Endogenous macrophage migration inhibitory factor modulates glucocorticoid sensitivity in macrophages via effects on MAP kinase phosphatase-1 and p38 MAP kinase

The pro-inflammatory cytokine macrophage migration inhibitory factor (MIF) is induced by glucocorticoids (GCs), but it was not previously known if MIF regulates cellular sensitivity to GC. Here we show in GC and LPS-treated peritoneal macrophages derived from MIF-/- and wt mice that the absence of endogenous MIF is associated with increased sensitivity to GC of TNF release. This is associated with increased expression of mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP-1), concomitant decreased phosphorylation of p38 MAPK, but no effect of MIF on nuclear factor kappaB (NF-kappaB). These results demonstrate that MIF regulates GC sensitivity by phosphorylation of p38, and provides a cellular mechanism for this observation, indicating that MKP-1 is a central target of this regulation.     

Probing Interactions between CLIP-170, EB1, and Microtubules

Cytoplasmic linker protein 170 (CLIP-170) is a microtubule (MT) plus-end tracking protein (+ TIP) that dynamically localizes to the MT plus end and regulates MT dynamics. The mechanisms of these activities remain unclear because the CLIP-170-MT interaction is poorly understood, and even less is known about how CLIP-170 and other + TIPs act together as a network. CLIP-170 binds to the acidic C-terminal tail of α-tubulin. However, the observation that CLIP-170 has two CAP-Gly (cytoskeleton-associated protein glycine-rich) motifs and multiple serine-rich regions suggests that a single CLIP-170 molecule has multiple tubulin binding sites, and that these sites might bind to multiple parts of the tubulin dimer. Using a combination of chemical cross-linking and mass spectrometry, we find that CLIP-170 binds to both α-tubulin and β-tubulin, and that binding is not limited to the acidic C-terminal tails. We provide evidence that these additional binding sites include the H12 helices of both α-tubulin and β-tubulin and are significant for CLIP-170 activity. Previous work has shown that CLIP-170 binds to end-binding protein 1 (EB1) via the EB1 C-terminus, which mimics the acidic C-terminal tail of tubulin. We find that CLIP-170 can utilize its multiple tubulin binding sites to bind to EB1 and MT simultaneously. These observations help to explain how CLIP-170 can nucleate MTs and alter MT dynamics, and they contribute to understanding the significance and properties of the + TIP network.     

Glucose metabolism,H+ production and Na+/H+- exchanger mRNA levels in ischemic hearts from diabetic rats

Glycolysis uncoupled from glucose oxidation is a major reason for the intracellular acidosis that occurs during severe myocardial ischemia. The imbalance between glycolysis and glucose oxidation, and the resultant H⁺ produced from glycolytically derived ATP hydrolysis in the diabetic rat heart is the focus of this study. Isolated working hearts from 6 week streptozotocin diabetic rat hearts were perfused with 11 mM glucose and 1.2 mM palmitate and subjected to a 25 min period of global ischemia. A second series of experiments were also performed in which hearts from control, diabetic, and islet-transplanted diabetic rats were subjected to a 30 min aerobic perfusion, followed by a 60 min period of low-flow ischemia (coronary flow = 0.5 ml/min) and 30 min of aerobic reperfusion. H⁺ production from glucose metabolism was measured throughout the two protocols by simultaneous measurement of glycolysis and glucose oxidation using perfusate labelled with [5-³H/U-¹⁴C]-glucose. Rates of H⁺ production were calculated by measuring the difference between glycolysis and glucose oxidation. The H⁺ production throughout the perfusion was generally lower in diabetic rat hearts compared to control hearts, while islet-transplantation of diabetic rats increased H⁺ production to rates similar to those seen in control hearts. This occurred primarily due to a dramatic increase in the rates of glycolysis. Despite the difference in H⁺ production between control, diabetic and islet-transplanted diabetic rat hearts, no difference in mRNA levels of the cardiac Na⁺/H⁺-exchanger (NHE-1) was seen. This suggests that alterations in the source of protons (i.e. glucose metabolism) are as important as alterations in the fate of protons, when considering diabetes-induced changes in cellular pH. Furthermore, our data suggests that alterations in Na⁺/H⁺-exchange activity in the diabetic rat heart occur at a post-translational level, possibly due to direct alterations in the sarcolemmal membranes.     

Divergent effect of proteasome inhibition on interleukin-1beta and tumor necrosis factor alpha signaling in human astroglial cells

Impaired functioning of the proteasome pathway is one of the molecular mechanism underlying neurodegenerative changes in Alzheimer's disease. In this study, we report that dysfunction of the proteasome pathway in astroglial cells leads to decreased survival and dysregulation of chemokines by differential regulation of the nuclear factor kappa B and c-jun N-terminal kinase (JNK) pathways. We further demonstrated that proteasome inhibition augmented interleukin-1 beta- and tumor necrosis factor-alpha-induced activation of the IkappaBalpha kinase and MKK4/JNK/c-Jun pathway along with TAK1 activation. These results suggest that impaired function of the proteasome pathway may potentiate the immuno-pathologic role of secondarily activated astrocytes in the brain.     

Transcriptional expression analysis of genes involved in regulation of calcium translocation and storage in finger millet (Eleusine coracana L. Gartn.)

Finger millet (Eleusine coracana) variably accumulates calcium in different tissues, due to differential expression of genes involved in uptake, translocation and accumulation of calcium. Ca^2 +/H⁺ antiporter (CAX1), two pore channel (TPC1), CaM-stimulated type IIB Ca^2 + ATPase and two CaM dependent protein kinase (CaMK1 and 2) homologs were studied in finger millet. Two genotypes GP-45 and GP-1 (high and low calcium accumulating, respectively) were used to understand the role of these genes in differential calcium accumulation. For most of the genes higher expression was found in the high calcium accumulating genotype. CAX1 was strongly expressed in the late stages of spike development and could be responsible for accumulating high concentrations of calcium in seeds. TPC1 and Ca^2 + ATPase homologs recorded strong expression in the root, stem and developing spike and signify their role in calcium uptake and translocation, respectively. Calmodulin showed strong expression and a similar expression pattern to the type IIB ATPase in the developing spike only and indicating developing spike or even seed specific isoform of CaM affecting the activity of downstream target of calcium transportation. Interestingly, CaMK1 and CaMK2 had expression patterns similar to ATPase and TPC1 in various tissues raising a possibility of their respective regulation via CaM kinase. Expression pattern of 14-3-3 gene was observed to be similar to CAX1 gene in leaf and developing spike inferring a surprising possibility of CAX1 regulation through 14-3-3 protein. Our results provide a molecular insight for explaining the mechanism of calcium accumulation in finger millet.     

Links between enhanced fatty acid flux, protein kinase C and NFkappaB activation, and apoB-lipoprotein production in the fructose-fed hamster model of insulin resistance

In the current study, we show evidence, in a fructose-fed hamster model of insulin resistance, that free fatty acid (FFA) can induce hepatic insulin resistance in part via PKC activation leading to increased production of atherogenic apoB100-containing lipoproteins. Interestingly, IκB-kinase β (IKKβ)-dependent NF-κB was activated in hepatocytes from the fructose-fed hamster as an indication for PKC activation. Treatment of hepatocytes with oleate for 16 h showed the activation of the PKC isoforms, PKCα/βII, in a dose dependent manner. Strikingly, the general PKC inhibitor, bisindolylmaleimide-I, Bis-I (5 μM) was found to ameliorate fructose-induced insulin resistance, restoring the phosphorylation status of PKB and suppressing apoB100 overproduction in ex vivo and in vivo. The data suggest that hepatic PKC activation, induced by increased circulating FFA may be an important factor in the development of insulin resistance and dyslipidemia seen in the fructose-fed hamster model.     

Interactions between anion exchange and other membrane proteins in rabbit kidney medullary collecting duct cells

Summary In separated outer medullary collecting duct (MCD) cells, the time course of binding of the fluorescent stilbene anion exchange inhibitor, DBDS (4,4-dibenzamido-2,2-stilbene disulfonate), to the MCD cell analog of band 3, the red blood cell (rbc) anion exchange protein, can be measured by the stopped-flow method and the reaction time constant, _DBDS, can be used to report on the conformational state of the band 3 analog. In order to validate the method we have now shown that the ID_50,DBDS,MCD (0.5±0.1 m) for the H₂-DIDS (4,4-diisothiocyano-2,2-dihydrostilbene disulfonate) inhibition of _DBDS is in agreement with the ID_50,Cl ^–,_MCD (0.94±0.07 m) for H₂-DIDS inhibition of MCD cell Cl^– flux, thus relating _DBDS directly to anion exchange. The specific cardiac glycoside cation transport inhibitor, ouabain, not only modulates DBDS binding kinetics, but also increases the time constant for Cl^– exchange by a factor of two, from _Cl=0.30±0.02 sec to 0.56±0.06 sec (30mm NaHCO₃). The ID_50,DBDS,MCD for the ouabain effect on DBDS binding kinetics is 0.003±0.001 m, so that binding is about an order of magnitude tighter than that for inhibition of rbc K⁺ flux (K _I,K ⁺,_rbc=0.017 m). These experiments indicate that the Na⁺,K^–-ATPase, required to maintain cation gradients across the MCD cell membrane, is close enough to the band 3 analog that conformational information can be exchanged. Cytochalasin E (CE), which binds to the spectrin/actin complex in rbc and other cells, modulates DBDS binding kinetics with a physiological ID_50,DBDS,MCD (0.076±0.005 m); 2 m CE also more than doubles the Cl^– exchange time constant from 0.20±0.04 sec to 0.50±0.08 sec (30mm NaHCO₃). These experiments indicate that conformational information can also be exchanged between the MCD cell band 3 analog and the MCD cell cytoskeleton.     

Crystal structure of human adenylate kinase 4 (L171P) suggests the role of hinge region in protein domain motion

It is well known that motion of LID and NMP-binding (NMP_bind) domains in adenylate kinase (AK) is important in ligand binding and catalysis. However, the nature of such domain motions is poorly characterized. One of the critical hinge regions is hinge IV, which connects the CORE and LID domains. In addition, the hinge IV contains a strictly conserved residue, L171, in the AK family. To investigate the role of hinge IV, crystal structure of human adenylate kinase 4 (AK4) L171P mutant was determined. This mutation dramatically changes the orientation of the LID domain, which could be described as a novel twisted-and-closed conformation in contrast to the open and closed conformations in other AKs. This mutant provides a new example of domain motions in AK family.     

Characterization of the CaMKKβ-AMPK signaling complex

The AMP-activated protein kinase (AMPK) is a critical regulator of energy homeostasis, and is a potential target for treatment of metabolic diseases as well as cancer. AMPK can be phosphorylated and activated by the tumor suppressor LKB1 or the Ca²⁺/CaM-dependent protein kinase kinase β (CaMKKβ). We previously identified a physical complex between CaMKKβ and AMPK (Anderson, K. A., Ribar, T. J., Lin, F., Noeldner, P. K., Green, M. F., Muehlbauer, M. J., Witters, L. A., Kemp, B. E., and Means, A. R. (2008) Cell Metabolism 7, 377–388). Here we expand our analysis of the CaMKKβ–AMPK signaling complex and show that whereas CaMKKβ can form a complex with and activate AMPK, CaMKKα cannot. In addition, we show that CaMKKβ and AMPK associate through their kinase domains, and CaMKKβ must be in an active conformation in order to bind AMPK but not to associate with an alternative substrate, Ca²⁺/Calmodulin-dependent protein kinase IV (CaMKIV). Our results demonstrate that CaMKKβ and AMPK form a unique signaling complex. This raises the possibility that the CaMKKβ–AMPK complex can be specifically targeted by small molecule drugs to treat disease.     

Crystal structure of human FAF1 UBX domain reveals a novel FcisP touch-turn motif in p97/VCP-binding region

UBX domain is a general p97/VCP-binding module found in an increasing number of proteins including FAF1, p47, SAKS1 and UBXD7. FAF1, a multi-functional tumor suppressor protein, binds to the N domain of p97/VCP through its C-terminal UBX domain and thereby inhibits the proteasomal protein degradation in which p97/VCP acts as a co-chaperone. Here we report the crystal structure of human FAF1 UBX domain at 2.9 Å resolution. It reveals that the conserved FP sequence in the p97/VCP-binding region adopts a rarely observed cis-Pro touch-turn structure. We call it an FcisP touch-turn motif and suggest that it is the conserved structural element of the UBX domain. Four FAF1 UBX molecules in an asymmetric unit of the crystal show two different conformations of the FcisP touch-turn motif. The phenyl ring of F⁶¹⁹ in the motif stacks partly over cis-Pro⁶²⁰ in one conformation, whereas it is swung out from cis-P⁶²⁰, in the other conformation, and forms hydrophobic contacts with the residues of the neighboring molecule. In addition, the entire FcisP touch-turn motif is pulled out in the second conformation by about 2 Å in comparison to the first conformation. Those conformational differences observed in the p97/VCP-binding motif caused by the interaction with neighboring molecules presumably represent the conformational change of the UBX domain on its binding to the N domain of p97/VCP.     

Protein kinase C-beta: An emerging connection between nutrient excess and obesity

There is currently a global epidemic of obesity as a result of recent changes in lifestyle. Excess body fat deposition is caused by an imbalance between energy intake and energy expenditure due to interactions between genetic and environmental factors. The signals and biological mechanisms that trigger fat accumulation by disrupting energy homeostasis are not well understood. There is considerable evidence now supporting a possible role of protein kinase C beta (PKCβ) in energy homeostasis. This review highlights recent findings on the role of PKCβ activation in the genesis and progression of obesity, and of PKCβ repression in mediating the beneficial effects of physical exercise. Available data support a model in which adipose PKCβ activation is among the initiating events that disrupt mitochondrial function through interaction with p66^shc and amplify fat accumulation and adipose dysfunction, with systemic consequences. Manipulation of PKCβ levels, activity, or signaling could provide a therapeutic approach to combat obesity and associated metabolic disorders.     

Interactions between valproate,glutamate, aspartate,and GABA with respect to uptake in astroglial primary cultures

Astrocytes have been proposed to regulate the extracellular space in the brain, even if rather little is known about their specific functions. One possibility for obtaining more knowledge on the functions of astroglial cells is to examine how they respond on exposure to pharmacological agents. Na⁺-valproate is an anticonvulsive drug which is used in the treatment of several types of epilepsy. The mechanisms of action of the drug are not fully understood, but the GABA-ergic system, both in neurons and astrocytes, has been shown to be affected. In the present study, the effects of valproate were investigated on astroglial cells in primary cultures from newborn rat cerebral cortex. The transport of the drug itself and its effects on the transport of the amino acid transmitters glutamate, aspartate and -aminobutyric acid (GABA) into astrocytes were examined. The [³H]valproate transport into the astrocytes was increased after exposure tol-glutamate but notl-aspartate. On the other hand, after acute exposure for the drug, the transport of [³H]l-glutamate and [³H]l-aspartate decreased, as also did the affinity but not the transport capacity for the [³H]GABA uptake. However, after 5 days chronic valproate exposure, no effects could be seen on the uptake kinetics ofl-glutamate orl-aspartate. For GABA, the affinity decreased, while the transport capacity remained unchanged compared with controls. The results showed that valproate, glutamate, aspartate and GABA were capable of interacting significantly with each others transport into the astrocytes.     

IKK regulates the deubiquitinase CYLD at the postsynaptic density

K63-linked polyubiquitination of proteins regulates their trafficking into specific cellular pathways such as endocytosis and autophagy. CYLD, a deubiquitinase specific for K63-linked polyubiquitins, is present in high quantities at the postsynaptic density (PSD). It was previously shown that, under excitatory conditions, CaMKII activates CYLD in a Ca²⁺-dependent manner. The observation that CYLD can also be phosphorylated in the absence of Ca²⁺ in isolated PSDs led us to further explore the regulation of CYLD under basal conditions. A possible involvement of the autonomous form of CaMKII and IKK, both kinases known to be localized at the PSD, was examined. A CaMKII inhibitor CN21 had no effect on CYLD phosphorylation in the absence of Ca²⁺, but two different IKK inhibitors, IKK16 and tatNEMO, inhibited its phosphorylation. Immuno-electron microscopy on hippocampal cultures, using an antibody for CYLD phosphorylated at S-418, revealed that the phosphorylated form of CYLD is present at the PSD under basal conditions. Phosphorylation of CYLD under basal conditions was inhibited by IKK16. NMDA treatment further promoted phosphorylation of CYLD at the PSD, but IKK16 failed to block the NMDA-induced effect. In vitro experiments using purified proteins demonstrated direct phosphorylation and activation of CYLD by the beta catalytic subunit of IKK. Activation of IKK in isolated PSDs also promoted phosphorylation of CYLD and an increase in endogenous deubiquitinase activity for K63-linked polyubiquitins. Altogether, the results suggest that in the absence of excitatory conditions, constitutive IKK activity at the PSD regulates CYLD and maintains basal levels of K63-linkage specific deubiquitination at the synapse.     

Importance for absorption of Na+ from freshwater of lysine, valine and serine substitutions in the alpha1a-isoform of Na,K-ATPase in the gills of rainbow trout (Oncorhynchus mykiss) and Atlantic salmon (Salmo salar)

In the gills of rainbow trout and Atlantic salmon, the alpha1a- and alpha1b-isoforms of Na,K-ATPase are expressed reciprocally during salt acclimation. The alpha1a-isoform is important for Na(+) uptake in freshwater, but the molecular basis for the functional differences between the two isoforms is not known. Here, three amino acid substitutions are identified in transmembrane segment 5 (TM5), TM8 and TM9 of the alpha1a-isoform compared to the alpha1b-isoform, and the functional consequences are examined by mutagenesis and molecular modeling on the crystal structures of Ca-ATPase or porcine kidney Na,K-ATPase. In TM5 of the alpha1a-isoform, a lysine substitution, Asn783 --> Lys, inserts the epsilon-amino group in cation site 1 in the E(1) form to reduce the Na(+)/ATP ratio. In the E(2) form the epsilon-amino group approaches cation site 2 to force ejection of Na(+) to the blood phase and to interfere with binding of K(+). In TM8, a Asp933 --> Val substitution further reduces K(+) binding, while a Glu961 --> Ser substitution in TM9 can prevent interaction of FXYD peptides with TM9 and alter Na(+) or K(+) affinities. Together, the three substitutions in the alpha1a-isoform of Na,K-ATPase act to promote binding of Na(+) over K(+) from the cytoplasm, to reduce the Na(+)/ATP ratio and the work done in one Na,K pump cycle of active Na(+) transport against the steep gradient from freshwater (10-100 microM: Na(+)) to blood (160 mM: Na(+)) and to inhibit binding of K(+) to allow Na(+)/H(+) rather than Na(+)/K(+) exchange.     

Involvement of Na+/H+ exchangers in induction of cyclooxygenase-2 by vacuolar-type (H+)-ATPase inhibitors in RAW 264 cells

In the mouse macrophage-like cell line RAW 264, vacuolar-type (H(+))-ATPase (V-ATPase) inhibitors, bafilomycin A(1) and concanamycin A, increased the level of cyclooxygenase (COX)-2 protein and its mRNA. The V-ATPase inhibitor-induced expression of COX-2 was suppressed by inhibitors of c-jun N-terminal kinase (JNK) and nuclear factor-kappaB, and by inhibitors of Na(+)/H(+) exchangers (NHEs). The bafilomycin A(1)-induced activation of JNK but not degradation of IkappaB-alpha was suppressed by NHE inhibitors and by an inhibitor of Na(+)/Ca(2+) exchanger SN-6. These results suggested that V-ATPase inhibitors induce the expression of COX-2 via NHE-dependent and -independent pathways.     

Nitrative and oxidative DNA damage caused by K-ras mutation in mice

Ras mutation is important for carcinogenesis. Carcinogenesis consists of multi-step process with mutations in several genes. We investigated the role of DNA damage in carcinogenesis initiated by K-ras mutation, using conditional transgenic mice. Immunohistochemical analysis revealed that mutagenic 8-nitroguanine and 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) were apparently formed in adenocarcinoma caused by mutated K-ras. 8-Nitroguanine was co-localized with iNOS, eNOS, NF-κB, IKK, MAPK, MEK, and mutated K-ras, suggesting that oncogenic K-ras causes additional DNA damage via signaling pathway involving these molecules. It is noteworthy that K-ras mutation mediates not only cell over-proliferation but also the accumulation of mutagenic DNA lesions, leading to carcinogenesis.