Multispecies-compatible antitumor effects of a cross-species small-interfering RNA against mammalian target of rapamycin

Successful development of sequence-specific siRNA (small interfering RNA)-based drugs requires an siRNA design that functions consistently in different organisms. Utilizing the CAPSID program previously developed by our group, we here designed siRNAs against mammalian target of rapamycin (mTOR) that are entirely complementary among various species and investigated their multispecies-compatible gene-silencing properties. The mTOR siRNAs markedly reduced mTOR expression at both the mRNA and protein levels in human, mouse, and monkey cell lines. The reduction in mTOR expression resulted in inactivation of both mTOR complex I and II signaling pathways, as confirmed by reduced phosphorylation of p70S6K (70-kDa ribosomal protein S6 kinase), 4EBP1 (eIF4E-binding protein 1), and AKT, and nuclear accumulation of FOXO1 (forkhead box O1), with consequent cell-cycle arrest, proliferation inhibition, and autophagy activation. Moreover, interfering with mTOR activity in vivo using mTOR small-hairpin RNA-expressing recombinant adeno-associated virus led to significant antitumor effects in xenograft and allograft models. Thus, the present study demonstrates that cross-species siRNA successfully silences its target and readily produces multispecies-compatible phenotypic alterations-antitumor effects in the case of mTOR siRNA. Application of cross-species siRNA should greatly facilitate the development of siRNA-based therapeutic agents.     

Transient and constitutive repression of cytoplasmic translation signaling in cells with mtDNA mutation

Cytoplasmic translation is under sophisticated control but how cells adapt its rate to constitutive loss of mitochondrial oxidative phosphorylation is unknown. Here we show that translation is repressed in cells with the pathogenic A3243G mtDNA mutation or in mtDNA-less ρ⁰ cells by at least two distinct pathways, one transiently targeting elongation factor eEF-2 and the other initiation factor eIF-2α constitutively. Under conditions of exponential cell growth and mammalian target of rapamycin (mTOR) activation, eEF-2 becomes transiently phosphorylated by an AMP-activated protein kinase (AMPK)-dependent pathway, especially high in mutant cells. Independent of AMPK and mTOR, eIF-2α is constitutively phosphorylated in mutant cells, likely a signature of endoplasmic reticulum (ER)-stress response induced by the loss of oxidative phosphorylation. While the AMPK/eEF-2K/eEF-2 pathway appears to function in adaptation to physiological fluctuations in ATP levels in the mutant cells, the ER stress signified by constitutive protein synthesis inhibition through eIF-2α-mediated repression of translation initiation may have pathobiochemical consequences. Received 29 October 2008; received after revision 11 December 2008; accepted 16 December 2008     

Akt-mediated GSK-3β inhibition prevents migration of polyamine-depleted intestinal epithelial cells via Rac1

The rapid migration of intestinal epithelial cells (IEC) is important for the healing of mucosal wounds. We have previously shown that polyamine depletion inhibits migration of IEC-6 cells. Akt activation and its downstream target GSK-3β have been implicated in the regulation of migration. Here we investigated the significance of elevated phosphatidylinositol 3-kinase (PI3K)/Akt signaling on migration of polyamine-depleted cells. Polyamine-depleted cells had high Akt (Ser473) and GSK-3β (Ser9) phosphorylation. Pretreatment with 20 μM LY294002 (PI3K inhibitor) for 30 min inhibited phosphorylation of Akt, increased migration by activating Rac1 in polyamine-depleted IEC-6 cells, and restored the actin structure similar to that in cells grown in control medium. Treatment of cells with a GSK-3β inhibitor (AR-A014418) altered the actin cytoskeleton and inhibited migration, mimicking the effects of polyamine depletion. Thus, our results indicate that sustained activation of Akt in response to polyamine depletion inhibits migration through GSK-3β and Rac1. Received 25 August 2006; received after revision 3 October 2006; accepted 16 October 2006     

MAP kinase signalling: interplays between plant PAMP- and effector-triggered immunity

In plants, mitogen-activated protein kinase (MAPK) cascades are involved in regulating many biological processes including immunity. They relay signals from membrane-residing immune receptors to downstream components for defense activation. Arabidopsis MPK3/6 and MPK4 are activated in two parallel MAPK cascades during PAMP-triggered immunity. MPK3/6 have been implicated in the activation of various immune responses and their inactivation leads to compromised defense against pathogens. On the other hand, the MEKK1-MKK1/2-MPK4 cascade plays critical roles in basal resistance. Disruption of this MAPK cascade results in constitutive defense responses mediated by the NB-LRR protein SUMM2. Interestingly, SUMM2 guards the MEKK1-MKK1/2-MPK4 cascade activity indirectly through monitoring the phosphorylation status of CRCK3, which is a substrate of MPK4. From the pathogens’ side, a number of effectors are shown to target various components of MAPK cascades in plants. Inactivation of MPK4 by the Pseudomonas effector HopAI1 triggers SUMM2-mediated immunity. Together, these findings suggest intricate interplays between PAMP-triggered immunity and effector-triggered immunity via MAPK signaling.     

Phosphorylation and activation of the atypical kinase p53-related protein kinase (PRPK) by Akt/PKB

p53-related protein kinase (PRPK), the human homologue of yeast Bud32, belonging to a small subfamily of atypical protein kinases, is inactive unless it is previously incubated with cell lysates. Here we show that such an activation of PRPK is mediated by another kinase, Akt/PKB, which phosphorylates PRPK at Ser250. We show that recombinant PRPK is phosphorylated in vitro by Akt and its phospho-form is recognized by a Ser250-phospho-specific antibody; that cell co-transfection with Akt along with wild-type PRPK, but not with its Ser250Ala mutant, results in increased PRPK phosphorylation; and that the phosphorylation of p53 at Ser15, the only known substrate of PRPK, is markedly increased by co-transfection of Akt with wild-type PRPK, but not PRPK dead mutant, and is abrogated by cell treatment with the Akt pathway inhibitor LY294002. Our data disclose an unanticipated mechanism by which PRPK can be activated and provide a functional link between this enigmatic kinase and the Akt signaling pathway.     

BRD7 regulates the insulin-signaling pathway by increasing phosphorylation of GSK3β

Reduced hepatic expression levels of bromodomain-containing protein 7 (BRD7) have been suggested to play a role in the development of glucose intolerance in obesity. However, the molecular mechanism by which BRD7 regulates glucose metabolism has remained unclear. Here, we show that BRD7 increases phosphorylation of glycogen synthase kinase 3β (GSK3β) in response to activation of the insulin receptor-signaling pathway shortly after insulin stimulation and the nutrient-sensing pathway after feeding. BRD7 mediates phosphorylation of GSK3β at the Serine 9 residue and this effect on GSK3β occurs even in the absence of AKT activity. Using both in vitro and in vivo models, we further demonstrate that BRD7 mediates phosphorylation of ribosomal protein S6 kinase (S6K) and leads to increased phosphorylation of the eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) and, therefore, relieves its inhibition of the eukaryotic translation initiation factor 4E (eIF4E). However, the increase in phosphorylation of 4E-BP1 with BRD7 overexpression is blunted in the absence of AKT activity. In addition, using liver-specific BRD7 knockout (LBKO) mice, we show that BRD7 is required for mTORC1 activity on its downstream molecules. These findings show a novel basis for understanding the molecular dynamics of glucose metabolism and suggest the unique function of BRD7 in the regulation of glucose homeostasis.     

Key factors in mTOR regulation

Mammalian target of rapamycin (mTOR) is a protein serine/threonine kinase that controls a wide range of growth-related cellular processes. In the past several years, many factors have been identified that are involved in controlling mTOR activity. Those factors in turn are regulated by diverse signaling cascades responsive to changes in intracellular and environmental conditions. The molecular connections between mTOR and its regulators form a complex signaling network that governs cellular metabolism, growth and proliferation. In this review, we discuss some key factors in mTOR regulation and mechanisms by which these factors control mTOR activity.     

Regulation of mitochondrial aconitase by phosphorylation in diabetic rat heart

Mitochondrial dysfunction and protein kinase C (PKC) activation are consistently found in diabetic cardiomyopathy but their relationship remains unclear. This study identified mitochondrial aconitase as a downstream target of PKC activation using immunoblotting and mass spectrometry, and then characterized phosphorylation-induced changes in its activity in hearts from type 1 diabetic rats. PKCβ₂ co-immunoprecipitated with phosphorylated aconitase from mitochondria isolated from diabetic hearts. Augmented phosphorylation of mitochondrial aconitase in diabetic hearts was found to be associated with an increase in its reverse activity (isocitrate to aconitate), while the rate of the forward activity was unchanged. Similar results were obtained on phosphorylation of mitochondrial aconitase by PKCβ₂ in vitro. These results demonstrate the regulation of mitochondrial aconitase activity by PKC-dependent phosphorylation. This may influence the activity of the tricarboxylic acid cycle, and contribute to impaired mitochondrial function and energy metabolism in diabetic hearts. Received 31 October 2008; received after revision 17 December 2008; accepted 2 January 2009     

Dephosphorylation and inactivation of Akt/PKB is counteracted by protein kinase CK2 in HEK 293T cells

Akt (PKB) is a critical kinase in cell-survival pathways. Its activity depends on the phosphorylation of Thr308 and Ser473, by PDK1 and mTORC2, respectively. We found that Akt can be further stimulated through phosphorylation of Ser129 by another kinase, CK2. Here we show that phosphorylation of Akt at Ser129 also facilitates its association with Hsp90 chaperone, thus preventing Thr308 dephosphorylation. This is supported by the following observations: (1) phospho-Thr308 decreases when Ser129 is mutated to alanine, (2) this decrease is abolished by cell treatment with okadaic acid (to inactivate PP2A) or geldanamycin (to inactivate Hsp90), (3) phosphorylation of Ser129 neither enhances the activity of PDK1 nor hampers the in vitro activity of PP2A on Thr308, but increases the Hsp90 association to Akt. These data support the view that the antiapoptotic potential of CK2 is at least in part mediated by its ability to maintain Akt in its active form.     

Developmental regulation of p70 S6 kinase by a G protein-coupled receptor dynamically modelized in primary cells

The mechanisms whereby G protein-coupled receptors (GPCR) activate signalling pathways involved in mRNA translation are ill-defined, in contrast to tyrosine kinase receptors (TKR). We compared a GPCR and a TKR, both endogenously expressed, for their ability to mediate phosphorylation of 70-kDa ribosomal S6 kinase p70S6K in primary rat Sertoli cells at two developmental stages. In proliferating cells stimulated with follicle-stimulating hormone (FSH), active p70S6K was phosphorylated on T389 and T421/S424, through cAMP-dependent kinase (PKA) and phosphatidyl-inositide-3 kinase (PI3K) antagonizing actions. In FSH-stimulated differentiating cells, active p70S6K was phosphorylated solely on T389, PKA and PI3K independently enhancing its activity. At both developmental stages, insulin-induced p70S6K regulation was consistent with reported data. Therefore, TKR and GPCR trigger distinct p70S6K active conformations. p70S6K developmental regulation was formalized in a dynamic mathematical model fitting the data, which led to experimentally inaccessible predictions on p70S6K phosphorylation rate.     

Two distinct phosphorylation events govern the function of muscle FHOD3

Posttranslational modifications such as phosphorylation are universally acknowledged regulators of protein function. Recently we characterised a striated muscle-specific isoform of the formin FHOD3 that displays distinct subcellular targeting and protein half-life compared to its non-muscle counterpart and which is dependent on phosphorylation by CK2 (formerly casein kinase 2). We now show that the two isoforms of FHOD3 are already expressed in the vertebrate embryonic heart. Analysis of CK2 alpha knockout mice showed that phosphorylation by CK2 is also required for proper targeting of muscle FHOD3 to the myofibrils in embryonic cardiomyocytes in situ. The localisation of muscle FHOD3 in the sarcomere varies depending on the maturation state, being either broader or restricted to the Z-disc proper in the adult heart. Following myofibril disassembly, such as that in dedifferentiating adult rat cardiomyocytes in culture, the expression of non-muscle FHOD3 is up-regulated, which is reversed once the myofibrils are reassembled. The shift in expression levels of different isoforms is accompanied by an increased co-localisation with p62, which is involved in autophagy, and affects the half-life of FHOD3. Phosphorylation of three amino acids in the C-terminus of FHOD3 by ROCK1 is sufficient for activation, which results in increased actin filament synthesis in cardiomyocytes and also a broader localisation pattern of FHOD3 in the myofibrils. ROCK1 can directly phosphorylate FHOD3, and FHOD3 seems to be the downstream mediator of the exaggerated actin filament formation phenotype that is induced in cardiomyocytes upon the overexpression of constitutively active ROCK1. We conclude that the expression of the muscle FHOD3 isoform is characteristic of the healthy mature heart and that two distinct phosphorylation events are crucial to regulate the activity of this isoform in thin filament assembly and maintenance.     

Structure and function of a calmodulin-dependent smooth muscle myosin light chain kinase

In smooth muscle the Mr 20,000 light chain of myosin is phosphorylated by a calmodulin-dependent protein kinase. It consists of 2 subunits: calmodulin, an acidic protein of Mr 17,000 that binds 4 moles of Ca2+; and a larger protein of Mr circa 130,000. Activation of the kinase is dependent upon their association in the presence of Ca2+. Cyclic AMP-dependent protein kinase phosphorylation of the myosin light chain kinase occurs at 2 sites. It decreases the affinity of the kinase for calmodulin and a reduction in the rate of light chain phosphorylation occurs. The kinase has an overall asymmetric shape composed of a globular head and tail region for the skeletal muscle enzyme. Trypsin digestion of this kinase releases a fragment of Mr 36,000 from the globular region that contains the catalytic and calmodulin binding sites. Chymotrypsin digestion of the kinase from smooth muscle generates a fragment of Mr 80,000 that does not contain the calmodulin binding or cyclic AMP-dependent protein kinase phosphorylation sites. It is a Ca2+-independent form of the kinase that phosphorylates the light chain of myosin. These structural features indicate a regulatory role for the kinase in smooth muscle phosphorylation and contraction.     

Velocity and myosin phosphorylation transients in arterial smooth muscle: effects of agonist diffusion

Transients in myoplasmic [Ca2+] and in phosphorylation of the 20,000 dalton light chain of myosin have been reported following stimulation of vascular smooth muscle by various agonists. Since these transients are rapid compared with the time required to attain a steady-state stress, agonist diffusion rates may be a significant limitation in activation. The purpose of this study was to estimate the effect of agonist diffusion rates on the time course of activation as assessed by mechanical measurements of stress development and isotonic shortening velocities and by determinations of the time course of myosin phosphorylation. The approach was to measure these parameters in K+ -stimulated preparations of the swine carotid media of varying thicknesses and to estimate the theoretical contributions imposed by diffusion rates and the presence of a diffusion boundary layer surrounding the tissue. The results show that the time course of parameters which are tissue averages such as stiffness, active stress, and myosin phosphorylation is dominated by agonist diffusion rates. The sequence of events involved in excitation-contraction coupling including agonist actions on the cell membrane, Ca2+ release, activation of myosin light chain kinase, and cross-bridge phosphorylation appear to be very rapid events compared with stress development. Estimates of unloaded or lightly loaded shortening velocities which are not simple tissue averages appear to provide an improved estimate of activation rates.     

Phosphorylation-dependent prolyl isomerization: a novel signaling regulatory mechanism

Protein phosphorylation on serine or threonine residues preceding proline (Ser/Thr-Pro) plays an essential role for regulating various cellular processes, including cell cycle progression. Although phosphorylation has been proposed to regulate the function of a protein by inducing conformational changes, much less is known about what phosphate additions actually do and how the functions of phosphoproteins are coordinated. Proline is important for determining protein structure because it exists in cis or trans conformation and can put kinks into a polypeptide chain. We have shown that phosphorylation on Ser/Thr-Pro motifs reduces the cis/trans isomerization rate of Ser/Thr-Pro bonds. At the same time, proteins containing phosphorylated Ser/Thr-Pro motifs are substrates for the prolyl isomerase Pin1. The WW domain of Pin1 acts as a phosphoserine/threonine-binding module binding a defined subset of mitosis-specific phosphoproteins, such as Cdc25 and tau. These interactions target the enzymatic activity of Pin1 close to its substrates. In contrast to other prolyl isomerases (peptidyl-prolyl isomerases, PPlases), Pin1 has an extremely high degree of substrate specificity, specifically isomerizing phosphorylated Ser/Thr-Pro bonds. Therefore, Pin1 binds and regulates the function of a defined subset of phosphoproteins. Furthermore, inhibiting Pin1 function is lethal for dividing cells. Interestingly, Pin1, which can restore the biological function of phosphorylated tau, is sequestered in the neurofibrillary tangles in Alzheimer's brains. Thus, we have proposed a novel signaling regulatory mechanism, where protein phosphorylation creates binding sites for Pin1, which can then latch on to and isomerize the phosphorylated Ser/Thr-Pro peptide bond. In turn, this may change the shape of the protein, regulating its activity, dephosphorylation, degradation or location in the cell. This new post-phosphorylation regulatory mechanism appears to play an important role in normal cell function, such as mitotic progression, and in the pathogenesis of some human pathologies, such as Alzheimer's disease.     

Structure and function of a calmodulin-dependent smooth muscle myosin light chain kinase

Summary In smooth muscle the M_r 20,000 light chain of myosin is phosphorylated by a calmodulin-dependent protein kinase. It consists of 2 subunits: calmodulin, an acidic protein of M_r 17,000 that binds 4 moles of Ca²⁺; and a larger protein of M_r circa 130,000. Activation of the kinase is dependent upon their association in the presence of Ca²⁺. Cyclic AMP-dependent protein kinase phosphorylation of the myosin light chain kinase occurs at 2 sites. It decreases the affinity of the kinase for calmodulin and a reduction in the rate of light chain phosphorylation occurs. The kinase has an overall asymmetric shape composed of a globular head and tail region for the skeletal muscle enzyme. Trypsin digestion of this kinase releases a fragment of M_r 36,000 from the globular region that contains the catalytic and calmodulin binding sites. Chymotrypsin digestion of the kinase from smooth muscle generates a fragment of M_r 80,000 that does not contain the calmodulin binding or cyclic AMP-dependent protein kinase phosphorylation sites. It is a Ca²⁺-independent form of the kinase that phosphorylates the light chain of myosin. These structural features indicate a regulatory role for the kinase in smooth muscle phosphorylation and contraction.     

Velocity and myosin phosphorylation transients in arterial smooth muscle: effects of agonist diffusion

Summary Transients in myoplasmic [Ca²⁺] and in phosphorylation of the 20,000 dalton light chain of myosin have been reported following stimulation of vascular smooth muscle by various agonists. Since these transients are rapid compared with the time required to attain a steady-state stress, agonist diffusion rates may be a significant limitation in activation. The purpose of this study was to estimate the effect of agonist diffusion rates on the time course of activation as assessed by mechanical measurements of stress development and isotonic shortening velocities and by determinations of the time course of myosin phosphorylation. The approach was to measure these parameters in K⁺-stimulated preparations of the swine carotid media of varying thicknesses and to estimate the theoretical contributions imposed by diffusion rates and the presence of a diffusion boundary layer surrounding the tissue. The results show that the time course of parameters which are tissue averages such as stiffness, active stress, and myosin phosphorylation is dominated by agonist diffusion rates. The sequence of events involved in excitation-contraction coupling including agonist actions on the cell membrane, Ca²⁺ release, activation of myosin light chain kinase, and cross-bridge phosphorylation appear to be very rapid events compared with stress development. Estimates of unloaded or lightly loaded shortening velocities which are not simple tissue averages appear to provide an imporoved estimate of activation rates.Supported by a grant from the National Institutes of Health 5-PO1-HL19242. K. E. Kamm was supported by a National Heart, Lung, and Blood Institute Research Service Award HL-05957.     

Regulation of muscle creatine kinase by phosphorylation in normal and diabetic hearts

Protein kinase C (PKC) is an important signaling molecule in the heart, but its targets remain unclear. Using a PKC substrate antibody, we detected a 40-kDa phosphorylated cardiac protein that was subsequently identified by tandem mass spectroscopy as muscle creatine kinase (M-CK) with phosphorylation at serine 128. The forward reaction using ATP to generate phosphocreatine was reduced, while the reverse reaction using phosphocreatine to generate ATP was increased following dephosphorylation of immunoprecipitated M-CK with protein phosphatase 2A (PP2A) or PP2C. Despite higher PKC levels in diabetic hearts, decreased phosphorylation of M-CK was more prominent than the reduction in its expression. Changes in CK activity in diabetic hearts were similar to those found following dephosphorylation of M-CK from control hearts. The decrease in phosphorylation may act as a compensatory mechanism to maintain CK activity at an appropriate level for cytosolic ATP regeneration in the diabetic heart. Received 15 September 2008; received after revision 30 September 2008; accepted 13 October 2008