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The molecular mechanisms underlying activity-dependent neural circuit growth and plasticity during early brain development remain poorly understood. Protein kinase Mζ (PKMz), an endogenous constitutively active kinase associated with late-phase long-term synaptic potentiation and memory in the mature brain, is expressed in the embryonic Xenopus retinotectal system with heightened levels during peak periods of dendrite growth and synaptogenesis. In vivo rapid time-lapse imaging of actively growing tectal neurons and comprehensive three-dimensional tracking of dynamic dendritic growth behavior finds that altered PKMz activity affects morphologic stabilization. Exogenous expression of PKMz within single neurons stabilizes dendritic filopodia by increasing dendritic filopodial lifetimes and decreasing filopodial additions, eliminations, and motility, whereas long-term in vivo imaging demonstrates restricted expansion of the dendritic arbor. Alternatively, blocking endogenous PKMz activity in individual growing tectal neurons with an inhibitory peptide (ζ-inhibitory peptide) destabilizes dendritic filopodia and over long periods promotes excessive arbor expansion. Furthermore, inhibiting endogenous PKMz throughout the tectum decreases colocalization of immunostained presynaptic and postsynaptic markers, SNAP-25 and PSD-95, respectively, suggesting impaired synapse maintenance. Together, these results implicate PKMz activity in restricting dendritic arborization during embryonic brain circuit development through synaptotropic stabilization of dynamic processes. [ABSTRACT FROM AUTHOR]

It is well documented that unliganded thyroid hormone receptor (TR) functions as a transcriptional repressor of specific cellular target genes by acting in concert with a corepressor complex harboring histone deacetylase (HDAC) activity. To fully explore the cofactors that interact with the transcriptionally repressive form of TR, we biochemically isolated a multiprotein complex that assembles on a TR-retinoid X receptor (RXR) heterodimer in HeLa nuclear extracts and identified its polypeptide components by mass spectrometry. A subset of TR-RXR-associated polypeptides included NCoR, SMRT, TBL1, and HDAC3, which represent the core components of a previously described NCoR/SMRT corepressor complex. We also identified several polypeptides that constitute a DNA-dependent protein kinase (DNA-PK) enzyme complex, a regulator of DNA repair, recombination, and transcription. These polypeptides included the catalytic subunit DNA-PKcs, the regulatory subunits Ku70 and Ku86, and the poly(ADP-ribose) polymerase 1. Density gradient fractionation and immunoprecipitation analyses provided evidence for the existence of a high molecular weight TR-RXR-corepressor holocomplex containing both NCoR/SMRT and DNA-PK complexes. Chromatin immunoprecipitation studies confirmed that unliganded TR-RXR recruits both complexes to the triiodothyronine-responsive region of growth hormone gene in vivo. Interestingly, DNA-PKcs, a member of the phosphatidylinositol 3-kinase family, was found to phosphorylate HDAC3 when the purified TR-RXR-corepressor holocomplex was incubated with ATP. This phosphorylation was accompanied by a significant enhancement of the HDAC activity of this complex. Collectively, our results indicated that DNA-PK promotes the establishment of a repressive chromatin at a TR target promoter by enhancing the HDAC activity of the receptor-bound NCoR/SMRT corepressor complex. [ABSTRACT FROM AUTHOR]

Activation of members of the protein kinase AGC (cAMP dependent, cGMP dependent, and protein kinase C) family is regulated primarily by phosphorylation at two sites: a conserved threonine residue in the activation loop and a serine/threonine residue in a hydrophobic motif (HM) near the COOH terminus. Although phosphorylation of these kinases in the activation loop has been found to be mediated by phosphoinositide-dependent protein kinase-1 (PDK1), the kinase(s) that catalyzes AGC kinase phosphorylation in the HM remains uncharacterized. So far, at least 10 kinases have been suggested to function as an HM kinase or the so-called ‘PDK2,’ including mitogen-activated protein (MAP) kinase-activated protein kinase-2 (MK2), integrin-linked kinase (ILK), p38 MAP kinase, protein kinase Cα (PKCα), PKCβ, the NIMA-related kinase-6 (NEK6), the mammalian target of rapamycin (mTOR), the double-stranded DNA-dependent protein kinase (DNK-PK), and the ataxia telangiectasia mutated (ATM) gene product. However, whether any or all of these kinases act as a physiological HM kinase remains to be established. Nonetheless, available data suggest that multiple systems may be used in cells to regulate the activation of the AGC family kinases. It is possible that, unlike activation loop phosphorylation, phosphorylation of the HM site in the different AGC family kinases is mediated by distinct kinases. In addition, phosphorylation of the AGC family kinase at the HM site could be cell type, signaling pathway, and substrate specific. Identification and characterization of the bonafide HM kinase(s) will be essential to verify these hypotheses. [ABSTRACT FROM AUTHOR]

Birukova, Anna A., Feng Liu, Joe G. N. Garcia, and Alexander D. Verin. Protein kinase A attenuates endothelial cell barrier dysfunction induced by microtubule disassembly. Am J Physiol Lung Cell Mol Physiol 287: L86-L93, 2004. First published March 5, 2004; 10.1152/ajplung.00441.2003.&ndsah;Cross between the actin cytoskeleton and the microyubule (MT) network plays a critical role in regulation of endothelial permeability. We have previously demonstrated that MT disruption by nocodazole results in increases in MLC phospholrylation, cardinal features of endothelial barrier dysfunction (Verin AD, Birukova A, Wang P, Liu F, Kaibuchi K, Ricks-cord A, Natarajan V, Alieva A, Garcia JG, and Verin AD. J Cell Physiol. In press.). Although activation of PKA opposes barrier-disrupting effects of edemagenic agents on confluent EC monolayers, information about the molecular mechanisms of PKA-mediated EC barrier protection is limited. Our results suggest that MT disassembly alters neither intracellular cAMP levels and PKA enzymatic activity; however, elevation of cAMP levels and PKA activation by either cholera toxin or forskolin dramatically attenuates the decline in transendothelial electrical resistance induced by nocodazole in human pulmonary EC. Barrier-protective effects of PKA on EC were associated with PKA-mediated inhibition of nocodazole-induced stress fiber formation, Rho activation, phosporylyzation of myosin phosphatase regulatory subunit at Thr696, and decreased MLC phosphorylation. In addition, forskolin pretreatment attenuated MT disassembly induced by nocodazole. These results suggest a critical role for PKA activity in stabilization of MT cytoskeleton and provide a novel mechanism for cAMP-mediated regu;ation of Rho-induced actin cytoskeletal remodeling, actomyosin contraction, and EC barrier dysfunction induced by MT disassembly. [ABSTRACT FROM AUTHOR]

Studies the regulation of cyclin-dependent kinase 5 (Cdk5) and casein kinase 1 (CK1) by metabotropic glutamate receptors (mGluRs). Effect of group 1 mGluR agonist on phosphorylation of DARPP-32 at Thr-75 and Ser-137; Effect of group 1 mGluR agonist on Cdk5 and CK1 activities; Effect of Cdk5 and CK1 inhibitors on phosphorylation of DARPP-32 at Thr-75 and Ser-137.

Investigates the impact of the cyclic adenylic acid-dependent protein kinase A (cAMP/PKA) inhibitors, Rp diastereomer of adenosine 3'-5'-cyclic monophosphorothioate and PKA inhibitor, on bovine pulmonary artery and lung microvascular endothelial cell cytoskeleton reorganization. Involvement of significant activity between cAMP- and extracellular signal-regulated kinase-dependent signaling pathways.

Focuses on a study which examined the role of the mitogen-activated protein kinase signaling cascade in phorbol ester-induced barrier dysfunction in bovine endothelium. Information on endothelial cell barrier dysfunction and electrocellular signal-regulated kinases; Accounts of related studies; Methodology; Analysis of the results.

Vascular smooth muscle (VSM) expresses calcium/calmodulin-dependent protein kinase II (CaMKII)-δ and -β isoforms. CaMKIIδ promotes VSM proliferation and vascular remodeling. We tested CaMKIIβ function in vascular remodeling after injury. CaMKIIβ protein decreased 90% 14 d after balloon injury in rat carotid artery. Intraluminal transduction of adenovirus encoding CaMKIIβC rescued expression to 35% of uninjured controls, inhibited neointima formation (>70%), inhibited VSM proliferation (>60%), and increased expression of the cell-cycle inhibitor p21 (>2-fold). Comparable doses of CaMKIIδ2 adenovirus had no effect. Similar dynamics in CaMKIIβ mRNA and protein expression were observed in ligated mouse carotid arteries, correlating closely with expression of VSM differentiation markers. Targeted deletion of CaMKIIβ in smooth muscle resulted in a 20-fold increase in neointimal area, with a 3-fold increase in the cell proliferation index, no change in apoptosis, and a 60% decrease in p21 expression. In cultured VSM, CaMKIIβ overexpression induced p53 mRNA (1.7 fold) and protein (1.8-fold) expression; induced the p53 target gene p21 (3-fold); decreased VSM cell proliferation (>50%); and had no effect on expression of apoptosis markers. We conclude that regulated CaMKII isoform composition is an important determinant of the injury-induced vasculoproliferative response and that CaMKIIβ and -δ isoforms have nonequivalent, opposing functions. [ABSTRACT FROM AUTHOR]

Background: Alzheimer's disease (AD) is the most general, chronic, and progressive neurodegenerative senile disorder characterized clinically by progressive cognitive deterioration and memory impairment. Neoline is effective against neuropathic pain models, but the effects of neoline against AD-like phenotypes have not been investigated.Objective: We offer the investigation of the effects of neoline in AD.Methods: In this study, a Tg-APPswe/PS1dE9 AD mouse model was treated orally with neoline at a concentration of 0.5 mg/kg or 0.1 mg/kg starting at 7.5 months and administered for three months, and its anti-AD effects were evaluated.Results: Neoline improved memory and cognition impairments and reduced the number of amyloid-beta plaque and the amount of amyloid-β in the brain of AD mice. Furthermore, neoline reduced the anxiety behavior in the AD mouse model. The chronic administration of neoline also induced AMPK phosphorylation and decreased tau, amyloid-β, and BACE1 expression in the hippocampus. These findings indicate that chronic administration of neoline has therapeutic effects via AMPK activation, and BACE1 downregulation resulted in a decrease in the amyloid-β levels in the brain of Tg-APPswe/PS1dE9 AD mice.Conclusion: Our results suggest that neoline is a therapeutic agent for the cure of neurodegenerative diseases like AD. [ABSTRACT FROM AUTHOR]

We have previously shown that thrombin induces endothelial cell barrier dysfunction via cytoskeleton activation and contraction and have determined the important role of endothelial cell myosin light chain kinase (MLCK) in this process. In the present study we explored p38 MAP kinase as a potentially important enzyme in thrombin-mediated endothelial cell contractile response and permeability. Thrombin induces significant p38 MAP kinase activation in a time-dependent manner with maximal effect at 30 min, which correlates with increased phosphorylation of actin- and myosin-binding protein, caldesmon. Both SB-203580 and dominant negative p38 adenoviral vector significantly attenuated thrombin-induced declines in transendothelial electrical resistance. Consistent with these data SB-203580 decreased actin stress fiber formation produced by thrombin in endothelium. In addition, dominant negative p38 had no effect on thrombin-induced myosin light chain diphosphorylation. Thrombin-induced total and site-specific caldesmon phosphorylation (Ser789) as well as dissociation of caldesmon-myosin complex were attenuated by SB-203580 pretreatment. These results suggest the involvement of p38 MAP kinase activities and caldesmon phosphorylation in the MLCK-independent regulation of thrombin-induced endothelial cell permeability. [ABSTRACT FROM AUTHOR]

The sphingolipid ceramide negatively regulates insulin action by inhibiting Akt/protein kinase B (PKB), a serine/threonine kinase that is a central regulator of glucose uptake and anabolic metabolism. Despite considerable attention, the molecular mechanism accounting for this action of ceramide has remained both elusive and controversial. Herein we utilized deletion constructs encoding two different functional domains of Akt/PKB to identify which region of the enzyme conferred responsiveness to ceramide. Surprisingly the findings obtained with these separate domains reveal that ceramide blocks insulin stimulation of Akt/PKB by two independent mechanisms. First, using the isolated pleckstrin homology domain, we found that ceramide specifically blocks the translocation of Akt/PKB, but not its upstream activator phosphoinositide-dependent kinase-1, to the plasma membrane. Second, using a construct lacking this pleckstrin homology domain, which does not require translocation for activation, we found that ceramide stimulates the dephosphorylation of Akt/PKB by protein phosphatase 2A. Collectively these findings identify at least two independent mechanisms by which excessive ceramide accumulation in peripheral tissues could contribute to the development of insulin resistance. Moreover the results obtained provide a unifying theory to account for the numerous dissenting reports investigating the actions of ceramide toward Akt/PKB. [ABSTRACT FROM AUTHOR]

Tests the hypothesis that pertussis toxin (PTX) directly activates endothelial cell p42/p44 mitogen-activated protein kinases via a novel signaling pathway. Adenosine 5'-diphosphate ribosylation of endothelial cell proteins; Effect of G protein inhibition on PTX-induced extracellular signal-regulated kinase activation.

Examines the activation of endothelial cell p42/p44 MAP kinases by pertussis toxin via a signaling pathway. Ability of pertussis toxin to ADP ribosylate specific G proteins; Disruption of monolayer integrity and and intercellular gap formation; Elimination of ADP ribosyltransferase activity.

The inhibitor kB protein kinase/nuclear factor kB (IKK/NF-kB) signaling pathway is critical for synaptic plasticity. However, the role of IKK/NF-kB in drug withdrawal-associated conditioned place aversion (CPA) memory is unknown. Here, we showed that inhibition of IKK/NF-kB by sulphasalazine (SSZ; 10 mM, i.c.v.) selectively blocked the extinction but not acquisition or expression of morphine-induced CPA in rats. The blockade of CPA extinction induced by SSZ was abolished by sodium butyrate, an inhibitor of histone deacetylase. Thus, the IKK/NF-kB signaling pathway might play a critical role in the extinction of morphine-induced CPA in rats and might be a potential pharmacotherapy target for opiate addiction. [ABSTRACT FROM AUTHOR]

The intense associative memories that develop between drug-paired contextual cues and rewarding stimuli or the drug withdrawal-associated aversive feeling have been suggested to contribute to the high rate of relapse. Various studies have elucidated the mechanisms underlying the formation and expression of drug-related cue memories, but how this mechanism is maintained is unknown. Protein kinase M ζ (PKMζ) was recently shown to be necessary and sufficient for long-term potentiation maintenance and memory storage. In the present study, we used conditioned place preference (CPP) and aversion (CPA) to examine whether PKMζ maintains both morphine-associated reward memory and morphine withdrawal-associated aversive memory in the basolateral amygdala (BLA). We also investigate the role of PKMζ in the infralimbic cortex in the extinction memory of morphine reward-related cues and morphine withdrawal-related aversive cues. We found that intra-BLA but not central nucleus of the amygdala injection of the selective PKMζ inhibitor ZIP 1 day after CPP and CPA training impaired the expression of CPP and CPA 1 day later, and the effect of ZIP on memory lasted at least 2 weeks. Inhibiting PKMζ activity in the infralimbic cortex, but not prelimbic cortex, disrupted the expression of the extinction memory of CPP and CPA. These results indicate that PKMζ in the BLA is required for the maintenance of associative morphine reward memory and morphine withdrawal-associated aversion memory, and PKMζ in the infralimbic cortex is required for the maintenance of extinction memory of morphine reward-related cues and morphine withdrawal-related aversive cues. [ABSTRACT FROM AUTHOR]

The natural polyphenol resveratrol (RSV) displays a wide spectrum of health beneficial activities, yet the precise mechanisms remain to be fully elucidated. Here we show that RSV promotes the multimerization and cellular levels of adiponectin in 3T3-L1 adipocytes. The stimulatory effect of RSV was not affected by knocking out Sirt1, but was diminished by suppressing the expression levels of DsbA-L, a recently identified adiponectin-interactive protein that promotes adiponectin multimerization. Suppression of the Akt signaling pathway resulted in an increase in the expression levels of DsbA-L and adiponectin. On the other hand, knocking out FOXO1 or suppressing the activity or expression levels of the AMP-activated protein kinase (AMPK) down-regulated DsbA-L and adiponectin. The stimulatory effect of RSV on adiponectin and DsbA-L expression was completely diminished in FOXO1-suppressed and AMPK-inactivated 3T3-L1 adipocytes. Taken together, our results demonstrate that RSV promotes adiponectin multimerization in 3T3-L1 adipocytes via a Sirt1-independent mechanism. In addition, we show that the stimulatory effect of RSV is regulated by both the Akt/FOXO1 and the AMPK signaling pathways. Last, we show that DsbA-L plays a critical role in the promoting effect of RSV on adiponectin multimerization and cellular levels. [ABSTRACT FROM AUTHOR]

The adaptor protein APPL1 mediates the stimulatory effect of adiponectin on p38 mitogen-activated protein kinase (MAPK) signaling, yet the underlying mechanism remains unclear. Here we show that, in C2C12 cells, overexpression or suppression of APPL1 enhanced or suppressed, respectively, adiponectin-stimulated p38 MAPK upstream kinase cascade, consisting of transforming growth factor-β-activated kinase 1 (TAK1) and mitogen-activated protein kinase kinase 3 (MKK3). In vitro affinity binding and coimmunoprecipitation experiments revealed that TAK1 and MKK3 bind to different regions of APPL1, suggesting that APPL1 functions as a scaffolding protein to facilitate adiponectin-stimulated p38 MAPK activation. Interestingly, suppressing APPL1 had no effect on TNFα-stimulated p38 MAPK phosphorylation in C2C12 myotubes, indicating that the stimulatory effect of APPL1 on p38 MAPK activation is selective. Taken together, our study demonstrated that the TAK1-MKK3 cascade mediates adiponectin signaling and uncovers a scaffolding role of APPL1 in regulating the TAK1-MKK3-p38 MAPK pathway, specifically in response to adiponectin stimulation. [ABSTRACT FROM AUTHOR]

Cocaine use and relapse involves learned associations between cocaine-associated environmental contexts and discrete stimuli and cocaine effects. Initially, these contextual and discrete cues undergo memory consolidation after being paired with cocaine exposure. During abstinence, cocaine cue memories can under go memory reconsolidation after cue exposure without the drug. We used a conditioned place preference (CPP) procedure in rats to study the role of neuronal protein kinase cyclin-dependent kinase 5 (Cdk5) in consolidation and reconsolidation of cocaine cue memories. We found that the expression of cocaine CPP in drug-free tests 1 d after CPP training (four pairings of 10 mg/kg cocaine with one context and four pairings of saline with a different context) increased Cdk5 activity, and levels of the Cdk5 activator p35 in basolateral but not central amygdala. We also found that basolateral (but not central) amygdala injections of the Cdk5 inhibitor β-butyrolactone (100 ng/side) immediately (but not 6 h) after cocaine-context pairings during training prevented subsequent cocaine CPP expression. After training, acute basolateral (but not central) amygdala β-butyrolactone injections immediately before testing prevented the expression of cocaine CPP; this effect was also observed on a second test performed 1 d later, suggesting an effect on reconsolidation of cocaine cue memories. In support, basolateral β-butyrolactone injections, given immediately (but not 6 h) after a single exposure to the cocaine-paired context, prevented cocaine CPP expression 1 and 14 d after the injections. Results indicate that basolateral amygdala Cdk5 activity is critical for consolidation and reconsolidation of the memories of cocaine-associated environmental cues. [ABSTRACT FROM AUTHOR]

Ethylene receptor is the first component of ethylene signaling that regulates plant growth, development and stress responses. Previously, we have demonstrated that tobacco subfamily 2 ethylene receptor NTHK1 had Ser/Thr kinase activity, and overexpression of NTHK1 caused large rosette, reduced ethylene sensitivity, and increased salt sensitivity in transgenic Arabidopsis plants. Here we found that N-box mutation in the NTHK1 kinase domain abolished the kinase activity and led to disruption of NTHK1 roles in conferring reduced ethylene sensitivity and salt sensitive response in transgenic Arabidopsis plants. However, N-box mutation had partial effects on NTHK1 regulation of rosette growth and expression of salt- and ethylene-responsive genes AtNAC2, AtERF1 and AtCor6.6. Mutation of conserved residues in the H box did not affect kinase activity, seedling growth, ethylene sensitivity or salt-induced epinasty in transgenic plants but did influence NTHK1 function in control of specific salt- and ethylene-responsive gene expression. Compared with NTHK1, the tobacco subfamily 1 ethylene receptor NtETR1 had His kinase activity and played a weak role in regulation of rosette growth, triple response and salt response. Mutation of the conserved His residue in the NtETR1 H box eliminated phosphorylation and altered the effect of Ntetr1-1 on reporter gene activity. These results imply that the Ser/Thr kinase activity of NTHK1 is differentially required for various responses, and NTHK1 plays a larger role than NtETR1. [ABSTRACT FROM PUBLISHER]

3-Phosphoinositide-dependent protein kinase-1 (PDK1) mediates phosphorylation and activation of members of the AGC protein kinase family and plays an essential role in insulin signaling and action. However, whether and how PDK1 activity is regulated in cells remains largely uncharacterized. In the present study, we show that PDK1 undergoes insulin-stimulated and phosphatidylinositol 3-kinase-dependent phosphorylation at Ser244 in the activation loop and at a novel site: Ser163 in the hinge region between the two lobes of the kinase domain. Sequence alignment studies revealed that the residue corresponding to Ser163 of PDK1 in all other AGC kinases is glutamate, suggesting that a negative charge at this site may be important for PDK1 function. Replacing Ser163 with a negatively charged residue, glutamate, led to a 2-fold increase in PDK1 activity. Molecular modeling studies suggested that phosphorylated Ser163 may form additional hydrogen bonds with Tyr149 and Gln223. In support of this, mutation of Tyr149 to Ala is sufficient to reduce PDK1 activity. Taken together, our results suggest that PDK1 phosphorylation of Ser163 may provide a mechanism to fine-tune PDK1 activity and function in cells. [ABSTRACT FROM AUTHOR]

Lung endothelial barrier function is regulated by multiple signaling pathways, including mitogen-activated protein kinases (MAPK) extracellular signal-regulated kinases (ERK) ½ and p38. We have recently shown involvement of microtubule (MT) disassembly in endothelial cell (EC) barrier failure. In this study, we examined potential involvement of ERK½ and p38 MAPK in lung EC barrier dysfunction associated with MT disassembly. MT inhibitors nocodazole (0.2 µM) and vinblastine (0.1 µM) induced sustained activation of Ras-RafMEK½-ERK½ and MKK3/6-p38-MAPKAPK2 MAPK cascades in human and bovine pulmonary EC, as detected by phosphospecific antibodies and in MAPK activation assays. These effects were linked to increased permeability assessed by measurements of transendothelial electrical resistance and cytoskeletal remodeling analyzed by morphometric analysis of EC monolayers. MT stabilization by taxol (5 µM, 1 h) attenuated nocodazole-induced ERK½ and p38 MAPK activation and phosphorylation of p38 MAPK substrate 27-kDa heat shock protein and regulatory myosin light chains, the proteins involved in actin polymerization and actomyosin contraction. Importantly, only pharmacological inhibition of p38 MAPK by SB-203580 (20 µM, 1 h) attenuated nocodazole-induced MT depolymerization, actin remodeling, and EC barrier dysfunction, whereas the MEK/ ERK½ inhibitor U0126 (5 µM, 1 h) exhibited no effect. These data suggest a direct link between p38 MAPK activation, remodeling of MT network, and EC barrier regulation. [ABSTRACT FROM AUTHOR]

Src tyrosine kinase suppresses KCNQ (M-type) K+ channels in a subunit-specific manner representing a mode of modulation distinct from that involving G protein-coupled receptors. We probed the molecular and biophysical mechanisms of this modulation using mutagenesis, biochemistry, and both whole-cell and single channel modes of patch clamp recording. Immunoprecipitation assays showed that Src associates with KCNQ2-5 subunits but phosphorylates only KCNQ3-5. Using KCNQ3 as a background, we found that mutation of a tyrosine in the amino terminus (Tyr-67) or one in the carboxyl terminus (Tyr-349) abolished Src-dependent modulation of heterologously expressed KCNQ2/3 heteromultimers. The tyrosine phosphorylation was much weaker for either the KCNQ3-Y67F or KCNQ3-Y349F mutants and wholly absent in the KCNQ3-Y67F/Y349F double mutant. Biotinylation assays showed that Src activity does not alter the membrane abundance of channels in the plasma membrane. In recordings from cell-attached patches containing a single KCNQ2/3 channel, we found that Src inhibits the open probability of the channels. Kinetic analysis was consistent with the channels having two discrete open times and three closed times. Src activity reduced the durations of the longest open time and lengthened the longest closed time of the channels. The implications for the mechanisms of channel regulation by the dual phosphorylations on both channel termini are discussed. [ABSTRACT FROM AUTHOR]

Activation of mouse 3-phosphoinositide-dependent protein kinase-1 (mPDK1) requires phosphorylation at a conserved serine residue, Ser[sup 244], in the activation loop. However, the mechanism by which mPDK1 is phosphorylated at this site remains unclear. We have found that kinase-defective mPDK1 (mPDK1[sup KD]), but not a kinasedefective mPDK1 in which Ser[sup 244] was replaced with alanine (mPDK1[sup KD/S244A]), is significantly phosphorylated in intact cells and is a direct substrate of wild-type mPDK1 fused to the yellow fluorescence protein. Phosphoamino acid analysis and phosphopeptide mapping studies revealed that mPDK1 trans-autophosphorylation occurred mainly on Ser[sup 244]. On the other hand, Ser[sup 399] and Thr[sup 516], two recently identified autophosphorylation sites of mPDK1, are phosphorylated primarily through a cis mechanism. In vivo labeling studies revealed that insulin stimulated both mPDK1[sup KD] and mPDK1[sup KD/S244A] phosphorylation in Chinese hamster ovary cells overexpressing the insulin receptor. However, Western blot analysis using a phosphospecific antibody revealed no increase in insulin-stimulated phosphorylation of Ser[sup 244] in these cells overexpressing mPDK1, mPDK1 undergoes dimerization in cells and this self-association is enhanced by kinase inactivation. Deletion of the extreme C terminus disrupts mPDK1 dimerization and Ser[sup 244] trans-phosphorylation, suggesting that dimerization is important for mPDK1 transphosphorylation. Taken together, our results show that mPDK1 autophosphorylation occurs at multiple sites through both cis and trans mechanisms and suggest that dimerization and trans-phosphorylation may serve as mechanisms to regulate PDK1 activity in cells. [ABSTRACT FROM AUTHOR]

AMP-activated kinase (AMPK) is a fuel-sensing enzyme present in most mammalian tissue. In response to a decrease in the energy state of a cell AMPK is phosphorylated and activated by still poorly characterized upstream events. Exposure of bovine aortic endothelial cells (BAEC) to chemically synthesized ONOO[sup -] acutely and significantly increased phosphorylation of c-Src, PDK1, AMPK, and its downstream target, acetyl-CoA carboxylase (ACC), without affecting cellular AMP. This novel pathway for AMPK activation was confirmed by the use of pharmacological inhibitors and dominant-negative mutants. Exposure of BAEC to hypoxia-reoxygenation (H/R) caused a biphasic increase in AMPK and ACC phosphorylation, which was prevented by adenoviral overexpression of superoxide dismutase (SOD) or inhibition of nitric-oxide synthase (NOS) implicating a role of ONOO[sup -] formed during H/R. Furthermore, dominant-negative mutants of c-Src or kinase-defective PDK1 also blocked H/R-induced AMPK activation indicating that, as with addition of exogenous ONOO[sup -], both c-Src and PI 3-kinase are upstream of AMPK. Moreover, H/R, like ONOO[sup -], significantly increased co-immunoprecipitation of AMPK with c-Src, suggesting that ONOO[sup -] favors physical association of AMPK with upstream kinases. Taken together, our results indicate a novel pathway by which H/R via ONOO[sup -] activates AMPK in a c-Src-mediated, PI 3-kinase-dependent mannet, and suggest that ONOO[sup -]-induced activation of AMPK might thereby regulate metabolic enzymes, such as ACC. [ABSTRACT FROM AUTHOR]

Examines the differential effect of myosin light chain (MLC) kinase in tumor necrosis factor (TNF)-alpha-induced endothelial cell apoptosis and barrier dysfunction. Description of the role played by the endothelial cell cytoskeleton in TNF-alpha-mediated apoptosis; Indication that the evolution of TNF-alpha-induced vascular permeability is independent of contractile tension generation.

Presents information on a study which examined whether calmodulin-dependent protein (CaM) kinase II activities are involved in thrombin-induced endothelial cell barrier dysfunction. Potential of filamin as a cytoskeletal target for CaM kinase II; Materials and methods used; Results and conclusions.

Reports the enhancement of endothelial cell barrier function through calcium and calmodulin-dependent protein kinase II. Association between cytoskeletal protein reorganization with endothelial cell barrier dysfunction; Elaboration on the importance of thrombin.

Studies the regulation of endothelial cell barrier function by calcium/calmodulin (CaM)-dependent protein kinase II in bovine pulmonary artery. Thrombin-induced CaM kinase II activation; Effect of thrombin-induced filamin phosphorylation and translocation on transendothelial electrical resistance.

Reports on the potential role of the phosphorylation of protein kinase N (PKN) by phosphoinositide-dependent protein kinase-1 (PDK1) in mediating insulin signaling to the actin cytoskeleton. Role of actin cytoskeleton in a variety of cellular functions; Interaction of PDK1 and PKN in mammalian cells; Effects of PDK1 and PKN on actin cytoskeleton reorganization.

Activation of the AMP-activated protein kinase (AMPK) mediates adiponectin signalling to regulate glucose and lipid metabolism in muscle. However, the molecular mechanism by which adiponectin activates AMPK remains largely unknown. We have recently found that the BAR, PH, and PTB domain-containing adaptor protein APPL1 binds directly to the intracellular part of adiponectin receptors and mediates adiponectin-stimulated AMPK activation (Mao et al. (2006) Nat Cell Biol. 8,516-523). Here we report that APPL1 directly binds to LKB1, an upstream kinase of AMPK, via the BAR domain. Under basal condition, LKBI is localized predominantly in the nucleus of C2C12 cells. Treating cells with either adiponectin or metformin significantly stimulates LKB1 translocation to the cytosol. Overexpression of LKB1SL-26, a mutant form of LKB1 which maintains intact kinase activity but localizes exclusively in the nucleus, inhibited adiponectin and metformin-stimulated AMPK activation in C2C12 cells. The regulation of LKB1 translocation is dependent on phosphorylation at Ser307, which is mediated by protein kinase Cz (PKCz). Taken together, our results reveal a novel mechanism by which adiponectin and metformin stimulate LKB1 cytosolic translocation, which is essential for AMPK activation in muscle cells. ADA-Funded Research [ABSTRACT FROM AUTHOR]