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Hepatocellular carcinoma (HCC) Is frequently associated with pathogen Infection-Induced chronic Inflammation. Large numbers of innate immune cells are present in HCCs and can influence disease outcome. Here, we demonstrated that the tumor suppressor serine/threonine-protein kinase 4 (STK4) differentially regulates TLR3/4/9-mediated inflammatory responses in macrophages and thereby is protective against chronic inflammation-associated HCC. STK4 dampened TLR4/9-induced proinflammatory cytokine secretion but enhanced TLR3/4-triggered IFN-β production via binding to and phosphorylating IL-1 receptor-associated kinase 1 (IRAK1), leading to IRAK1 degradation. Notably, macrophage-specific Stk4 deletion resulted in chronic inflammation, liver fibrosis, and HCC in mice treated with a combination of diethylnitrosamine (DEN) and C[Cl.sub.4], along with either LPS or E. coli infection. STK4 expression was markedly reduced in macrophages isolated from human HCC patients and was inversely associated with the levels of IRAK1, IL-6, and phospho-p65 or phospho-STAT3. Moreover, serum STK4 levels were specifically decreased in HCC patients with high levels of IL-6. In STK4-deficient mice, treatment with an IRAK1/4 inhibitor after DEN administration reduced serum IL-6 levels and liver tumor numbers to levels similar to those observed in the control mice. Together, our results suggest that STK4 has potential as a diagnostic biomarker and therapeutic target for inflammation-induced HCC., Introduction The persistent presence and amplification of inflammation can predispose cells to oncogenic transformation and contribute to tumor development. Up to 25% of cancers are linked to the persistent presence [...]

Tissue inflammation in several autoimmune diseases, including SLE and MS, has been linked to an imbalance of IL-17-producing Th (Th17) cells and Tregs; however, the factors that promote Th17-driven autoimmunity are unclear. Here, we present evidence that the calcium/calmodulin-dependent protein kinase IV (CaMK4) is increased and required during Th17 cell differentiation. Isolation of naive T cells from a murine model of lupus revealed increased levels of CaMK4 following stimulation with Th17-inducing cytokines but not following Treg, Th1, or Th2 induction. Furthermore, naive T cells from mice lacking CaMK4 did not produce IL-17. Genetic or pharmacologic inhibition of CaMK4 decreased the frequency of IL-17-producing T cells and ameliorated EAE and lupus-like disease in murine models. Inhibition of CaMK4 reduced Il17 transcription through decreased activation of the cAMP response element modulator α (CREM-α) and reduced activation of the AKT/mTOR pathway, which is known to enhance Th17 differentiation. Importantly, silencing CaMK4 in T cells from patients with SLE and healthy individuals inhibited Th17 differentiation through reduction of IL17A and IL17FmRNA. Collectively, our results suggest that CaMK4 inhibition has potential as a therapeutic strategy for Th17-driven autoimmune diseases., Introduction IL-17-producing [CD4.sup.+] Th 17 (Th17) cells are defined by specific developmental and functional features that are distinct from those of 'classical' Th1 and Th2 cells (1, 2). Th17 cells [...]

Genome-wide analyses determined previously that the receptor tyrosine kinase (RTK) EPHA2 is commonly overexpressed in non-small cell lung cancers (NSCLCs). EPHA2 overexpression is associated with poor clinical outcomes; therefore, EPHA2 may represent a promising therapeutic target for patients with NSCLC. In support of this hypothesis, here we have shown that targeted disruption of EphA2 in a murine model of aggressive Kras-mutant NSCLC impairs tumor growth. Knockdown of EPHA2 in human NSCLC cell lines reduced cell growth and viability, confirming the epithelial cell autonomous requirements for EPHA2 in NSCLCs. Targeting EPHA2 in NSCLCs decreased S6K1-mediated phosphorylation of cell death agonist BAD and induced apoptosis. Induction of EPHA2 knockdown within established NSCLC tumors in a subcutaneous murine model reduced tumor volume and induced tumor cell death. Furthermore, an ATP-competitive EPHA2 RTK inhibitor, ALW-II-41-27, reduced the number of viable NSCLC cells in a time-dependent and dose-dependent manner in vitro and induced tumor regression in human NSCLC xenografts in vivo. Collectively, these data demonstrate a role for EPHA2 in the maintenance and progression of NSCLCs and provide evidence that ALW-II-41-27 effectively inhibits EPHA2-mediated tumor growth in preclinical models of NSCLC., Introduction Genome-wide expression analyses of human lung cancer have identified a number of receptor tyrosine kinases (RTKs) as overexpressed and potentially representing drivers of non-small cell lung cancer (NSCLC) (1-4). [...]

Activation of self-reactive T cells and their trafficking to target tissues leads to autoimmune organ destruction. Mice lacking the co-inhibitory receptor cytotoxic T lymphocyte antigen-4 (CTLA-4) develop fatal autoimmunity characterized [...]

[T.sub.H]17 cells (interleukin-17 (IL-17)-producing helper T cells) are highly proinflammatory cells that are critical for clearing extracellular pathogens and for inducing multiple autoimmune diseases (1). IL-23 has a critical role [...]

Myocardial cell death is initiated by excessive mitochondrial [Ca.sup.2+] entry causing [Ca.sup.2+] overload, mitochondrial permeability transition pore (mPTP) opening and dissipation of the mitochondrial inner membrane potential (ΔΨm) (1,2). However, [...]

Overcoming metabolic stress is a critical step for solid tumour growth (1,2). However, the underlying mechanisms of cell death and survival under metabolic stress are not well understood. A key signalling pathway involved in metabolic adaptation is the liver kinase B1 (LKB1)-AMP-activated protein kinase (AMPK) pathway (2,3). Energy stress conditions that decrease intracellular ATP levels below a certain level promote AMPK activation by LKB1. Previous studies showed that LKB1-deficient or AMPK-deficient cells are resistant to oncogenic transformation and tumorigenesis (4-6), possibly because of the function of AMPK in metabolic adaptation. However, the mechanisms by which AMPK promotes metabolic adaptation in tumour cells are not fully understood. Here we show that AMPK activation, during energy stress, prolongs cell survival by redox regulation. Under these conditions, NADPH generation by the pentose phosphate pathway is impaired, but AMPK induces alternative routes to maintain NADPH and inhibit cell death. The inhibition of the acetyl-CoA carboxylases ACC1 and ACC2 by AMPK maintains NADPH levels by decreasing NADPH consumption in fatty-acid synthesis and increasing NADPH generation by means of fatty-acid oxidation. Knockdown of either ACC1 or ACC2 compensates for AMPK activation and facilitates anchorage-independent growth and solid tumour formation invivo, whereas the activation of ACC1 or ACC2 attenuates these processes. Thus AMPK, in addition to its function in ATP homeostasis, has a key function in NADPH maintenance, which is critical for cancer cell survival under energy stress conditions, such as glucose limitations, anchorage-independent growth and solid tumour formation in vivo., The lack of LKB1 or AMPK activation rendered cancer cells more sensitive to cell death induced by glucose deprivation (5,7-9) (Supplementary Fig. 1). However, the mechanism by which the failure [...]

Mechanical hyperalgesia is a common and potentially disabling complication of many inflammatory and neuropathic conditions. Activation of the enzyme PKC ζ in primary afferent nociceptors is a major mechanism that underlies mechanical hyperalgesia, but the PKC ζ substrates involved downstream are not known. Here, we report that in a proteomic screen we identified the [Na.sub.V] 1.8 sodium channel, which is selectively expressed in nociceptors, as a PKC ζ substrate. PKC ζ -mediated phosphorylation increased [Na.sub.V] 1.8 currents, lowered the threshold voltage for activation, and produced a depolarizing shift in inactivation in wild-type--but not in PKC ζ -null - sensory neurons. PKC ζ phosphorylated NaV1.8 at S1452, and alanine substitution at this site blocked PKC ζ modulation of channel properties. Moreover, a specific PKC ζ activator peptide, ψ ζ RACK, produced mechanical hyperalgesia in wild-type mice but not in Scn10a-/- mice, which lack NaV1.8 channels. These studies demonstrate that [Na.sub.V] 1.8 is an important, direct substrate of PKC ζ that mediates PKC ζ -dependent mechanical hyperalgesia., Introduction Tissue damage, inflammation, and neuropathic disorders often produce hyperalgesia, a state of increased sensitivity to painful stimuli. Sensitization of primary afferent nociceptors by inflammatory mediators or by nerve damage [...]

Blood pressure regulation is crucial for the maintenance of health, and hypertension is a risk factor for myocardial infarction, heart failure, stroke and renal disease. Nitric oxide (NO) and prostacyclin [...]

doi: 10.1016/j.phymed.2011.03.006 ABSTRACT Danshen-Gegen (DG) decoction, an herbal formulation comprising Radix Salvia Miltiorrhiza and Radix Puerariae Lobatae, is prescribed for the treatment of coronary heart disease in Chinese medicine. Experimental [...]

How the biochemical reactions that lead to the repair of DNA damage are controlled by the diffusion and availability of protein reactants within the nucleoplasm is poorly understood. Here, we use gene targeting to replace Brca2 (a cancer suppressor protein essential for DNA repair) with a functional enhanced green fluorescent protein (EGFP)-tagged form, followed by fluorescence correlation spectroscopy to measure Brca2-EGFP diffusion in the nucleoplasm of living cells exposed to DNA breakage. Before damage, nucleoplasmic Brca2 molecules exhibit complex states of mobility, with long dwell times within a sub-fL observation volume, indicative of restricted motion. DNA damage significantly enhances the mobility of Brca2 molecules in the S/G2 phases of the cell cycle, via signaling through damage-activated protein kinases. Brca2 mobilization is accompanied by increased binding within the nucleoplasm to its cargo, the Rad51 recombinase, measured by fluorescence cross-correlation spectroscopy. Together, these results suggest that DNA breakage triggers the redistribution of soluble nucleoplasmic Brca2 molecules from a state of restricted diffusion, into a mobile fraction available for Rad51 binding. Our findings identify signal-regulated changes in nucleoplasmic protein diffusion as a means to control biochemical reactions in the cell nucleus. DNA damage response I protein dynamics I fluorescence spectroscopy I single-molecule imaging doi: 1073/pnas.1009577107

Apoptosis has been shown to be a significant form of cell loss in many diseases. Detachment of photoreceptors from the retinal pigment epithelium, as seen in various retinal disorders, causes photoreceptor loss and subsequent vision decline. Although caspase-dependent apoptotic pathways are activated after retinal detachment, caspase inhibition by the pan-caspase inhibitor Z-VAD fails to prevent photoreceptor death; thus, we investigated other pathways leading to cell loss. Here, we show that receptor interacting protein (RIP) kinase-mediated necrosis is a significant mode of photoreceptor cell loss in an experimental model of retinal detachment and when caspases are inhibited, RIP-mediated necrosis becomes the predominant form of death. RIP3 expression, a key activator of RIP1 kinase, increased more than 10-fold after retinal detachment. Morphological assessment of detached retinas treated with Z-VAD showed decreased apoptosis but significantly increased necrotic photoreceptor death. RIP1 kinase inhibitor necrostatin-1 or Rip3 deficiency substantially prevented those necrotic changes and reduced oxidative stress and mitochondrial release of apoptosis-inducing factor. Thus, RIP kinase-mediated programmed necrosis is a redundant mechanism of photoreceptor death in addition to apoptosis, and simultaneous inhibition of RIP kinases and caspases is essential for effective neuroprotection and may be a novel therapeutic strategy for treatment of retinal disorders. degenerations | necroptosis doi/ 10.1073/pnas.1009179107

Serum and glucocorticoid-regulated kinase 2 (sgk2) is 80% identical to the kinase domain of sgk1, an important mediator of mineralocorticoid-regulated sodium ([Na.sup.+]) transport in the distal nephron of the kidney. The expression pattern and role in renal function of sgk2 are virtually uncharacterized. In situ hybridization and immunohistochemistry of rodent kidney coupled with real-time RT-PCR of microdissected rat kidney tubules showed robust sgk2 expression in the proximal straight tubule and thick ascending limb of the loop of Henle. Sgk2 expression was minimal in distal tubule cells with aquaporin-2 immunostaining but significant in proximal tubule cells with [Na.sup.+]/[H.sup.+] exchanger 3 (NHE3) immunostaining. To ascertain whether mineralocorticoids regulate expression of sgk2 in a manner similar to sgkl, we examined sgk2 mRNA expression in the kidneys of adrenalectomized rats treated with physiological doses of aldosterone together with the glucocorticoid receptor antagonist RU486. Northern blot analysis and in situ hybridization showed that, unlike sgkl, sgk2 expression in the kidney was not altered by aldosterone treatment. Based on the observation that sgk2 is expressed in proximal tubule cells that also express NHE3, we asked whether sgk2 regulates NHE3 activity. We heterologously expressed sgk2 in opossum kidney (OKP) cells and measured [Na.sup.+]/[H.sup.+] exchange activity by Na[+ or -]dependent cell pH recovery. Constitutively active sgk2, but not sgkl, stimulated [Na.sup.+]/[H.sup.+] exchange activity by >30%. Moreover, the sgk2-mediated increase in Na+/H+ exchange activity correlated with an increase in cell surface expression of NHE3. Together, these results suggest that the pattern of expression, regulation, and role of sgk2 within the mammalian kidney are distinct from sgkl and that sgk2 may play a previously unrecognized role in the control of transtubular [Na.sup.+] transport through NHE3 in the proximal tubule. sgk; OKP; kidney tubule; sodium/hydrogen exchanger doi: 10.1152/ajprenal.00075.2010

Hypertonicity increases urea transport independently of, as well as synergistically with, vasopressin in the inner medullary collect duct (IMCD). We previously showed that hypertonicity does not increase the level of cAMP in the IMCD, but it does increase the level of intracellular calcium. Since we also showed that hypertonicity increases both the phosphorylation and biotinylation of the urea transporters UT-A1 and UT-A3, this would suggest involvement of a calcium-dependent protein kinase in the regulation of urea transport in the inner medulla. In this study, we investigated whether protein kinase C (PKC), which is present in the IMCD, is a regulator of urea permeability. We tested the effect of PKC inhibitors and activators on urea permeability in the isolated, perfused rat terminal IMCD. Increasing osmolality from 290 to 690 mosmol/kg[H.sub.2]O significantly stimulated (doubled) urea permeability; it returned to control levels on inhibition of PKC with either 10 [micro]M chelerythrine or 50 [micro]M rottlerin. To determine the potential synergy between vasopressin and PKC, phorbol dibutyrate (PDBu) was used to stimulate PKC. Vasopressin stimulated urea permeability 247%. Although PDBu alone did not change basal urea permeability, in the presence of vasopressin, it significantly increased urea permeability an additional 92%. The vasopressin and PDBu-stimulated urea permeability was reduced to AVP alone levels by inhibition of PKC. We conclude that hypertonicity stimulates urea transport through a PKC-mediated phosphorylation. Whether PKC directly phosphorylates UT-A1 and/or UT-A3 or phosphorylates it as a consequence of a cascade of activations remains to be determined. tubule perfusion; calcium signaling; urine concentration; vasopressin; hypertonicity doi: 10.1152/ajprenal.00322.2010

Salt reabsorption by the loop of Henle controls NaC1 handling and blood pressure regulation. Increased oxidative stress stimulates NaC1 transport in one specific segment of the loop of Henle called the thick ascending limb (TAL). The isoprostane 8-iso-prostaglandin-F2[alpha] (8iso-PGF2[alpha]) is one of the most abundant nonenzymatic lipid oxidation products and has been implicated in the development of hypertension. However, it is not known whether 8-iso-PGF2[alpha] regulates transport or the mechanisms involved. Because protein kinase A (PKA) stimulates NaCI transport in several nephron segments, we hypothesized that 8-iso-PGF2[alpha] increases NaC1 transport in the cortical TAL (cTAL) via a PKA-dependent mechanism. We examined the effect of luminal 8-iso-PGF2[alpha] on NaC1 transport by measuring chloride absorption ([J.sub.C1]) in isolated microperfused cTALs. Adding 8-iso-PGF2[alpha] to the lumen increased [J.sub.CI] by 54% (from 288.7 [+ or -] 30.6 to 446.5 [+ or -] 44.3 pmol x [min.sup.-1] x [mm.sup.-1]; P < 0.01), while adding it to the bath enhanced [J.sub.C1] by 35% (from 236.3 [+ or -] 35.3 to 319.2 [+ or -] 39.8 pmol x [min.sup.-1] x [mm.sup.-1]; P < 0.05). This stimulation was blocked by Na-K-2C1 cotransporter inhibition. Next, we tested the role of cAMP. Basal cAMP in the cTAL was 18.6 [+ or -] 1.6 fmol x [min.sup.-1] x [mm.sup.-1], and 8-iso-PGF2[alpha] raised it to 35.1 [+ or -] 1.4 fmol x [min.sup.-1] x [mm.sup.-1], an increase of 94% (P < 0.01). Because cAMP stimulates PKA, we measured [J.sub.C1] using the PKA-selective inhibitor H89. In the presence of H89 (10 [micro]M), 8-iso-PGF2[alpha] failed to increase transport regardless of whether it was added to the lumen (216.1 [+ or -] 16.7 vs. 209.7 [+ or -] 23.8 pmol x [min.sup.-1] x [mm.sup.-1]; NS) or the bath (150.4 [+ or -] 32.9 vs. 127.1 [+ or -] 28.6 pmol x [min.sup.-1] x [mm.sup.-1]; NS). We concluded that 8-iso-PGF2[alpha] stimulates cAMP and increases C1 transport in cTALs via a PKA-dependent mechanism. salt reabsorption; cortico-medullary osmotic gradient doi: 10.1152/ajprenal.00225.2010

Acid-sensing ion channels (ASICs) extensively exist in both central and peripheral neuronal systems and contribute to many physiological and pathological processes. The protein that interacts with C kinase 1 (PICK1) was cloned as one of the proteins interacting with protein kinase C (PKC) and colocalized with ASIC1 and ASIC2. Here, we used PICK1 knockout (PICK1-KO) C57/BL6 mice together with the whole cell patch clamp, calcium imaging, RT-PCR, Western blot, and immunocytochemistry techniques to explore the possible change in ASICs and the regulatory effects of PKC on ASICs. The results showed that PICK1 played a key role in regulation of ASIC functions. In PICK1-KO mouse cortical neurons, both the amplitude of ASIC currents and elevation of [[[Ca.sup.2+]].sub.i] mediated by acid were decreased, which were attributable to the decreased expression of ASIC1a and ASIC2a proteins in the plasma membrane. PKC, a partner protein of PICK1, regulated ASIC functions via PICK1. The agonist and antagonist of PKC only altered ASIC currents and acid-induced increase in [[[Ca.sup.2+]].sub.i] in wild-type, but not in KO mice. In conclusion, our data provided the direct evidence from PICK1-KO mice that a novel target protein, PICK1, would regulate ASIC function and membrane expression in the brain. In addition, PICK1 played the bridge role between PKC and ASICs. protein interacting with C kinase 1; acid-sensing ion channels; protein kinase C; knockout; cortical neurons doi: 10.1152/ajpcell.00569.2009.

Individuals often eat calorically dense, highly palatable 'comfort' foods during stress for stress relief. This article demonstrates that palatable food intake (limited intake of Sucrose drink) reduces neuroendocrine, cardiovascular, and behavioral responses to stress in rats. Artificially sweetened (saccharin) drink reproduces the stress dampening, whereas oral intragastric gavage of sucrose is without effect. Together, these results suggest that the palatable/rewarding properties of sucrose are necessary and sufficient for stress dampening. In support of this finding, another type of natural reward (sexual activity) similarly reduces stress responses. Ibotenate lesions of the basolateral amygdala (BLA) prevent stress dampening by sucrose, suggesting that neural activity in the BLA is necessary for the effect. Moreover, sucrose intake increases mRNA and protein expression in the BLA for numerous genes linked with functional and/or structural plasticity. Lastly, stress dampening by sucrose is persistent, which is consistent with long-term changes in neural activity after synaptic remodeling. Thus, natural rewards, such as palatable foods, provide a general means of stress reduction, likely via structural and/or functional plasticity in the BLA. These findings provide a clearer understanding of the motivation for consuming palatable foods during times of stress and influence therapeutic strategies for the prevention and/or treatment of obesity and other stress-related disorders. corticosterone | anxiety-related behavior | synaptophysin | cAMP response element-binding protein | calcium/calmodulin-dependent protein kinase doi/ 10.1073/pnas.1007740107

The AMP-activated protein kinase (AMPK) is an [alpha][beta][gamma] heterotrimer that acts as a master metabolic regulator to maintain cellular energy balance following increased energy demand and increases in the AMP/ATP ratio. This regulation provides dynamic control of energy metabolism, matching energy supply with demand that is essential for the function and survival of organisms. AMPK is inactive unless phosphorylated on Thr172 in the [alpha]-catalytic subunit activation loop by upstream kinases (LKB1 or calcium-calmodulin-dependent protein kinase kinase [beta]). How a rise in AMP levels triggers AMPK [alpha]-Thr172 phosphorylation and activation is incompletely understood. Here we demonstrate unequivocally that AMP directly stimulates [alpha]-Thr172 phosphorylation provided the AMPK [beta]-subunit is myristoylated. Loss of the myristoyl group abolishes AMP activation and reduces the extent of [alpha]-Thr172 phosphorylation. Once AMPK is phosphorylated, AMP further activates allosterically but this activation does not require [beta]-subunit myristoylation. AMP and glucose deprivation also promote membrane association of myristoylated AMPK, indicative of a myristoyl-switch mechanism. Our results show that AMP regulates AMPK activation at the initial phosphorylation step, and that [beta]-subunit myristoylation is important for transducing the metabolic stress signal. myristome | signal transduction | adenylate charge | [gamma]-subunit doi/ 10.1073/pnas.1009705107

The glutamine transporter SNAT3 is involved in the uptake and release of glutamine in the brain, liver, and kidney. Substrate transport is accompanied by [Na.sup.+] cotransport and [H.sup.+] antiport. In this study, treatment of Xenopus laevis oocytes expressing rat SNAT3 with the phorbol ester PMA resulted in a rapid downregulation of glutamine uptake in less than 20 min. PMA treatment of oocytes coexpressing SNAT3 and the monocarboxylate transporter MCT1 reduced SNAT3 activity only, demonstrating the specificity of the regulatory mechanism. Single or combined mutations of seven putative phosphorylation sites in the SNAT3 sequence did not affect the regulation of SNAT3 by PMA. Expression of an EGFP-SNAT3 fusion protein in oocytes established that the downregulation was caused by the retrieval of the transporter from the plasma membrane. Coexpression of SNAT3 with dominant-negative mutants of dynamin or caveolin revealed that SNAT3 trafficking occurs in a dynamin-independent manner and is influenced by caveolin. Although system N activity was not affected by PMA in cultured astrocytes, a downregulation was observed in HepG2 cells. glutamine transport; SLC38A3; protein kinase C; phorbol ester; protein trafficking doi: 10.1152/ajpcell.00209.2010.

[Cav.sub.v]1 channels initiate excitation-contraction coupling in skeletal and cardiac muscle. During the fight-or-flight response, epinephrine released by the adrenal medulla and norepinephrine released from sympathetic nerves increase muscle contractility by activation of the [beta]-adrenergic receptor/cAMP-dependent protein kinase pathway and up-regulation of [ca.sub.v]1 channels in skeletal and cardiac muscle. Although the physiological mechanism of this pathway is well defined, the molecular mechanism and the sites of protein phosphorylation required for [Ca.sub.v]1 channel regulation are unknown. To identify the regulatory sites of phosphorylation under physiologically relevant conditions, [Cav.sub.v]1.1 channels were purified from skeletal muscle and sites of phosphorylation on the [alpha]1 subunit were identified by mass spectrometry. Two phosphorylation sites were identified in the proximal C-terminal domain, serine 1575 (S1575) and threonine 1579 (T1579), which are conserved in cardiac [Ca.sub.v]1.2 channels (S1700 and T1704, respectively). In vitro phosphorylation revealed that [Ca.sub.v]1.1-S1575 is a substrate for both cAMP-dependent protein kinase and calcium/calmodulin-dependent protein kinase II, whereas [ca.sub.v]1.1-T1579 is a substrate for casein kinase 2. Treatment of rabbits with isoproterenol to activate [beta]-adrenergic receptors increased phosphorylation of S1575 in skeletal muscle [ca.sub.v]1.1 channels in vivo, and treatment with propranolol to inhibit [beta]-adrenergic receptors reduced phosphorylation. As S1575 and T1579 in [ca.sub.v]1.1 channels and their homologs in [ca.sub.v]1.2 channels are located at a key regulatory interface between the distal and proximal C-terminal domains, it is likely that phosphorylation of these sites in skeletal and cardiac muscle is directly involved in calcium channel regulation in response to the sympathetic nervous system in the fight-or-flight response. adrenalin | calcium channels | cyclic AMP | mass spectometry | protein kinase A doi/ 10.1073/pnas.1012384107

The ataxia-telangiectasia mutated (ATM) protein kinase is activated by DNA double-strand breaks (DSBs) through the Mre11-Rad50-Nbs1 (MRN) DNA repair complex and orchestrates signaling cascades that initiate the DNA damage response. Cells lacking ATM are also hypersensitive to insults other than DSBs, particularly oxidative stress. We show that oxidation of ATM directly induces ATM activation in the absence of DNA DSBs and the MRN complex. The oxidized form of ATM is a disulfide--cross-linked dimer, and mutation of a critical cysteine residue involved in disulfide bond formation specifically blocked activation through the oxidation pathway. Identification of this pathway explains observations of ATM activation under conditions of oxidative stress and shows that ATM is an important sensor of reactive oxygen species in human cells. 10.1126/science.1192912

Intracellular cAMP is compartmentalized to near membrane domains in endothelium, where it strengthens endothelial cell barrier function. Phosphodiesterase 4D4 (PDE4D4) interacts with the spectrin membrane skeleton and prevents cAMP from accessing microtubules. Expression of a dominant-negative PDE4D4 peptide enables cAMP to access microtubules, where it results in phosphorylation of the nonneuronal microtubule-associated protein tau at serine 214. Presently, we sought to determine whether PKA is responsible for tau-Ser214 phosphorylation and furthermore whether PKA phosphorylation of tau-Ser214 is sufficient to reorganize microtubules and induce endothelial cell gaps. In cells expressing the dominant-negative PDE4D4 peptide, forskolin activated transmembrane adenylyl cyclases, increased cAMP, and induced tau-Ser214 phosphorylation that was accompanied by microtubule reorganization. PKA catalytic and regulatory I subunits, but not the regulatory II subunit, coassociated with reorganized microtubules. To determine the functional consequence of tau-Ser214 phosphorylation, wild-type human tau40 and tau40 engineered to possess an alanine point mutation (S214A) were stably expressed in endothelium. In cells expressing the dominant-negative PDE4D4 peptide and tauS214A, PKA-dependent phosphorylation of both the endogenous and heterologously expressed tau were abolished. Expression of tau-S214A prevented forskolin from depolymerizing microtubules, inducing intercellular gaps, and increasing macromolecular permeability. These findings therefore identify nonneuronal tau as a critical cAMP-responsive microtubule-associated protein that controls microtubule architecture and endothelial cell barrier function. adenylyl cyclase; cytoskeleton; phosphodiesterase doi: 10.1152/ajplung.00431.2009.

Hepatic glucagon action increases in response to accelerated metabolic demands and is associated with increased whole body substrate availability, including circulating lipids. The hypothesis that increases in hepatic glucagon action stimulate AMP-activated protein kinase (AMPK) signaling and peroxisome proliferator-activated receptor-[alpha] (PPAR[alpha] and fibroblast growth factor 21 (FGF21) expression in a manner modulated by fatty acids was tested in vivo. Wild-type ([gcgr.sup.+/+]) and glucagon receptor-null ([gcgr.sup.-/-]) littermate mice were studied using an 18-h fast, exercise, and hyperglucagonemic-euglycemic clamps plus or minus increased circulating lipids. Fasting and exercise in [gcgr.sup.+/+], but not [gcgr.sup.-/-] mice, increased hepatic phosphorylated AMPK[alpha] at threonine 172 ([MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII]) and PPAR[alpha] and FGF21 mRNA. Clamp results in [gcgr.sup.+/+] mice demonstrate that hyperlipidemia does not independently impact or modify glucagon-stimulated increases in hepatic AMP/ATP, [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII], or PPAR[alpha] and FGF21 mRNA. It blunted glucagon-stimulated acetyl-CoA carboxylase phosphorylation, a downstream target of AMPK, and accentuated PPAR[alpha] and FGF21 expression. All effects were absent in [gcgr.sup.-/-] mice. These findings demonstrate that glucagon exerts a critical regulatory role in liver to stimulate pathways linked to lipid metabolism in vivo and shows for the first time that effects of glucagon on PPARa and FGF21 expression are amplified by a physiological increase in circulating lipids. adenosine 5'-monophosphate-activated protein kinase; peroxisome proliferator-activated receptor-[alpha]; fibroblast growth factor 21 doi: 10.1152/ajpendo.00263.2010.

Protein kinase C-[delta] (PKC[delta]) exerts important cardiac actions as a lipid-regulated kinase. There is limited evidence that PKC[delta] also might exert an additional kinase-independent action as a regulator of the subcellular compartmentalization of binding partners such as Shc (Src homologous and collagen), a family of adapter proteins that play key roles in growth regulation and oxidative stress responses. This study shows that native PKC[delta] forms complexes with endogenous Shc proteins in [H.sub.2][O.sub.2]-treated cardiomyocytes; [H.sub.2][O.sub.2] treatment also leads to the accumulation of PKC[delta] and Shc in a detergent-insoluble cytoskeletal fraction and in mitochondria. [H.sub.2][O.sub.2]-dependent recruitment of Shc isoforms to cytoskeletal and mitochondrial fractions is amplified by wild-type-PKC[delta] overexpression, consistent with the notion that PKC[delta] acts as a signal-regulated scaffold to anchor Shc in specific subcellular compartments. However, overexpression studies with kinase-dead (KD)PKC[delta]-K376R (an ATP-binding mutant of PKC[delta] that lacks catalytic activity) are less informative, since KD-PKC[delta]-K376R aberrantly localizes as a constitutively tyrosine-phosphorylated enzyme to detergent-insoluble and mitochondrial fractions of resting cardiomyocytes; relatively little KD-PKC[delta]-K376R remains in the cytosolic fraction. The aberrant localization and tyrosine phosphorylation patterns for KD-PKC[delta]-K376R do not phenocopy the properties of native PKC[delta], even in cells chronically treated with GF109203X to inhibit PKC[delta] activity. Hence, while KD-PKC[delta]-K376R overexpression increases Shc localization to the detergent-insoluble and mitochondrial fractions, the significance of these results is uncertain. Our studies suggest that experiments using KD-PKC[delta]-K376R overexpression as a strategy to competitively inhibit the kinase-dependent actions of native PKC[delta] or to expose the kinase-independent scaffolding functions of PKC[delta] should be interpreted with caution. oxidative stress; mitochondria; cytoskeleton doi: 10.1152/ajpcell.00170.2010.

The phosphodie sterase type-5 inhibitor sildenafil has powerful cardioprotective effects against ischemia-reperfusion injury. PKG-mediated signaling has been implicated in this protection, although the mechanism and the downstream targets of this kinase remain to be fully elucidated. In this study we assessed the role of phospholemman (PLM) phosphorylation, which activates the [Na.sup.+]/[K.sup.+]-ATPase, in cardioprotection afforded by sildenafil administered during reperfusion. Isolated perfused mouse hearts were optimally protected against infarction (indexed by tetrazolium staining) by 0.1 [micro]M sildenafil treatment during the first 10 min of reperfusion. Extended sildenafil treatment (30, 60, or 120 min at reperfusion) did not alter the degree of protection provided. This protection was PKG dependent, since it was blocked by KT-5823. Western blot analysis using phosphospecific antibodies to PLM showed that sildenafil at reperfusion did not modulate PLM Ser63 or Set68 phosphorylation but significantly increased Ser69 phosphorylation. The treatment of isolated rat ventricular myocytes with sildenafil or 8-bromo-cGMP (PKG agonist) enhanced PLM Ser69 phosphorylation, which was bisindolylmaleimide (PKC inhibitor) sensitive. Patch-clamp studies showed that sildenafil treatment also activated the [Na.sup.+]/[K.sup.-]-ATPase, which is anticipated in light of PLM Ser69 phosphorylation. [Na.sup.+]/[K.sup.-]-ATPase activation during reperfusion would attenuate [Na.sup.+] overload at this time, providing a molecular explanation of how sildenafil guards against injury at this time. Indeed, using flame photometry and rubidium uptake into isolated mouse hearts, we found that sildenafil enhanced [Na.sup.+]/[K.sup.-]-ATPase activity during reperfusion. In this study we provide a molecular explanation of how sildenafil guards against myocardial injury during postischemic reperfusion. protein kinase G; sodium/potassium-adenosine 5'-triphosphatase; ischemia; heart doi: 10.1152/ajpheart.00129.2010.

Insulin resistance (IR) is a condition where different organs are refractory to insulin stimulation of glucose uptake. ANG II has been suggested to be involved in the development of IR in the heart. The precise mechanism by which this occurs is still unknown. Here we have used dynamic fluorescent imaging techniques to show that ANG II inhibits insulin production of phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P3] in cardiac myocytes. Fluorophore (Venus)-conjugated cAMP-dependent protein kinase-pleckstrin homology domain, which specifically binds to PI(3,4,5)P3, was transfected in neonatal rat cardiac myocytes. Insulin induced a robust increase in the fluorescence intensity at the cell surface, which was diminished by application of ANG II. The inhibitory action of ANG II was antagonized by RNH-6270 (an angiotensin type 1 receptor antagonist) but not by PD-122370 (an angiotensin type 2 receptor antagonist). BAPTA-AM ([Ca.sup.2+] chelator) largely attenuated the ANG II effect, whereas K-252b (PKC inhibitor) did not. Furthermore, an elevation of intracellular [Ca.sup.2+] induced by ionomycin mimicked the ANG II effect. Therefore, it is suggested that ANG II antagonizes insulin-mediated production of PI(3,4,5)P3 via a [Ca.sup.2+]-dependent but PKC-independent pathway in cardiac myocytes. protein kinase C; angiotensin type 1 receptor; heart; adenosine 3',5'cyclic monophosphate-dependent protein kinase; real-time imaging; fluorophore doi: 10.1152/ajpheart.00220.2009.

Atypical protein kinase C (aPKC) isoforms ([lambda] and [zeta]) have been implicated in the control of insulin-stimulated glucose uptake in adipose and skeletal muscle, but their precise role in this process remains unclear, especially in light of accumulating evidence showing that, in response to numerous stimuli, including insulin and lipids such as ceramide, activation of aPKCs acts to negatively regulate key insulin-signaling molecules, such as insulin receptor substrate-1 (IRS-1) and protein kinase B (PKB)/ cAMP-dependent PKC (Akt). In this study, we have depleted PKC[lambda] in L6 skeletal muscle cells using RNA interference and assessed the effect this has upon insulin action. Muscle cells did not express detectable amounts of PKC[zeta]. Depletion of PKC[lambda] (>95%) had no significant effect on the expression of proteins participating in insulin signaling [i.e., insulin receptor, IRS-1, phosphatidylinositol 3-kinase (PI 3-kinase), PKB, or phosphate and tensin homolog deleted on chromosome 10] or those involved in glucose transport [Akt substrate of 160 kDa, glucose transporter (GLUT)l, or GLUT4]. However, PKC[lambda]-depleted muscle cells exhibited greater activation of PKB/Akt and phosphorylation of its downstream target glycogen synthase kinase 3, in the basal state and displayed greater responsiveness to submaximal doses of insulin with respect to p85-PI 3-kinase/IRS-1 association and PKB activation. The increase in basal and insulin-induced signaling resulted in an associated enhancement of basal and insulin-stimulated glucose transport, both of which were inhibited by the PI 3-kinase inhibitor wortmannin. Additionally, like RNAi-mediated depletion of PKC[lambda], overexpression of a dominant-negative mutant of PKC[zeta] induced a similar insulin-sensitizing effect on PKB activation. Our findings indicate that aPKCs are likely to play an important role in restraining proximal insulin signaling events but appear dispensable with respect to insulin-stimulated glucose uptake in cultured L6 muscle cells. protein kinase C[lambda]; glucose uptake; insulin receptor substrate 1; ribonucleic acid interference; protein kinase B/adenosine 3',5'-cyclic monophosphate-associated kinase doi: 10.1152/ajpendo.00171.2010.

The sympathetic nervous system increases heart rate by activating [beta] adrenergic receptors and increasing cAMP levels in myocytes in the sinoatrial node. The molecular basis for this response is not well understood; however, the cardiac funny current ([I.sub.f]) is thought to be among the end effectors for cAMP signaling in sinoatrial myocytes. If is produced by hyperpolarization-activated cyclic nucleotide-sensitive (HCN4) channels, which can be potentiated by direct binding of cAMP to a conserved cyclic nucleotide binding domain in the C terminus of the channels. [beta] adrenergic regulation of If in the sinoatrial node is thought to occur via this direct binding mechanism, independent of phosphorylation. Here, we have investigated whether the cAMP-activated protein kinase (PKA) can also regulate sinoatrial HCN4 channels. We found that inhibition of PKA significantly reduced the ability of [beta] adrenergic agonists to shift the voltage dependence of If in isolated sinoatrial myocytes from mice. PKA also shifted the voltage dependence of activation to more positive potentials for heterologously expressed HCN4 channels. In vitro phosphorylation assays and mass spectrometry revealed that PKA can directly phosphorylate at least 13 sites on HCN4, including at least three residues in the N terminus and at least 10 in the C terminus. Functional analysis of truncated and alanine-substituted HCN4 channels identified a PKA regulatory site in the distal C terminus of HCN4, which is required for PKA modulation of It. Collectively, these data show that native and expressed HCN4 channels can be regulated by PKA, and raise the possibility that this mechanism could contribute to sympathetic regulation of heart rate. doi/10.1085/jgp.201010488

Learning theories hypothesize specific circuits encode essential information for performance. For simple tasks in invertebrates and mammals, the essential circuits are known, but for cognitive functions, the essential circuits remain unidentified. Here, we show that some essential information for performing a choice task is encoded in a specific circuit in a neocortical area. Rat postrhinal (POR) cortex is required for visual shape discriminations, protein kinase C (PKC) pathways mediate changes in neuronal physiology that support learning, and specific PKC genes are required for multiple learning tasks. We used direct gene transfer of a constitutively active PKC to prime a specific POR cortex circuit for learning visual shape discriminations. In the experiment, rats learned a discrimination, received gene transfer, learned new discriminations, received a small lesion that ablated [approximately equal to] 21% of POR cortex surrounding the gene transfer site, and were tested for performance for discriminations learned either before or after gene transfer. Lesions of the genetically targeted circuit selectively interfered with performance for discriminations learned after gene transfer. Activity-dependent gene imaging confirmed increased activity in the genetically targeted circuit during learning and showed the essential information was sparse-coded in [approximately equal to] 500 neurons in the lesioned area. Wild-type rats contained circuits with similar increases in activity during learning, but these circuits were located at unpredictable, different positions in POR cortex. These results establish that some essential information for performing specific visual discriminations can be encoded in a small, identified, neocortical circuit and provide a foundation for characterizing the circuit and essential information. learning | vision | protein kinase C | neocortex | herpes simplex virus vector doi/ 10.1073/pnas.0912950107

Here we show that the functional human ortholog of Greatwall protein kinase (Gwl) is the microtubule-associated serine/threonine kinase-like protein, MAST-L. This kinase promotes mitotic entry and maintenance in human cells by inhibiting protein phosphatase 2A (PP2A), a phosphatase that dephosphorylates cyclin B-Cdc2 substrates. The complete depletion of Gwl by siRNA arrests human cells in G2. When the levels of this kinase are only partially depleted, however, cells enter into mitosis with multiple defects and fail to inactivate the spindle assembly checkpoint (SAC). The ability of cells to remain arrested in mitosis by the SAC appears to be directly proportional to the amount of Gwl remaining. Thus, when Gwl is only slightly reduced, cells arrest at prometaphase. More complete depletion correlates with the premature dephosphorylation of cyclin B-Cdc2 substrates, inactivation of the SAC, and subsequent exit from mitosis with severe cytokinesis defects. These phenotypes appear to be mediated by PP2A, as they could be rescued by either a double Gwl/PP2A knockdown or by the inhibition of this phosphatase with okadaic acid. These results suggest that the balance between cyclin B-Cdc2 and PP2A must be tightly regulated for correct mitotic entry and exit and that Gwl is crucial for mediating this regulation in somatic human cells. spindle assembly checkpoint | cell cycle | phosphatase | kinase | slippage doi/10.1073/pnas.0914191107

The metabolic sensor AMP-activated protein kinase (AMPK) regulates several transport proteins, potentially coupling transport activity to cellular stress and energy levels. The creatine transporter (CRT; SLC6AS) mediates creatine uptake into several cell types, including kidney epithelial cells, where it has been proposed that CRT is important for reclamation of filtered creatine, a process critical for total body creatine homeostasis. Creatine and phosphocreatine provide an intracellular, high-energy phosphate-buffering system essential for maintaining ATP supply in tissues with high energy demands. To test our hypothesis that CRT is regulated by AMPK in the kidney, we examined CRT and AMPK distribution in the kidney and the regulation of CRT by AMPK in cells. By immunofluorescence staining, we detected CRT at the apical pole in a polarized mouse S3 proximal tubule cell line and in native rat kidney proximal tubules, a distribution overlapping with AMPK. Two-electrode voltage-clamp (TEV) measurements of [Na.sup.+]-dependent creatine uptake into CRT-expressing Xenopus laevis oocytes demonstrated that AMPK inhibited CRT via a reduction in its Michaelis-Menten [V.sub.max] parameter. [[sup.14]C]creatine uptake and apical surface biotinylation measurements in polarized S3 cells demonstrated parallel reductions in creatine influx and CRT apical membrane expression after AMPK activation with the AMP-mimetic compound 5-aminoimidazole-4-carboxamide-l-[beta]-D-ribofuranoside. In oocyte TEV experiments, rapamycin and the AMPK activator 5-aminoimidazole-4-carboxamide- l -[beta]-D-ribofuranosyl 5'-monophosphate (ZMP) inhibited CRT currents, but there was no additive inhibition of CRT by ZMP, suggesting that AMPK may inhibit CRT indirectly via the mammalian target of rapamycin pathway. We conclude that AMPK inhibits apical membrane CRT expression in kidney proximal tubule cells, which could be important in reducing cellular energy expenditure and unnecessary creatine reabsorption under conditions of local and whole body metabolic stress. proximal tubule; metabolism; Xenopus oocytes; target of rapamycin; SLC6A8 doi: 10.1152/ajprenal.00162.2010.

Adrenergic stimulation of isolated guinea pig distal colonic mucosa produced transient [Cl.sup.-] and sustained [K.sup.+] secretion. Transient short-circuit current ([I.sub.sc]) depended on [[beta].sub.2]-adrenergic receptors ([[beta].sub.2]-AdrR), and sustained [I.sub.sc] relies on a [[beta].sub.1]-AdrR/[[beta].sub.2]-AdrR complex. Epinephrine (epi) increased cAMP content with a biphasic time course similar to changes in epi-activated [I.sub.sc] ([sup.epi][I.sub.sc]). Inhibition of transmembrane adenylyl cyclases (tmACs) reduced peak [sup.epi][I.sub.sc] and cAMP to near zero without decreasing sustained [sup.epi][I.sub.sc], consistent with cAMP from tmAC signaling for only [Cl.sup.-] secretion. Inhibition of soluble adenylyl cyclase (sAC) reduced sustained [sup.epi][I.sub.sc] and cAMP to near zero without decreasing peak [sup.epi][I.sub.sc] or cAMP, consistent with cAMP from sAC signaling for [K.sup.+] secretion. Sensitivity to phosphodiesterase (PDE) inhibitors and peptide YY (PYY) stimulation further supported separate signaling for the two components. PDE3 or PDE4 inhibitors enhanced peak [sup.epi][I.sub.sc] but not sustained [sup.epi][I.sub.sc], consistent with these PDEs as part of the [[beta].sub.2]-AdrR signaling domain. PYY suppressed peak [sup.epi][I.sub.sc] in a pertussis toxin (PTx)-sensitive manner, supporting G[[alpha].sub.i]-dependent inhibition of tmACs producing cAMP for [Cl.sup.-] secretion. Since PYY or PTx did not alter sustained [sup.epi][I.sub.sc], signaling for [K.sup.+] secretion occurred via a G[[alpha].sub.i]-independent mechanism. Presence of multiple sAC variants in colonic epithelial cells was supported by domain-specific antibodies. Responses to specific activators and inhibitors suggested that protein kinase A was not involved in activating peak or sustained components of [sup.epi][I.sub.sc], but the cAMP-dependent guanine nucleotide exchange factor, Epac, may contribute. Thus [beta]-adrenergic activation of electrogenic [Cl.sup.-] and [K.sup.+] secretion, respectively, required tmAC- and sAC-dependent signaling pathways. epinephrine; norepinephrine; soluble adenylyl cyclase; protein kinase A; Epac doi: 10.1152/ajpgi.00035.2010.

The effect of 5-aminoimidazole-4-carboxamide-ribonucleoside (AICAR) activation of the AMP-activated protein kinase (AMPK) on the transport of the model radiolabeled dipeptide [[sup.3]H]-D-Phe-L-Gln was investigated in the human epithelial colon cancer cell line Caco-2. Uptake and transepithelial fluxes of [[sup.3]H]-D-Phe-L-Gln were carried out in differentiated Caco-2 cell monolayers, and hPepT1 and glucose transporter 2 (GLUT2) protein levels were quantified by immunogold electron microscopy. AICAR treatment of Caco-2 cells significantly inhibited apical [[sup.3]H]-D-Phe-L-Gln uptake, matched by a decrease in brush-border membrane hPepT1 protein but with a concomitant increase in the facilitated glucose transporter GLUT2. A restructuring of the apical brush-border membrane was seen by electron microscopy. The hPepT1-mediated transepithelial (A-to-B) peptide flux across the Caco-2 monolayers showed no significant alteration in AICAR-treated cells. The electrical resistance in the AICAR-treated monolayers was significantly higher compared with control cells. Inhibition of the sodium/hydrogen exchanger 3 (NHE3) had an additive effect to AICAR, suggesting that the AMPK effect is not via NHE3. Fluorescence measurement of intracellular pH showed no reduction in the proton gradient driving PepT1-mediated apical uptake. The reduction in apical hPepT1 protein and dipeptide uptake after AICAR treatment in Caco-2 cells demonstrates a regulatory effect of AMPK on hPepT1, along with an influence on both the microvilli and tight junction structures. The absence of an associated reduction in transepithelial peptide movement implies an additional stimulatory effect of AICAR on the basolateral peptide transport system in these cells. These results provide a link between the hPepT1 transporter and the metabolic state of this model enterocyte. SLC15a1; 5-aminoimidazole-4-carboxamine ribonucleoside; glucose transporter 2; intestinal epithelium; membrane transport doi: 10.1152/ajpgi.00014.2010.

How persistent synaptic and spine modification is achieved is essential to our understanding of developmental refinement of neural circuitry and formation of memory. Within a short period after their induction, both types of modifications can eitherC be stabilized or reversed, but how this reversibility is controlled is largely unknown. We have shown previously that AMPA receptors (AMPARs) are delivered to perisynaptic regions after the induction of long-term potentiation (LTP) but are absent from perisynaptic regions after the full expression of LTP. Here, we report that perisynaptic AMPARs are GluR2-lacking and they translocate to synapses in a protein kinase C (PKC)-dependent manner. Once entering synapses, these AMPARs quickly switch to GluR2-containing in an activity-dependent manner. Absence of postinduction activity or blocking interactions between GluR2 and NSF, or GluR2 and GRIP/ PICK1 results in LTP mediated by GluR2-1acking AMPARs. However, these synaptic GluR2-1acking AMPARs are not sufficient to allow reversibility of LTP. On the other hand, postsynaptic inhibition of PKC activity hords AMPARs at perisynaptic regions. As long as perisynaptic AMPARs are present, both LTP and spine expansion remain labile: they can be reverted to the baseline state together with removal of perisynaptic AMPARs, or they can enter a stabilized state of persistent increase together with synaptic incorporation of perisynaptic AMPARs. Thus, perisynaptic GluR2-1acking AMPARs play a critical role in controlling the reversibility of both synaptic and spine modifications, dendritic spine | long-term potentiation | two-photon imaging doi/ 10.1073/pnas.0913004107

Smac mimetics target cancer cells in a TNF[alpha]-dependent manner, partly via proteasome degradation of cellular inhibitor of apoptosis I (clAP1) and clAP2. Degradation of cIAPs triggers the release of receptor interacting protein kinase (RIPK1) from TNF receptor I (TNFR1) to form a caspase-8 activating complex together with the adaptor protein Fas-associated death domain (FADD). We report here a means through which cancer cells mediate resistance to Smac mimetic/TNF[alpha]-induced apoptosis and corresponding strategies to overcome such resistance. These human cancer cell lines evades Smac mimetic-induced apoptosis by up-regulation of clAP2, i which although initially degraded, rebounds and is refractory to subsequent degradation, clAP2 is induced by TNF[alpha] via NF-[kappa]B and modulation of the NF-[kappa]B signal renders otherwise resistant cells : sensitive to Smac mimetics. In addition, other signaling pathways, including phosphatidyl inositol-3 kinase (PI3K), have the potential to concurrently regulate clAP2. Using the PI3K inhibitor, LY294002, cIAP2 up-regulation was suppressed and resistance to Smac mimetics-induced apoptosis was also overcome. TNF[alpha] | NF-[kappa]B | PI3K | Caspase-8 | PRIK1 doi/ 10.1073/pnas.1005667107

To what extent the secretory pathway is regulated by cellular signaling is unknown. In this study, we used RNA interference to explore the function of human kinases and phosphatases in controlling the organization of and trafficking within the secretory pathway. We identified 122 kinases/phosphatases that affect endoplasmic reticulum (ER) export, ER exit sites (ERESs), and/or the Golgi apparatus. Numerous kinases/phosphatases regulate the number of ERESs and ER to Golgi protein trafficking. Among the pathways identified, the Raf-MEK (MAPK/ERK [extracellular signal-regulated kinase] kinase)-ERK cascade, including its regulatory proteins CNK1 (connector enhancer of the kinase suppressor of Ras-1) and neurofibromin, controls the number of ERESs via ERK2, which targets Sec16, a key regulator of ERESs and COPII (coat protein II) vesicle biogenesis. Our analysis reveals an unanticipated complexity of kinase/ phosphatase-mediated regulation of the secretory pathway, uncovering a link between growth factor signaling and ER export. doi/10.1083/jcb.200912082

PIK3CA mutations are reported to be present in approximately 25% of breast cancer (BC), particularly the estrogen receptor--positive (ER+) and HER2-overexpressing (HER2+) subtypes, making them one of the most common genetic aberrations in BC. In experimental models, these mutations have been shown to activate AKT and induce oncogenic transformation, and hence these lesions have been hypothesized to render tumors highly sensitive to therapeutic PI3K/ mTOR inhibition. By analyzing gene expression and protein data from nearly 1,800 human BCs, we report that a PIK3CA mutation-associated gene signature (PIK3CA-GS) derived from exon 20 (kinase domain) mutations was able to predict PIK3CA mutation status in two independent datasets, strongly suggesting a characteristic set of gene expression-induced changes. However, in ER+/HER2-BC despite pathway activation, PIK3CA mutations were associated with a phenotype of relatively low mTORC1 signaling and a good prognosis with tamoxifen monotherapy. The relationship between clinical outcome and the PIK3CA-GS was also assessed. Although the PIK3CA-GS was not associated with prognosis in ER- and HER2+ BC, it could identify better clinical outcomes in ER+/HER2--disease. In ER+ BC cell lines, PIK3CA mutations were also associated with sensitivity to tamoxifen. These findings could have important implications for the treatment of PIK3CA-mutant BCs and the development of PI3K/mTOR inhibitors. gene expression profiling | PI3 kinase doi/ 10.1073/pnas.0907011107

The tumor suppressor liver kinase B1 (LKB1), also called STK11, is a protein kinase mutated in Peutz-Jeghers syndrome. LKB1 phosphorylates AMP-activated protein kinase (AMPK) and several related protein kinases. Whereas deletion of both catalytic isoforms of AMPK from the pancreatic [beta]-cell and hypothalamic neurons using the rat insulin promoter (RIP2).Cre transgene ([beta]AMPKdKO) diminishes insulin secretion in vivo, deletion of LKB1 in the [beta]-cell with an inducible Pdx-1.CreER transgene enhances insulin secretion in mice. To determine whether the differences between these models reflect genuinely distinct roles for the two kinases in the [beta]-cell or simply differences in the timing and site(s) of deletion, we have therefore created mice deleted for LKB1 with the RIP2.Cre transgene. In marked contrast to [beta]AMPKdKO mice, [beta]LKB1KO mice showed diminished food intake and weight gain, enhanced insulin secretion, unchanged insulin sensitivity, and improved glucose tolerance. In line with the phenotype of Pdx1-CreER mice, total [beta]-cell mass and the size of individual islets and [beta]-cells were increased and islet architecture was markedly altered in [beta]LKB1KO islets. Signaling by mammalian target of rapamycin (mTOR) to eIF4-binding protein-1 and ribosomal S6 kinase was also enhanced. In contrast to Pdx1-CreER-mediated deletion, the expression of Glut2, glucose-induced changes in membrane potential and intracellular [Ca.sup.2+] were sharply reduced in [beta]LKB1KO mouse islets and the stimulation of insulin secretion was modestly inhibited. We conclude that LKB1 and AMPK play distinct roles in the control of insulin secretion and that the timing of LKB1 deletion, and/or its loss from extrapancreatic sites, influences the final impact on [beta]-cell function. AMP-activated protein kinase; [beta]-cell; insulin secretion; food intake; liver kinase B1; pancreas doi: 10.1152/ajpendo.00100.2010.

Inositol phosphates (InsPs) are signaling molecules with multiple roles in cells. In particular Ins(1,2,3,4,5,6)[P.sub.6] ([InsP.sub.6]) is involved in mRNA export and editing or chromatin remodeling among other events. [InsP.sub.6] accumulates as mixed salts (phytate) in storage tissues of plants and plays a key role in their physiology. Human diets that are exclusively grain-based provide an excess of [InsP.sub.6] that, through chelation of metal ions, may have a detrimental effect on human health. Ins (1,3,4,5,6)[P.sub.5] 2-kinase ([InsP.sub.5] 2-kinase or Ipk1) catalyses the synthesis of [InsP.sub.6] from [InsP.sub.5] and ATP, and is the only enzyme that transfers a phosphate group to the axial 2-OH of the myo-inositide. We present the first structure for an [InsP.sub.5] 2-kinase in complex with both substrates and products. This enzyme presents a singular structural region for inositide binding that encompasses almost half of the protein. The key residues in substrate binding are identified, with Asp368 being responsible for recognition of the axial 2-OH. This study sheds light on the unique molecular mechanism for the synthesis of the precursor of inositol pyrophosphates. [InsP.sub.5] 2-kinase | IP5 | IP6 | ipkl | crystal structure www.pnas.org/cgi/doi/10.1073/pnas.0912979107

Aurora A is a spindle pole--associated protein kinase required for mitotic spindle assembly and chromosome segregation. In this study, we show that Drosophila melanogaster aurora A phosphorylates the dynactin subunit [p150.sup.glued] on sites required for its association with the mitotic spindle. Dynactin strongly accumulates on microtubules during prophase but disappears as soon as the nuclear envelope breaks down, suggesting that its spindle localization is tightly regulated. If aurora A's function is compromised, dynactin and dynein become enriched on mitotic spindle microtubules. Phosphorylation sites are localized within the conserved microtubule-binding domain (MBD) of the [p150.sup.glued]. Although wild-type [p150.sup.glued] binds weakly to spindle microtubules, a variant that can no longer be phosphorylated by aurora A remains associated with spindle microtubules and fails to rescue depletion of endogenous [p150.sup.glued]. Our results suggest that aurora A kinase participates in vivo to the phosphoregulation of the [p150.sup.glued] MBD to limit the microtubule binding of the dynein--dynactin complex and thus regulates spindle assembly. doi/ 10.1083/jcb.201001144

A population of stromal cells that retains osteogenic capacity in adult bone (adult bone stromal cells or aBSCs) exists and is under intense investigation. Mice heterozygous for a null allele of prkar1a ([Prkar1a.sup.+/-]), the primary receptor for cyclic adenosine monophosphate (cAMP) and regulator of protein kinase A (PKA) activity, developed bone lesions that were derived from cAMP-responsive osteogenic cells and resembled fibrous dysplasia (FD). [Prkar1a.sup.+/-] mice were crossed with mice that were heterozygous for catalytic subunit C[alpha] ([Prkaca.sup.+/-]), the main PKA activity-mediating molecule, to generate a mouse model with double heterozygosity for prkar1a and prkaca ([Prkar1a.sup.+/-][Prkaca.sup.+/-]). Unexpectedly, [Prkar1a.sup.+/-][Prkaca.sup.+/-] mice developed a greater number of osseous lesions starting at 3 months of age that varied from the rare chondromas in the long bones and the ubiquitous osteochondrodysplasia of vertebral bodies to the occasional sarcoma in older animals. Cells from these lesions originated from an area proximal to the growth plate, expressed osteogenic cell markers, and showed higher PKA activity that was mostly type II (PKA-II) mediated by an alternate pattern of catalytic subunit expression. Gene expression profiling confirmed a preosteoblastic nature for these cells but also showed a signature that was indicative of mesenchymal-to-epithelial transition and increased Wnt signaling. These studies show that a specific subpopulation of aBSCs can be stimulated in adult bone by alternate PKA and catalytic subunit activity; abnormal proliferation of these cells leads to skeletal lesions that have similarities to human FD and bone tumors. catalytic subunit | mesenchymal cells | regulatory subunit | tumor | sarcoma doi/10.1073/pnas.1003680107

Phosphoinositides are essential lipid regulators of trafficking and signaling pathways of all eukaryotic cells. Phosphatidylinositol 4-phosphate (Ptdlns4P) is an intermediate in the synthesis of several important phosphoinositide species but also serves as a regulatory molecule in its own right. Phosphatidylinositol 4-kinases are most abundant in the Golgi but are also found in the plasma membrane and in endocytic compartments. To investigate the role of Golgi Ptdlns4P in orchestrating trafficking events, we used a unique drug-inducible molecular approach to rapidly deplete Ptdlns4P from Golgi membranes by a recruitable Sac1 phosphatase enzyme. The utility of the system was shown by the rapid loss of Golgi localization of PH domains known to bind Ptdlns4P after Sac1 recruitment to the Golgi. Acute Ptdlns4P depletion prevented the exit of cargo from the Golgi destined to both the plasma membrane and the late endosomes and led to the loss of some but not all clathrin adaptors from the Golgi membrane. Rapid Ptdlns4P depletion in the Golgi also impaired but did not eliminate the replenishment of the plasma membrane Ptdlns(4,5)[P.sub.2] during phospholipase C activation revealing a hitherto unrecognized contribution of Golgi Ptdlns4P to this process. This unique approach will allow further studies on the role of phosphoinositides in endocytic compartments that have evaded detection using the conventional longterm manipulations of inositide kinase and phosphatase activities. phosphatidylinositol 4-phosphate | PI 4-kinase | Sac1 phosphatase | vesicular trafficking | green fluorescent protein doi/ 10.1073/pnas.1000157107

PhoQ is the transmembrane sensor histidine kinase of the bacterial phoPQ two-component system, which detects and responds to divalent cations and to antimicrobial peptides, and can trigger virulence. Despite their ubiquitous importance in bacterial signaling, the structure and mechanism of the sensor kinases are not fully understood. In particular, the mechanism by which the signal is propagated through the transmembrane (TM) region remains unclear. We have identified a critical asparagine residue in the second TM helix of PhoQ. Replacement of this Asn202 with a variety of hydrophobic amino acids results in a protein that is blind to signal, fails to activate transcription of PhoQ-dependent genes, and abrogates transcription when coexpressed with wild-type PhoQ. Analysis of other two-component kinase sequences indicated that many such proteins contain similarly conserved polar residues, and the structure of one such domain shows a polar residue proximal to an extended cavity near the center of the TM bundle. We therefore examined the role of Asn202 in PhoQ. Our analysis indicated that its kinase function is dependent on the polarity of Asn202, rather than its precise structure or position in the TM region; it can be displaced up or down one turn of TM helix 2, or even moved to the adjacent TM helix 1. The presence of polar TM amino acids among many diverse sensor kinases suggest a widespread mechanism of two-component signal transduction; we speculate that they might stabilize underpacked water-containing cavities that can accommodate conformational changes required for switching from phosphatase to kinase-competent conformations. histidine kinase | membrane proteins | signal transduction mechanism | two-component signaling doi/ 10.1073/pnas.1003166107

Pioglitazone (PIO) and glucagon-like peptide-1 (GLP-1) analogs limit infarct size (IS) in experimental models. The effects of the dipeptidyl-peptidase-IV inhibitors, which increase the endogenous levels of GLP-1, on myocardial protection, are unknown. We studied whether sitagliptin (SIT) and PIO have additive effects on IS limitation in the mouse. Mice received 3-day or 14-day oral SIT (300 mg x [kg.sup.-1] x [day.sup.-1]), PIO (5 mg x [kg.sup.-1] x [day.sup.-1]), SIT + PIO, or vehicle. In addition, mice received intravenous H-89 [20 mg/kg, a protein kinase A (PKA) inhibitor] or vehicle 1 h before ischemia. Rats underwent 30 min myocardial ischemia and 4 h reperfusion. SIT, PIO, and SIT + PIO for 3 days significantly reduced IS (24.3 [+ or -] 2.7, 23.0 [+ or -] 0.8, and 14.7 [+ or -] 0.9%) compared with controls (46.2 [+ or -] 2.8%). H-89 completely blocked the effect of SIT and partially blocked the PIO effect. SIT, but not PIO, increased cAMP levels. PKA activity was increased by PIO and to a greater extent by SIT. PIO, but not SIT, increased cytosolic phospholipase [A.sub.2] and cyclooxygenase-2 activity. Accordingly, 6-keto-[PGF.sub.1a] and 15-deoxy-[PGJ.sub.2] increased by PIO but not SIT. In contrast, SIT, and to a lesser extent PIO, increased 15-epi-lipoxin [A.sub.4] levels. H-89 completely blocked the effect of SIT and PIO on 15-epi-lipoxin [A.sub.4] levels. PIO, and to a greater extent SIT, increased endothelial nitric oxide synthase and cAMP response element-binding protein phosphorylation, an effect that was blocked by H-89. With a 14-day pretreatment experiment, IS was 46.4 [+ or -] 1.0% in the control group, 16.9 [+ or -] 0.6% in SIT (P < 0.001), 19.1 [+ or -] 1.1% in PIO (P = 0.014), and 12.9 [+ or -] 0.7% in SIT + PIO (P < 0.001). We found that SIT and PIO limit IS using different pathways. The protective effect of SIT is via cAMP-dependent PKA activation, whereas PIO mediates its effects via both PKA-dependent and -independent pathways. protein kinase A; adenosine 3'-5'-cyclic monophosphate doi: 10.1152/ajpheart.00867.2009.

L-type voltage-gated [Ca.sup.2+] channels (LVGCs) are functionally downregulated in arterial smooth muscle (SM) cells (ASMCs) of mice with SM-specific knockout of [Na.sup.+]/[Ca2.sup.+] exchanger type-1 ([NCX1.sup.SM-/-]) (32). Here, using activators and inhibitors of protein kinase C (PKC), we explore the regulation of these channels by a PKC-dependent mechanism. In both wild-type (WT) and [NCX1.sup.SM-/-] myocytes, the PKC activator phorbol 12,13-dibutyrate (PDBu) increases LVGC conductance, decreases channel closing rate, and shifts the voltage dependence of channel opening to more negative potentials. Three different PKC inhibitors, bisindolylmaleimide, Ro-31-8220, and PKC 19-31, all decrease LVGC currents in WT myocytes and prevent the PDBu-induced increase in LVGC current. Dialysis of WT ASMCs with activated PKC increases LVGC current and decreases channel closing rate. These results demonstrate that PKC activates LVGCs in ASMCs. The phosphatase inhibitor calyculin A increases LVGC conductance by over 50%, indicating that the level of LVGC activation is a balance between phosphatase and PKC activities. PDBu causes a larger increase in LVGC conductance and a larger shift in voltage dependence in [NCX1.sup.SM-/-] myocytes than in WT myocytes. The inhibition of PKC with PKC 19-31 decreased LVGC conductance by 65% in WT myocytes but by only 37% in [NCX1.sup.SM-/-] myocytes. These results suggest that LVGCs are in a state of low PKC-induced phosphorylation in [NCX1.sup.SM-/-] myocytes. We conclude that in [NCX1.sup.SM-/-] myocytes, reduced [Ca2.sup.+] entry via NCX1 lowers cytosolic [Ca2.sup.+], thereby reducing PKC activation that lowers LVGC activation. sodium/calcium exchanger type-1 knockdown; calcium channels doi: 10.1152/ajpheart.00965.2009.

Diabetes is a major predictor of in-stent restenosis, which is associated with fibroproliferative remodeling of the vascular wall due to increased transforming growth factor-[beta] (TGF-[beta]) action. It is well established that thrombospondin1 (TSP1) is a major regulator of TGF-[beta] activation in renal and cardiac complications of diabetes. However, the role of the TSP1-TGF-[beta] pathway in macrovascular diabetic complications, including restenosis, has not been addressed. In mesangial cells, high glucose concentrations depress protein kinase G (PKG) activity, but not PKG-I protein, thereby downregulating transcriptional repression of TSP1. Previously, we showed that high glucose downregulates PKG-I protein expression by vascular smooth muscle cells (VSMCs) through altered NADPH oxidase signaling. In the present study, we investigated whether high glucose regulation of PKG protein and activity in VSMCs similarly regulates TSP1 expression and downstream TGF-[beta] activity. These studies showed that high glucose stimulates both TSP1 expression and TGF-[beta] bioactivity in primary murine aortic smooth muscle cells (VSMCs). TSP1 is responsible for the increased TGF-[beta] bioactivity under high glucose conditions, because treatment with anti-TSP1 antibody, small interfering RNA-TSP1, or an inhibitory peptide blocked glucose-mediated increases in TGF-[beta] activity and extracellular matrix protein (fibronectin) expression. Overexpression of constitutively active PKG, but not the PKG-I protein, inhibited glucose-induced TSP1 expression and TGF-[beta] bioactivity, suggesting that PKG protein expression is insufficient to regulate TSP1 expression. Together, these data establish that glucose-mediated downregulation of PKG levels stimulates TSP1 expression and enhances TGF-[beta] activity and matrix protein expression, which can contribute to vascular remodeling in diabetes. transforming growth factor-[beta]; fibronectin doi: 10.1152/ajpcell.00330.2009.

A-kinase (PKA) anchoring proteins (AKAPs) are essential for targeting type II PKA to specific locales in the cell to control function. In the present study, AKAP5 (formerly AKAP150) and AKAP6 were identified in mouse parotid acini by type II PKA regulatory subunit (RII) overlay assay and Western blot analysis of mouse parotid cellular fractions, and the role of AKAP5 in mouse parotid acinar cell secretion was determined. Mice were euthanized with C[O.sub.2]. Immunofluorescence staining of acinar cells localized AKAP5 to the basolateral membrane, whereas AKAP6 was associated with the perinuclear region. In functional studies, amylase secretion from acinar cells of AKAP5 mutant [knockout (KO)] mice treated with the [beta]-adrenergic agonist, isoproterenol, was reduced overall by 30-40% compared with wild-type (WT) mice. In contrast, amylase secretion in response to the adenylyl cyclase (AC) activator, forskolin, and the cAMP-dependent protein kinase (PKA) activator, [N.sup.6]-phenyl-cAMP, was not statistically different in acini from WT and AKAP5 KO mice. Treatment of acini with isoproterenol mimicked the effect of the Epac activator, 8-(4-methoxyphenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate (8-pMeOPT-2'-O-Me-cAMP), in stimulating Rap1. However, in contrast to isoproterenol, treatment of acini with 8-pMeOPT-2'-O-Me-cAMP resulted in stimulation of amylase secretion from both AKAP5 KO and WT acinar cells. As a scaffolding protein, AKAP5 was found to coimmunoprecipitate with AC6, but not AC8. Data suggest that isoproterenol-stimulated amylase secretion occurs via both an AKAP5/AC6/PKA complex and a PKA-independent, Epac pathway in mouse parotid acini. A-kinase anchoring protein 5; knockout mice; isoproterenol; Epac; 8-(4-methoxyphenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate; Rap1 doi: 10.1152/ajpcell.00382.2009.

Laminin is the first extracellular matrix (ECM) component to be expressed in the developing mammalian embryo. However, the roles of laminin or the related signal pathways are not well known in mouse embryonic stem cells (mESCs). Presently, we examined the effect of laminin on mESC migration. Laminin (10 [micro]g/ml) decreased cell aggregation, whereas migration was increased. Laminin bound [alpha]6[beta]1 integrin and laminin receptor 1 (LR1), decreasing their mRNA levels. Laminin increased focal adhesion kinase (FAK) and paxillin phosphorylation, cAMP intracellular concentration, and the protein levels of exchange factor directly activated by cAMP (Epac1) and Rap1. These increases were completely blocked by [alpha]6[beta]1 integrin and LR1 neutralizing antibody, indicating that laminin-bound LR1 assists laminin-induced [alpha]6[beta]1 integrin activity and initiates signal. As a downstream signal molecule, laminin activated small G protein such as Rac1/cdc42 and its effector protein p21-activated kinase (PAK). Subsequently, laminin stimulated E-cadherin complex disruption. Inhibition of each pathway such as those for [alpha]6[beta]1 integrin and LR1, FAK, Rap1, and PAK1 blocked laminin-induced migration. We conclude that laminin binds both [alpha]6[beta]1 integrin and LR1 and induces signaling FAK/paxillin and cAMP/Epac1/Rap1. These signaling merge at Rac1/cdc42 subsequently activate PAK1. Activated PAK1 enhances E-cadherin complex disruption and finally increases mESCs migration. cAMP/Epac1/Rap1; Rac1/cdc42/p21-activated kinase; E-cadherin doi: 10.1152/ajpcell.00496.2009.

The Mycobacterium tuberculosis genome encodes 11 serine/threonine protein kinases (STPKs) that are structurally related to eukaryotic kinases. To gain insight into the role of Ser/Thr phosphorylation in this major global pathogen, we used a phosphoproteomic approach to carry out an extensive analysis of protein phosphorylation in M. tuberculosis. We identified more than 500 phosphorylation events in 301 proteins that are involved in a broad range of functions. Bioinformatic analysis of quantitative in vitro kinase assays on peptides containing a subset of these phosphorylation sites revealed a dominant motif shared by six of the M. tuberculosis STPKs. Kinase assays on a second set of peptides incorporating targeted substitutions surrounding the phosphoacceptor validated this motif and identified additional residues preferred by individual kinases. Our data provide insight into processes regulated by STPKs in M. tuberculosis and create a resource for understanding how specific phosphorylation events modulate protein activity. The results further provide the potential to predict likely cognate STPKs for newly identified phosphoproteins. signal transduction | phosphorylation motif | phosphoproteomics doi/ 10.1073/pnas.0913482107

Autophagy is important for many cellular processes such as innate immunity, neurodegeneration, aging, and cancer. Although the signaling events triggering autophagy have been studied, little is known regarding the signaling mechanisms by which autophagy is redirected to achieve selective removal of cellular components. We have used the degradation of a peroxisomal marker to investigate the role of protein kinases in selective autophagy of peroxisomes (pexophagy) in Saccharomyces cerevisiae. We show that the Slt2p mitogen-activated protein kinase (MAPK) and several upstream components of its signal transduction pathway are necessary for pexophagy but not for pexophagosome formation or other nonselective and selective forms of autophagy. Other extracellular signals that activate this pathway do not trigger pexophagy on their own, suggesting that this MAPK cascade is necessary but not sufficient to trigger pexophagy. We propose that pexophagy requires the simultaneous activation of this MAPK pathway and a hexose-sensing mechanism acting through protein kinase A and cyclic adenosine monophosphate. doi/10.1083/jcb.200909154