation of PTEN/Akt signalling has been recently implicated in the pathogenesis of Alzheimer's disease (AD), but the effects on the molecular processes underlying AD pathology have not yet been fully described. Here we report that overexpression of PTEN reduces tau phosphorylation in CHO cells. This effect was abrogated by mutant PTEN constructs with either a catalytically inactive point mutation (C124S) or with only inactive lipid phosphatase activity (G129E), suggesting an indirect, lipid phosphatase-dependent process. The predominant effects of PTEN on tau appeared to be mediated by reducing ERK1/2 activity, but were independent of Akt, GSK-3, JNK and the tau phosphatases PP1 and PP2A. Our studies provide evidence for an effect of PTEN on the phosphorylation of tau in AD pathogenesis, and provide some insight into the mechanisms through which deregulation of PTEN may contribute towards the progression of tauopathy.     

A positive role of mammalian Tip41-like protein,TIPRL, in the amino-acid dependent mTORC1-signaling pathway through interaction with PP2A

Target of rapamycin complex 1 (TORC1) has a key role in cellular regulations in response to environmental conditions. In yeast, Tip41 downregulates TORC1 signaling via activation of PP2A phosphatase. We show here that overexpression of TIPRL, a mammalian Tip41, suppressed dephosphorylation of mechanistic TORC1 (mTORC1) substrates under amino acid withdrawal, and knockdown of TIPRL conversely attenuated phosphorylation of those substrates after amino acid refeeding. TIPRL associated with the catalytic subunit of PP2A (PP2Ac), which was required for the TIPRL action on mTORC1 signaling. Collectively, unlike yeast TIP41, TIPRL has a positive effect on mTORC1 signaling through the association with PP2Ac.     

Sindbis virus replication, is insensitive to rapamycin and torin1, and suppresses Akt/mTOR pathway late during infection in HEK cells

Genetically engineered Sindbis viruses (SIN) are excellent oncolytic agents in preclinical models. Several human cancers have aberrant Akt signaling, and kinase inhibitors including rapamycin are currently tested in combination therapies with oncolytic viruses. Therefore, it was of interest to delineate possible cross-regulation between SIN replication and PI3K/Akt/mTOR signaling. Here, using HEK293T cells as host, we report the following key findings: (a) robust SIN replication occurs in the presence of mTOR specific inhibitors, rapamycin and torin1 or Ly294002 – a PI3K inhibitor, suggesting a lack of requirement for PI3K/Akt/mTOR signaling; (b) suppression of phosphorylation of Akt, mTOR and its effectors S6, and 4E-BP1 occurs late during SIN infection: a viral function that may be beneficial in counteracting cellular drug resistance to kinase inhibitors; (c) Ly294002 and SIN act additively to suppress PI3K/Akt/mTOR pathway with little effect on virus release; and (d) SIN replication induces host translational shut off, phosphorylation of eIF2α and apoptosis. This first report on the potent inhibition of Akt/mTOR signaling by SIN replication, bolsters further studies on the development and evaluation of engineered SIN genotypes in vitro and in vivo for unique cytolytic functions.     

Specific interaction between S6K1 and CoA synthase: a potential link between the mTOR/S6K pathway, CoA biosynthesis and energy metabolism

Ribosomal protein S6 kinase (S6K) is a key regulator of cell size and growth. It is regulated via phosphoinositide 3-kinases (PI3K) and the mammalian target of rapamycin (mTOR) signaling pathways. We demonstrate for the first time that CoA synthase associates specifically with S6K1. The association was observed between native and transiently overexpressed proteins in vivo, as well as by BIAcore analysis in vitro. The sites of interaction were mapped to the C-terminal regions of both CoA synthase and S6K1. In vitro studies indicated that the interaction does not affect their enzymatic activities and that CoA synthase is not a substrate for S6 kinase. This study uncovers a potential link between mTor/S6K signaling pathway and energy metabolism through CoA and its thioester derivatives, but its physiological relevance should be further elucidated.     

mTOR/S6K1 and MAPK/RSK signaling pathways coordinately regulate estrogen receptor α serine 167 phosphorylation

Resistance to anti-estrogen therapy is a major clinical concern in treatment of breast cancer. Estrogen-independent phosphorylation of estrogen receptor α, specifically on Ser167, is one of the contributing causes to development of resistance, and a prognostic marker for the disease. Here, we dissect the signaling pathways responsible for Ser167 phosphorylation. We report that the mTOR/S6K1 and MAPK/RSK contribute non-overlapping inputs into ERα activation via Ser167 phosphorylation. This cooperation may be targeted in breast cancer treatment by a combination of mTOR and MAPK inhibitors.     

克隆、表达及纯化核糖体蛋白S6用于体外激酶活性检测

目的:构建40S核糖体蛋白S6的原核表达载体,表达并纯化S6蛋白,将其作为底物用于S6激酶(S6K)的体外活性测定。方法:采用RT-PCR方法从人胚肾细胞HEK293中获取S6 cDNA,将扩增产物克隆至大肠杆菌表达载体中,进行酶切及测序鉴定;IPTG诱导GST-S6融合蛋白在大肠杆菌中表达,用谷胱甘肽亲和层析纯化GST-S6,免疫沉淀法检测该蛋白是否可作为底物用于S6K的体外激酶活性测定。结果:酶切及测序鉴定表明构建了S6原核表达载体,并表达及纯化出GST-S6融合蛋白,相对分子质量为55×103。该蛋白可用于S6K的体外激酶活性测定,特异性强。结论:S6蛋白的克隆、表达与纯化成功,可用于S6K的体外激酶活性测定,为研究S6K的功能奠定了基础。     

AMPKalpha2 counteracts the development of cardiac hypertrophy induced by isoproterenol

As AMP-activated protein kinase (AMPK) controls protein translation, an anti-hypertrophic effect of AMPK has been suggested. However, there is no genetic evidence to confirm this hypothesis. We investigated the contribution of AMPKα2 in the control of cardiac hypertrophy by using AMPKα2−/− mice submitted to isoproterenol. The isoproterenol-induced cardiac hypertrophy, measured by left ventricular mass and histological examination, was significantly higher in AMPKα2−/− than in WT animals. Moreover, the intensification of cardiac hypertrophy found in AMPKα2−/− mice can be linked to the abnormal basal overstimulation of the p70 ribosomal S6 protein kinase, an enzyme known to regulate protein translation and cell growth. In conclusion, this work shows that AMPKα2 plays a role of brake for the development of cardiac hypertrophy.     

Identification of different specificity requirements between SGK1 and PKBalpha

NDRG1 is phosphorylated by SGK1 (but not PKB) in vivo at three residues each contained within three nonapeptide repeats. Here, we demonstrate that this nonapeptide, like the NDRG1 protein, is phosphorylated by SGK1, but not by PKBalpha or RSK1 in vitro. The inability of PKBalpha and RSK1 to phosphorylate the nonapeptide was traced to residues n+1, n+2 and n-4 (where n is the phosphorylation site). Changing them from Ser, Glu and Ser to Phe, Ala and Pro, respectively, transformed the nonapeptide into an excellent substrate for PKBalpha and RSK1. Our results identify a specific substrate for SGK1 and may facilitate detection of additional physiological substrates for this enzyme.     

Hepatic insulin resistance in ob/ob mice involves increases in ceramide,aPKC activity,and selective impairment of Akt-dependent FoxO1 phosphorylation

Pathogenesis of insulin resistance in leptin-deficient ob/ob mice is obscure. In another form of diet-dependent obesity, high-fat-fed mice, hepatic insulin resistance involves ceramide-induced activation of atypical protein kinase C (aPKC), which selectively impairs protein kinase B (Akt)-dependent forkhead box O1 protein (FoxO1) phosphorylation on scaffolding protein, 40 kDa WD(tryp-x-x-asp)-repeat propeller/FYVE protein (WD40/ProF), thereby increasing gluconeogenesis. Resultant hyperinsulinemia activates hepatic Akt and mammalian target of rapamycin C1, and further activates aPKC; consequently, lipogenic enzyme expression increases, and insulin signaling in muscle is secondarily impaired. Here, in obese minimally-diabetic ob/ob mice, hepatic ceramide and aPKC activity and its association with WD40/ProF were increased. Hepatic Akt activity was also increased, but Akt associated with WD40/ProF was diminished and accounted for reduced FoxO1 phosphorylation and increased gluconeogenic enzyme expression. Most importantly, liver-selective inhibition of aPKC decreased aPKC and increased Akt association with WD40/ProF, thereby restoring FoxO1 phosphorylation and reducing gluconeogenic enzyme expression. Additionally, lipogenic enzyme expression diminished, and insulin signaling in muscle, glucose tolerance, obesity, hepatosteatosis, and hyperlipidemia improved. In conclusion, hepatic ceramide accumulates in response to CNS-dependent dietary excess irrespective of fat content; hepatic insulin resistance is prominent in ob/ob mice and involves aPKC-dependent displacement of Akt fromWD40/ProF and subsequent impairment of FoxO1 phosphorylation and increased expression of hepatic gluconeogenic and lipogenic enzymes; and hepatic alterations diminish insulin signaling in muscle.     

Association of dystrobrevin and regulatory subunit of protein kinase A: a new role for dystrobrevin as a scaffold for signaling proteins

The dystrophin-related and -associated protein dystrobrevin is a component of the dystrophin-associated protein complex, which directly links the cytoskeleton to the extracellular matrix. It is now thought that this complex also serves as a dynamic scaffold for signaling proteins, and dystrobrevin may play a role in this context. Since dystrobrevin involvement in signaling pathways seems to be dependent on its interaction with other proteins, we sought new insights and performed a two-hybrid screen of a mouse brain cDNA library using beta-dystrobrevin, the isoform expressed in non-muscle tissues, as bait. Among the positive clones characterized after the screen, one encodes the regulatory subunit RIalpha of the cAMP-dependent protein kinase A (PKA). We confirmed the interaction by in vitro and in vivo association assays, and mapped the binding site of beta-dystrobrevin on RIalpha to the amino-terminal region encompassing the dimerization/docking domain of PKA regulatory subunit. We also found that the domain of interaction for RIalpha is contained in the amino-terminal region of beta-dystrobrevin. We obtained evidence that beta-dystrobrevin also interacts directly with RIIbeta, and that not only beta-dystrobrevin but also alpha-dystrobrevin interacts with PKA regulatory subunits. We show that both alpha and beta-dystrobrevin are specific phosphorylation substrates for PKA and that protein phosphatase 2A (PP2A) is associated with dystrobrevins. Our results suggest a new role for dystrobrevin as a scaffold protein that may play a role in different cellular processes involving PKA signaling.     

Ceramide 1-phosphate/ceramide, a switch between life and death

Ceramide is a well-characterized sphingolipid metabolite and second messenger that participates in numerous biological processes. In addition to serving as a precursor to complex sphingolipids, ceramide is a potent signaling molecule capable of regulating vital cellular functions. Perhaps its major role in signal transduction is to induce cell cycle arrest, and promote apoptosis. In contrast, little is known about the metabolic or signaling pathways that are regulated by the phosphorylated form of ceramide. It was first demonstrated that ceramide-1-phosphate (C1P) had mitogenic properties, and more recently it has been described as potent inhibitor of apoptosis and inducer of cell survival. C1P and ceramide are antagonistic molecules that can be interconverted in cells by kinase and phosphatase activities. An appropriate balance between the levels of these two metabolites seems to be crucial for cell and tissue homeostasis. Switching this balance towards accumulation of one or the other may result in metabolic dysfunction, or disease. Therefore, the activity of the enzymes that are involved in C1P and ceramide metabolism must be efficiently coordinated to ensure normal cell functioning.     

Rapamycin preserves the follicle pool reserve and prolongs the ovarian lifespan of female rats via modulating mTOR activation and sirtuin expression

To maintain the normal length of female reproductive life, the majority of primordial follicles must be maintained in a quiescent state for later use. In this study, we aimed to study the effects of rapamycin on primordial follicle development and investigate the role of mTOR and sirtuin signaling. Rats were treated every other day with an intraperitoneal injection of rapamycin (5 mg/kg) or vehicle. After 10 weeks of treatment, ovaries were harvested for hematoxylin and eosin (HE) staining, and analysis by immunohistochemistry and Western blotting. HE staining showed that the number and percentage of primordial follicles in the rapamycin-treated group were twice the control group (P < 0.001). Immunohistochemical analysis showed that mTOR and phosphorylated-p70S6K were extensively expressed in surviving follicles with strong staining observed in the cytoplasm of the oocyte. Western blotting showed decreased expression of phosphorylated mTOR and phosphorylated p70S6K in the rapamycin-treated group, and increased the expression of both SIRT1 and SIRT6 compared to the control group (P < 0.05). Taken together, these results suggest that rapamycin may inhibit the transition from primordial to developing follicles and preserve the follicle pool reserve, thus extending the ovarian lifespan of female rats via the modulation of mTOR and sirtuin signalings.