Liposome-mediated gene transfer to fetal human ventral mesencephalic explant cultures

To clarify how Abeta deposits induce secondary tauopathy, the presence of phosphorylated tau, glycogen synthase kinase 3alpha (GSK3alpha), GSK3beta, cyclin-dependent kinase 5 (CDK5), mitogen-activated protein kinase (MAPK) and fyn were examined in the Tg2576 brain showing substantial brain Abeta amyloidosis and behavioral abnormalities. Phosphorylated tau at Ser199, Thr231/Ser235, Ser396 and Ser413 accumulated in the dystrophic neurites of senile plaques. The major kinase for tau phosphorylation was GSK3beta. Smaller contributions of GSK3alpha, CDK5 and MAPK were suggested. Thus, brain Abeta amyloidosis has a potential role in the induction of tauopathy leading to the mental disturbances of Alzheimer's disease.     

Protein levels of glycogen synthase 3 kinase are normal in progressive supranuclear palsy

Progressive supranuclear palsy (PSP) is a neurodegenerative disorder characterized by pure neurofibrillary tau pathology involving mainly basal ganglia and brain stem nuclei. One of the kinases involved in tau phosphorylation is glycogen synthase 3 kinase (GSK3). In mammals GSK3 is present in two isoforms, alpha and beta regulated by phosphorylation: phosphorylation of Ser9 in GSK3beta or Ser21 in GSK3alpha leads to inactivation while phosphorylation of Tyr216 in GSK3beta or Tyr279 in GSK3alpha leads to activation. We analyzed the protein levels of GSK3alpha/beta and of the phosphorylated forms GSK3beta S(9), GSK3beta Y(216), GSK3alpha Y(279) in brain tissues of subjects with PSP. The analysis failed to show significant differences of all GSK3 isoforms in PSP in comparison to age-matched control cases. This negative result argues against the role of GSK3 in the pathogenesis of PSP.     

Regulation of the interaction of Disabled-1 with CIN85 by phosphorylation with Cyclin-dependent kinase 5

Disabled-1 (Dab1) is an adaptor protein mediating Reelin signaling in neuronal migration during brain development. Cyclin-dependent kinase 5 (Cdk5)-p35 is a proline-directed Ser/Thr kinase also involved in neuronal migration. The interaction between Dab1 and Cdk5 is in need of investigation. Dab1 was phosphorylated at Ser400 and Ser491 by Cdk5 in vivo. We search for proteins that interact with Dab1 in a phosphorylation-dependent manner at these sites, and identified CIN85, an SH3-containing adaptor protein involved in endocytosis, and CPalpha/CPbeta, which are subunits of barbed end F-actin-capping proteins (CP), as proteins bound to unphosphorylated Dab1 by mass spectrometric analysis. It was shown that the PTPAPR sequence of Dab1, conforming to the PxxxPR atypical SH3-binding motif, was the binding site for SH3 domains of CIN85. The results that phosphorylation at Ser491 close to the PTPAPR sequence inhibited association with CIN85 may provide a mechanism regulating the interaction between the PxxxPR motif proteins and SH3 domains of CIN85 family proteins. Together with previous results that CIN85 regulates actin assembly, present results raise the possibility that Cdk5 modulates actin dynamics through regulation of CIN85-Dab1 interaction by the Dab1 phosphorylation.     

Adrenaline potentiates insulin-stimulated PKB activation in the rat fast-twitch epitrochlearis muscle without affecting IRS-1-associated PI 3-kinase activity

We have previously shown in the rat slow-twitch soleus muscle that adrenaline greatly potentiates insulin-stimulated protein kinase B (PKB) phosphorylation without having an effect alone. However, insulin signalling capacity through the PKB pathway is higher in soleus than in fast-twitch muscles, whereas adrenaline activates phosphorylase more strongly in epitrochlearis. Therefore, the aim of the present study was to investigate the interaction between adrenaline and insulin signalling in the fast-twitch epitrochlearis muscle. Insulin increased insulin receptor substrate-1 (IRS-1)-associated phosphoinositide (PI) 3-kinase activity threefold, and adrenaline did not influence basal or insulin-stimulated PI 3-kinase activity. Insulin but not adrenaline increased PKB activity and phosphorylation of Ser(473) and Thr(308). It is interesting to note that adrenaline potentiated insulin-stimulated PKB activity and PKB Ser(473) and Thr(308) phosphorylation. These effects were mimicked by dibutyryl-cyclic adenosine monophosphate (db-cAMP). Adrenaline and db-cAMP increased glycogen synthase kinase (GSK)-3beta Ser(9) phosphorylation independently of PKB activation and enhanced insulin-stimulated GSK-3beta Ser(9) phosphorylation. Although adrenaline increased GSK-3 phosphorylation (inhibiting activity), phosphorylation of its target sites on glycogen synthase was increased, and adrenaline blocked insulin-stimulated glycogen synthase dephosphorylation of Ser(641) and Ser(645,649,653,657), glycogen synthase activation and glycogen synthesis. Insulin-stimulated glucose transport was not influenced by adrenaline despite the increased PKB activation. In conclusion, as in the slow-twitch soleus muscle, adrenaline potentiates insulin-stimulated PKB activation in the fast-twitch glycolytic epitrochlearis muscle without increasing IRS-1-associated PI 3-kinase activity. Furthermore, adrenaline induces phosphorylation of a pool of GSK-3 that is not involved in the regulation of glycogen metabolism. These results indicate that the combination of adrenaline and insulin may activate novel signalling molecules rather than just summing up their effects on linear pathways.     

SH-SY5Y细胞高表达糖原合成激酶3β对tau蛋白磷酸化和微管稳定性的影响

目的：构建高表达糖原合成激酶3β（GSK3β）的SH-SY5Y（人骨髓神经母细胞瘤细胞）转基因细胞模型,观察GSK3β高表达对宿主细胞tau蛋白磷酸化、微管稳定性的影响。 方法：构建GSK3β真核表达质粒,转染SH-SY5Y细胞,使GSK3β在SH-SY5Y细胞中得到高表达,应用蛋白免疫印迹方法,观察tau蛋白磷酸化、总tau蛋白、微管蛋白乙酰化水平的变化情况。 结果：GSK3β真核表达质粒转染SH-SY5Y细胞36 h后,GSK3β在SH-SY5Y细胞中的表达达到高峰,tau蛋白Ser199/202、Thr231、Thr205等位点的磷酸化水平在转染后48 h达到高峰; tau蛋白总量未有明显变化。转染后60 h,微管蛋白乙酰化水平明显减低。 结论：高表达GSK3β的SH-SY5Y细胞可使tau蛋白的磷酸化水平增高,从而降低tau蛋白结合和稳定微管蛋白的能力,导致微管蛋白乙酰化水平明显减低。     

A mechanism for the coordination of proliferation and differentiation by spatial regulation of Fus2p in budding yeast

Yeast cells induce the genes required for mating prior to the completion of mitosis. To ensure proper cell cycle progression prior to mating differentiation, a key cytoplasmic regulator of cell fusion, Fus2p, is sequestered in the nucleus by cyclin-dependent kinase (Cdk). In response to pheromone signaling, the mitogen-activated protein kinase Fus3p phosphorylates Ser 84 in Fus2p to drive nuclear export. We found that Fus3p becomes active and phosphorylates S84 as early as S phase, raising the question of how Cdk prevents inappropriate activation of Fus2p. Countering Fus3p, Cdk and a p21-activated kinase, Cla4p, maintain Fus2p's nuclear localization by phosphorylating Ser 67, which drives nuclear import and inhibits nuclear export. When Cdk and Cla4p activities drop after cell division, Fus3p promotes Fus2p export both via S84 phosphorylation and by down-regulating S67 phosphorylation. Thus, potential premature activation of Fus2p in mitosis is prevented by cell cycle-dependent phosphorylation that overrides the mating pheromone-induced phosphorylation that drives nuclear export.     

Distinct regulatory mechanism for p70 S6 kinase β from that for p70 S6 kinase α

BACKGROUND: A novel ribosomal S6 kinase, termed p70 S6 kinase beta (p70beta), has a highly homologous amino acid sequence to that of p70/p85 S6 kinase (p70alpha). This includes the critical phosphorylation sites, Thr252, Ser394 and Thr412 in p70alpha1, which correspond to Thr241, Ser383 and Thr401 in p70beta1, respectively. However, the regulatory mechanism for p70beta remains to be elucidated. RESULTS: We report here the expression and the mechanism of in vivo regulation of p70beta. Two isoforms, p70beta1 and p70beta2, were expressed in a variety of tissues at a different level. p70beta1 was mainly targeted to the nucleus, whereas p70beta2 dispersed throughout the cytoplasm including nucleoplasm. The kinase activity of p70beta1 was less sensitive to the inhibition induced by rapamycin, wortmannin and amino acid withdrawal than that of p70alpha. The portion of p70beta activity inhibited by rapamycin was rescued by the rapamycin-resistant mutant of the mammalian target of rapamycin (mTOR). Mutational analysis revealed that the phosphorylation of Thr241 and Thr401 in p70beta1 was indispensable for the kinase activity. In contrast, a p70beta1 mutant in which Ser383 was substituted with Gly (S383G) still retained nearly the half maximal activity. Sequential phosphorylation of wild-type and S383G mutant of p70beta1 with mTOR and 3-phosphoinositide-dependent protein kinase 1 (PDK1) in vitro synergistically activated their kinase activities. CONCLUSION: These results indicate that p70beta is regulated by the mTOR- and PDK1-signalling pathways through a synergistic interaction between phosphorylated Thr241 and Thr401, while Ser383 plays minor role in their activation mechanism. Activated p70beta may be less sensitive to dephosphorylation mediated by putative phosphatases activated by rapamycin, amino acid withdrawal, and probably wortmannin.     

Genetic suppression of collapsin response mediator protein 2 phosphorylation improves outcome in methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine‐induced Parkinson’s model mice

Parkinson's disease (PD) is a common neurodegenerative disorder characterized by slow and progressive degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNc). Levodopa (l ‐Dopa), the current main treatment for PD, supplies dopamine, but it does not prevent neurodegeneration. There is thus no promising remedy for PD. Recent in vitro study showed the increase in the phosphorylation levels of Collapsin Response Mediator Protein 2 (CRMP2) is involved in dopaminergic axon degeneration. In the present study, we report elevation of CRMP2 phosphorylation in dopaminergic neurons in SNc after challenge with the dopaminergic neurotoxin 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP), a common model for PD. Genetic suppression of CRMP2 phosphorylation by mutation of the obligatory Cyclin‐dependent kinase 5 (Cdk5)‐targeted serine‐522 site prevented axonal degradation in the nigrostriatal pathway of transgenic mice. As a result, the degree of MPTP‐induced motor impairment in the rotarod test was suppressed. These results suggest that suppression of CRMP2 phosphorylation may be a novel therapeutic target for PD.     

AATYK1A phosphorylation by Cdk5 regulates the recycling endosome pathway

Trafficking of recycling endosomes (REs) is regulated by the small GTPase, Rab11A; however, the regulatory mechanism remains elusive. Apoptosis‐associated tyrosine kinase 1A (AATYK1A) is a Ser/Thr kinase expressed highly in brain. We have recently shown that AATYK1A localizes to Rab11A‐positive RE and is phosphorylated at Ser34 by cyclin‐dependent kinase 5 (Cdk5). Here, we have investigated a role of AATYK1A and its phosphorylation in recycling endosomal trafficking using Chinese hamster ovary‐K1 (CHO‐K1) cells. AATYK1A localizes predominantly to Rab11A‐positive pericentrosomal endocytic recycling compartment (ERC). Phosphorylation at Ser34 of AATYK1A disrupts its accumulation in the pericentrosomal ERC. Consistently, phosphorylation‐mimic mutant (AATYK1A‐S34D) did not accumulate in the ERC and additionally attenuated ERC formation. ERC formation suppression can be reversed by constitutively active Rab11A‐Q70L, suggesting a functional link between AATYK1A phosphorylation and Rab11A activity. Although no direct interaction between AATYK1A and Rab11A could be detected, the exchange of guanine nucleotides bound to Rab11A was significantly reduced in the presence of the phosphorylation‐mimic AATYK1A‐S34D. Together, our results reveal a regulatory role for AATYK1A in the formation of pericentrosomal ERC. They furthermore indicate that Cdk5 can disrupt ERC formation via Ser34 phosphorylation of AATYK1A. Finally, our data suggest a mechanism by which AATYK1A signaling couples Cdk5 to Rab11A activity.     

Identification of an N-terminal glycogen synthase kinase 3 phosphorylation site which regulates the functional localization of polycystin-2 in vivo and in vitro

PKD2 is mutated in 15% of patients with autosomal dominant polycystic kidney disease. Polycystin-2 (PC2), the PKD2 protein, is a non-selective Ca(2+)-permeable cation channel which may function at the cell surface and ER. Nevertheless, the factors that regulate the dynamic translocation of PC2 between the ER and other compartments are not well understood. Constitutive phosphorylation of PC2 at a single C-terminal site (Ser(812)) has been previously reported. As we were unable to abolish phospholabelling of PC2 in HEK293 cells by site-directed mutagenesis of Ser(812) or all five predicted phosphorylation sites in the C-terminus, we hypothesized that PC2 could also be phosphorylated at the N-terminus. In this paper, we report the identification of a new phosphorylation site for PC2 within its N-terminal domain (Ser(76)) and demonstrate that this residue is phosphorylated by glycogen synthase kinase 3 (GSK3). The consensus recognition sequence for GSK3 (Ser(76)/Ser(80)) is evolutionarily conserved down to lower vertebrates. In the presence of specific GSK3 inhibitors, the lateral plasma membrane pool of endogenous PC2 redistributes into an intracellular compartment in MDCK cells without any change in primary cilia localization. Finally, co-injection of wild-type but not a S76A/S80A mutant PKD2 capped mRNA could rescue the cystic phenotype induced by an antisense morpholino oligonucleotide to pkd2 in zebrafish pronephric kidney. We conclude that surface localization of PC2 is regulated by phosphorylation at a unique GSK3 site in its N-terminal domain in vivo and in vitro. This site is functionally significant for the maintenance of normal glomerular and tubular morphology.     

Phosphorylation of tau at THR212 and SER214 in human neuronal and glial cultures: the role of AKT

We have reported recently that the microtubule-associated protein tau is phosphorylated in vitro by Akt, an important kinase in anti-apoptotic signaling regulated by insulin and growth factors. We also established that Akt phosphorylates tau separately at T212 and S214, two sites previously shown to be phosphorylated by glycogen synthase kinase 3beta (GSK3beta) and protein kinase A (PKA), respectively. In the present studies, we examined the relationship between Akt and T212/S214 in primary cultures of human neurons and astrocytes, and evaluated the contribution of two other kinases. In intact cells, we found a very low content of active (phospho-S473) form of Akt. We also found a low content of phospho-S214 but not phospho-T212 of tau, suggesting that only phospho-S212 may depend on Akt activity in situ. We upregulated Akt activity using two experimental models: treatment with a protein phosphatase inhibitor, okadaic acid, and transfection with a constitutively active Akt gene construct (c-Akt). Under these conditions, phosphorylation of tau at T212 and S214 was regulated independently, with little change or downregulation of phospho-T212 and dynamic upregulation of phospho-S214. Our studies revealed that Akt may influence the phospho-S214 content in a meaningful manner. They also revealed that PKA may only partially contribute to the phosphorylation of S214. In comparison, okadaic acid treatment severely depleted the content of GSK3beta and downregulated the remaining GSK3beta activity by Akt-dependent inhibition, consistent with minimal changes in phospho-T212. In summary, these results strongly suggest that in primary cultures, Akt selectively phosphorylates tau at S214 rather than T212. Our studies raise the possibility that tau S214 may participate in Akt-mediated anti-apoptotic signaling.     

Alpha-synuclein phosphorylation controls neurotoxicity and inclusion formation in a Drosophila model of Parkinson disease

Alpha-synuclein is phosphorylated at serine 129 (Ser129) in intracellular protein aggregates called Lewy bodies. These inclusion bodies are the characteristic pathologic lesions of Parkinson disease. Here we define the role of phosphorylation of Ser129 in alpha-synuclein toxicity and inclusion formation using a Drosophila model of Parkinson disease. Mutation of Ser129 to alanine to prevent phosphorylation completely suppresses dopaminergic neuronal loss produced by expression of human alpha-synuclein. In contrast, altering Ser129 to the negatively charged residue aspartate, to mimic phosphorylation, significantly enhances alpha-synuclein toxicity. The G protein-coupled receptor kinase 2 (Gprk2) phosphorylates Ser129 in vivo and enhances alpha-synuclein toxicity. Blocking phosphorylation at Ser129 substantially increases aggregate formation. Thus Ser129 phosphorylation status is crucial in mediating alpha-synuclein neurotoxicity and inclusion formation. Because increased number of inclusion bodies correlates with reduced toxicity, inclusion bodies may protect neurons from alpha-synuclein toxicity.     

Inhibition of neuronal calcium oscillations by cell surface APP phosphorylated on T668

Adenoviral expression of human APP (hAPP), but not of hAPP deleted from its C-terminal intracellular domain, in rat cortical neurons abolishes spontaneous synchronous calcium oscillations. The intracellular domain of APP695 contains several residues that can be phosphorylated. Contrary to non-neuronal cells, a very high phosphorylation of APP on T668 is observed in neurons, which is mediated by JNK, GSK3 and Cdk5 protein kinases. JNK activity, modulated by GSK3, enhances the traffic of phosphorylated APP to nerve terminals, contrary to Cdk5. Here we show that inhibition of GSK3 and JNK restores calcium oscillations in an hAPP expressing neuronal network, whereas inhibition of Cdk5 does not. Expression of mutant hAPPT668A does not inhibit calcium oscillations, and the proportion of hAPPT668A at the plasma membrane is reduced by more than 50%. Altogether, these results indicate that the intracellular domain of APP is needed to inhibit neuronal calcium oscillations because GSK3/JNK phosphorylation of T668 controls APP trafficking at the plasma membrane.     

Cdk5-CRMP通路在七氟醚抑制新生大鼠前额叶皮层树突发育中的作用

目的:探究七氟醚对新生大鼠前额叶皮层(prefrontal cortex,PFC)树突发育的影响及与细胞周期依赖性蛋白激酶5(cyclin dependent kinase 5,Cdk5)-坍塌反应调节蛋白(collapsin response mediator protein,CRMP)通路的关系。方法:88只出生后7 d的SD大鼠随机分为4组,每组22只:空白对照组(Air+NS组),Cdk5抑制剂roscovitine(Ros)对照组(Air+Ros组),Sevo+NS组,Sevo+Ros组。前2组吸入空气,后2组吸入2.8%七氟醚4 h。Air+Ros组和Sevo+Ros组在麻醉前15 min经腹腔注射10 mg/kg的Cdk5抑制剂roscovitine。其余2组则在麻醉前15min经腹腔注射150μL生理盐水。麻醉结束后,每组各取5只幼鼠,取出新鲜皮质组织,Western blot检测P35、P25、Cdk5,CRMP1、2、4的蛋白表达以及CRMP2 Ser 522的磷酸化水平。剩余幼鼠继续饲养,出生后30 d每组各取6只幼鼠,取脑进行高尔基染色,制备冰冻切片,镜下观察神经元形态。其余幼鼠在出生后25~27 d进行旷场试验,31~32 d进行情景依赖性恐惧记忆检测。结果:(1)与Air+NS组比较,Sevo+NS组的P35减少了47.32%,同时P25增加了173.77%(P0.05);Sevo+Ros组的P35比Sevo+NS组增加了78.81%,其P25则减少了60.22%(P0.05)。Cdk5表达组间差异尚无统计学意义。(2)与Air+NS组比较,Sevo+NS组的总CRMP1、2和4表达分别下降了36.22%、53.74%和51.28%(P0.05),同时p-CRMP2 Ser522/CRMP2比值增加了96.20%(P0.05);Sevo+Ros组的总CRMP1和4与Sevo+NS组比较分别增加了56.95%和88.91%(P0.05),而pCRMP2 Ser522/CRMP2比值下降了43.58%(P0.05)。(3)与Air+NS组比较,Sevo+NS组的树突总长度、第二级树突长度以及60和80μm直径圆环上的交点数均显著下降(P0.01)。Sevo+Ros组的树突总长度、第二级树突长度以及60和80μm直径圆环上的交点数均比Sevo+NS组显著增加(P0.05)。(4)在恐惧记忆实验中,与Air+NS组比较,Sevo+NS组大鼠的"木僵"时间比Air+NS组减少约21%(P0.05)。Sevo+Ros组的"木僵"时间比Sevo+NS组显著增加(P0.05)。各组幼鼠在旷场实验的结果均无统计学差异。结论:七氟醚抑制了新生大鼠前额叶皮层神经元树突的正常生长发育和幼鼠学习记忆能力,而Cdk5-CRMP通路激活可能是七氟醚引起神经发育毒性的机制之一。     

Up-regulation of phosphorylated/activated p70 S6 kinase and its relationship to neurofibrillary pathology in Alzheimer's disease

The ribosomal S6 protein kinase p70 S6 kinase is known for its role in modulating cell-cycle progression, cell size, and cell survival. In response to mitogen stimulation, p70 S6 kinase activation up-regulates ribosomal biosynthesis and enhances the translational capacity of the cell. In Alzheimer's disease (AD), there is a marked increase in total tau protein in the form of abnormally hyperphosphorylated tau (PHF-tau) in neurons with neurofibrillary tangles (NFTs). In the present study, we investigated whether p70 S6 kinase activation is associated with PHF-tau accumulation in AD. By immunohistochemistry, we found that the levels of phosphorylated p70 S6 kinase (at Thr389 or at Thr421/Ser424) were increased in accordance with the progressive sequence of neurofibrillary changes according to Braak's criteria. Confocal microscopy showed that in AD brain, phosphorylated p70 S6 kinase appeared especially in neurons that are known to later develop NFTs. This pattern of neurons showed dot-like structures of phosphorylated p70 S6 kinase and hyperphosphorylated tau, which partially correlated with rab5 (endosome marker), lamp-1 (lysosome marker), and ubiquitin (ubiquitin-proteasomal system marker). By indirect enzyme-linked immunosorbent assay, phosphorylated p70 S6 kinase (Thr389 or Thr421/Ser424), total tau, and PHF-tau were found to be significantly increased in AD brain as compared to control cases. The levels of total p70 S6 kinase and p70 S6 kinase phosphorylated at Thr421/Ser424 showed significant correlations with the levels of both total tau and PHF-tau. Regression analyses revealed a significant dependence of total tau or PHF-tau on p70 S6 kinase phosphorylated at Thr421/Ser424 rather than at Thr389. The levels of ribosomal protein S6 as well as the levels of markers for the proteolytic system were also significantly increased in AD as compared to control brain. Using a SH-SY5Y neuroblastoma cell model, we found that 100 micro mol/L zinc sulfate could induce p70 S6 kinase phosphorylation and activation, in particular at Thr421/Ser424. This up-regulation of the activated kinase resulted in an increased expression and phosphorylation of tau. Pretreatment of cells with rapamycin (an inhibitor of FRAP/mTOR which is the immediate upstream kinase of the p70 S6 kinase) attenuated the effects induced by zinc. In primary cultured neurons of rat cortical cortex, zinc sulfate treatment could repeat p70 S6 kinase phosphorylation and activation at Thr421/Ser424, followed by increased expression and phosphorylation of tau. Taken together, these data suggest that activated p70 S6 kinase could mediate an up-regulation of tau translation. The partial co-localization of phosphorylated p70 S6 kinase with rab5, lamp-1 and ubiquitin, or PHF-tau with ubiquitin suggests that the activated proteolytic system might not be sufficient to degrade the over-produced and over-phosphorylated tau protein. A p70 S6 kinase modulated up-regulation of tau translation might contribute to PHF-tau accumulation in neurons with neurofibrillary changes.     

In vitro phosphorylation of SV40 large T antigen

Phosphorylation of simian virus 40 large T antigen (large T) was investigated in vitro. "Autophosphorylation" of large T resulted in the modification of Ser106, Ser112, Ser123, Thr124, either Ser676, Ser677, or Ser679, and Thr701. All of these residues were also found to be phosphorylated in vivo. Reaction of large T with purified casein kinase I resulted in phosphorylation of Ser123, possibly Thr124, and either Ser676, Ser677, or Ser679, while purified casein kinase II phosphorylated Ser106 and possibly Ser112. Submolar amounts of phosphate were transferred to large T indicating that only a fraction of large T served as substrate for the casein kinases. Removal of serine-bound phosphate did not affect the subsequent autophosphorylation or phosphorylation by casein kinase I and II. No phosphorylation at in vivo sites was observed with the cAMP-, cGMP-, or Ca2+/phospholipid-dependent protein kinases, or with the protease-activated kinase I and II.     

Effects of contraction and insulin on protein synthesis, AMP-activated protein kinase and phosphorylation state of translation factors in rat skeletal muscle

In rat epitrochlearis skeletal muscle, contraction inhibited the basal and insulin-stimulated rates of protein synthesis by 75 and 70%, respectively, while increasing adenosine monophosphate-activated protein kinase (AMPK) activity. Insulin, on the other hand, stimulated protein synthesis (by 30%) and increased p70 ribosomal protein S6 kinase (p70S6K) Thr389, 40S ribosomal protein S6 (rpS6) Ser235/236, rpS6 Ser240/244 and eukaryotic initiation factor-4E-binding protein-1 (4E-BP1) Thr37/46 phosphorylation over basal values. Electrical stimulation had no effect on mammalian target of rapamycin complex 1 (mTORC1) signalling, as reflected by the lack of reduction in basal levels of p70S6K, rpS6 Ser235/236, rpS6 Ser240/244 and 4E-BP1 phosphorylation, but did antagonize mTORC1 signalling after stimulation of the pathway by insulin. Eukaryotic elongation factor-2 (eEF2) Thr56 phosphorylation increased rapidly on electrical stimulation reaching a maximum at 1 min, whereas AMPK Thr172 phosphorylation slowly increased to reach threefold after 30 min. Eukaryotic elongation factor-2 kinase (eEF2K) was not activated after 30 min of contraction when AMPK was activated. This could not be explained by the expression of a tissue-specific isoform of eEF2K in skeletal muscle lacking the Ser398 AMPK phosphorylation site. Therefore, in this skeletal muscle system, the contraction-induced inhibition of protein synthesis could not be attributed to a reduction in mTORC1 signalling but could be due to an increase in eEF2 phosphorylation independent of AMPK activation.     

Estradiol prevents the injury-induced decrease of Akt/glycogen synthase kinase 3beta phosphorylation

Estradiol prevents neuronal cell death through the activation of cell survival signals and the inhibition of apoptotic signals. This study investigated whether estradiol modulates the anti-apoptotic signal through the phosphorylation of Akt and its downstream target, glycogen synthase kinase 3beta (GSK3beta). Adult female rats were ovariectomized and treated with estradiol prior to middle cerebral artery occlusion (MCAO). Brains were collected 24 h after MCAO and infarct volumes were analyzed. Estradiol administration significantly reduced infarct volume and decreased the positive cells of TUNEL staining in the cerebral cortex. Potential activation was measured by phosphorylation of Akt at Ser(473) and GSK3beta at Ser(9) using Western blot analysis and immunohistochemistry. Estradiol prevented the injury-induced decrease of pAkt and pGSK3beta. Furthermore, pretreatment with estradiol decreased glutamate toxicity-induced cell death in a hippocampal cell line (HT22). Also, estradiol prevented the glutamate toxicity-induced decrease of pAkt and pGSK3beta in HT22 cells. Our findings suggest that estradiol plays a potent protective role against brain injury and that phosphorylation of Akt and GSK3beta by estradiol mediated these protective effects.