![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
TOXICOLOGY
Toxicology and Drug Disposition, Lilly Research Laboratories, Eli Lilly and Company, Greenfield, Indiana (M.A.D., S.J., Y.-H.H., N.H.H.); University of Massachusetts Medical School, Proteomic Consortium, Shrewsbury, Massachusetts (D.H., J.L., S.W.T.); and Medical University of South Carolina, Department of Pharmacology, Charleston, South Carolina (J.R.-B.)
Received for publication
December 19, 2005
Accepted
May 8, 2006.
 |
Abstract |
|---|
 Top Abstract Materials and MethodsResultsDiscussionReferences |
|---|
H89 (N-(2-[p-bromocinnamylamino-]ethyl)-5-isoquinolinesulfonamide) is a commercially available isoquinolinesulfonyl kinase inhibitor (Hidaka et al., 1984
). It is purportedly a potent inhibitor of protein kinase A (PKA), although it may have additional kinase inhibitory activities (Davies et al., 2000; Choi et al., 2001; Makaula et al., 2005). H89 has been widely used as a tool to inhibit PKA in cell-based or biochemical assays; however, mammalian studies using H89 in vivo are limited. Short-term studies that used intrathecal, topical, s.c., or intracerebroventricular administration have been reported with durations of treatment ranging from 5 to 120 min (Hua et al., 1999; Dolan and Nolan, 2001; Vargas et al., 2001; Fang et al., 2003; Li and Chen, 2003; Sun et al., 2004; Lim et al., 2005). Recently, H89 administration to isolated rat hearts has been reported (Makaula et al., 2005). We used H89 as a tool to determine whether differential profiling of tissue phosphoproteins can be used to detect the effects of PKA inhibitor treatment in vivo. The data presented support the application of differential proteomic methods to measure the effects of kinase inhibitor treatment on protein phosphorylation in vivo. We used standard two-dimensional gel MALDI-TOF MS analysis technologies in combination with two recently commercialized reagents: a QIAGEN phosphoprotein enrichment column and the phosphoprotein specific dye, Pro-Q Diamond stain, to analyze proteins from H89-treated rat liver tissues (Schulenberg et al., 2004; Makrantoni et al., 2005; Wu et al., 2005). Using this method, we successfully identified a decrease in phosphorylation of kinase substrates in liver protein extracts from animals treated with the kinase inhibitor H89. The results presented support the possibility that kinase inhibitor activity can be examined from whole animal studies via differential profiling of phosphoproteins isolated from tissues.
|
Materials and Methods |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Experimental Animals, Treatment Groups, and Pharmacokinetic Study. All protocols and animal care were approved and performed according to the Institutional Animal Care and Use Committee of Eli Lilly Research Laboratories (Greenfield, IN). Adult male Sprague-Dawley rats (250 ± 40 g b.wt.; Harlan, Indianapolis, IN) were housed in a temperature-controlled room with a 12:12-h light/dark cycle and fed a standard chow diet throughout their stay in the animal facility.
Animals were randomly assigned to one of three treatment groups comprising six animals per group: 5% DMSO in 0.9% sterile saline (vehicle) only; 20 mg/kg H89 in vehicle, or 200 mg/kg H89 in vehicle. Animals were dosed twice daily on test days 0 to 2 at approximately 8:00 AM and 1:00 PM. A single dose was administered at approximately 8:30 AM on test day 3 to accommodate the 12:30 PM necropsy on day 3. Animals were euthanized by CO2 chamber, and sections from the left lateral lobe of the liver from each animal were clamp frozen with metal clamps prechilled in liquid nitrogen. Tissue samples were stored at -80°C until processed for protein analysis.
A separate group of animals was used for pharmacokinetic measurements. Approximately 1.0 ml of whole blood was collected into heparinized collection tubes at 0.5, 1, 4, and 24 h after a single dose of 20 or 200 mg/kg H89. Whole blood was processed to plasma, and plasma samples were stored at below -60°C immediately after processing until analyzed. Plasma levels of H89 were determined by a standard protein precipitation and LC-tandem mass spectrometry detection. Stock solutions (1 mg/ml) of H89 were prepared in ethanol and were serially diluted with pooled rat plasma to prepare duplicate standards ranging from 1 to 4000 ng/ml. One set of standards was analyzed at the beginning and one set at the end of each sample batch. Plasma samples (0.05 ml) in 96-well plates were treated by protein precipitation with the addition of methanol solution containing an analog internal standard (0.1 ml). The resulting mixtures were centrifuged at 4000 rpm and 4°C for 10 min, and supernatants (0.05 ml) were transferred to a second 96-well plate for MS injection. The samples were analyzed with a Sciex API 4000 triple quadrupole mass spectrometer (Sciex Division of MDS Inc., Toronto, ON, Canada) coupled to a Shimadzu high-performance liquid chromatography system (LC-IOAD; Shimadzu Biotech, Tokyo, Japan) and a HTS PAL Leap autosampler (Leap Technology, Carrboro, NC). Samples (0.01 ml) were injected onto a high-performance liquid chromatography column (5-µm Merck Chromolith SpeedRod, 4.6 x 50 mm) and eluted with the gradient mode at 45°C. The chromatographic conditions consisted of a mobile phase A (1000:1, water-88% formic acid, v/v), and mobile phase B (1000:1, MeOH-88% formic acid, v/v) that was run over a 1-min gradient at a flow rate of 1.5 ml/min. A positive ion mode with turbo spray, an ion source temperature of 550°C, and a dwell time of 100 ms were used for mass spectrometric detection. Quantitation was performed using multiple reaction monitoring at the 445.92 to 194.83 transitions. The calibration curves were obtained by plotting the peak area ratio of parent drug to the internal standard versus drug concentration using linear regression with 1/x2 weighting. Pharmacokinetic parameters were calculated using a noncompartmental model.
Enrichment for Phosphorylated Rat Liver Protein. Phosphoproteins from rat liver were enriched on a QIAGEN PhosphoProtein Purification column by the manufacturer's protocol. In brief, rat liver protein was extracted by homogenization in lysis buffer containing 0.25% (w/v) CHAPS, protease/phosphatase inhibitors, and benzonase as described in the manufacturer's protocol (Phosphoprotein Purification Kit; QIAGEN, Valencia, CA) for 30 min at 4°C and centrifuged at 10,000g at 4°C for 30 min to remove insoluble material.
Total extracted rat liver protein was diluted to a concentration of 0.1 mg/ml in a total of 25 ml of lysis buffer (described above) and was applied to a lysis buffer-equilibrated PhosphoProtein purification column at room temperature. After the column was washed with 6.0 ml of lysis buffer, the phosphoproteins were eluted with 2 ml of PhosphoProtein Elution Buffer. The yield of phosphorylated protein was determined by the Bradford assay. The flow-through samples were passed through two additional columns to ensure complete removal of phosphoproteins from the sample. The phosphoproteins were then concentrated by ultrafiltration in a 10-kDa cutoff Amicon Ultra column (Millipore Corporation, Billerica, MA).
QIAGEN PhosphoProtein Purification Column and Pro-Q Stain Validation. The QIAGEN phosphoprotein column recovery was validated using a standard phosphopeptide [Pp60 c-src (521-533), no. 86-3-11; American Peptide Company (Sunnyvale, CA). A seven-point standard curve was prepared, ranging from 12.5 to 1000 ng, and analyzed by LC/UV with the LC Packings capillary LC system with a 100 µm C18 column, and 250 ng of the standard peptide was spiked into a 250-mg liver lysate and loaded onto the QIAGEN column. The flow-through, wash, and eluent were collected and analyzed by LC/UV to determine the amount of peptide retained and recovered from the phosphoprotein enrichment procedure. Standard curves were obtained before and after the flow-through, wash, and eluent fractions were injected.
To validate the Pro-Q Diamond stain specificity, 4 µg of the liver protein was solublized in NuPAGE LDS buffer (Invitrogen, Carlsbad CA), reduced with 50 mM dithiothreitol, and electrophoresed on a 4 to 12% bis-Tris SDS NuPAGE gel for 1 h at 200 V. The gel was fixed and stained in Pro-Q Diamond by the manufacturer's protocol (Invitrogen) and imaged on an Amersham (GE Healthcare, Fairfield, CT) Typhoon laser scanner and then counterstained in Coomassie Blue and imaged on a flat-bed scanner.
Two-Dimensional Electrophoresis. Before isoelectric focusing (IEF), samples were acetone-precipitated and solubilized in 40 mM Tris, 7 M urea, 2 M thiourea, and 2% CHAPS, reduced with tributylphosphine, and alkylated with 10 mM acrylamide for 90 min at room temperature. After a second round of acetone precipitation, the pellet was solubilized in 7 M urea, 2 M thiourea, and 2% CHAPS (resuspension buffer), and the buffer was run through a 10-kDa cutoff Amicon Ultra device with at least 2 volumes of the resuspension buffer to reduce the conductivity of the sample. Seventy-five micrograms of protein were subjected to IEF on 11-cm pH 3 to 10 and pH 4 to 7 immobilized pH gradient strips (Proteome Systems, Sydney, NSW, Australia). All gels were run in triplicate. After IEF, immobilized pH gradient strips were equilibrated in 6 M urea, 2% SDS, 50 mM Tris-acetate buffer, pH 7.0, and 0.01% bromphenol blue and subjected to SDS-polyacrylamide gel electrophoresis on 6 to 15% Gel Chips (Proteome Systems). All gels were fixed and stained in Pro-Q Diamond, 60 ml/gel, imaged on the Typhoon scanner and then stained in SYPRO Ruby, 70 ml/gel (Invitrogen) and imaged again on the Typhoon scanner.
Image Analysis. Analysis of all gel images was performed using Progenesis Discovery and Pro (Nonlinear Dynamics Inc., Newcastle upon Tyne, UK). After spot detection, matching, background subtraction, normalization, and filtering, data for both Pro-Q Diamond and SYPRO Ruby gel images was exported to the Pro Informatics package. Normalized volumes for all spots were subjected to principle component analysis (PCA). Pairwise comparisons between groups was performed using the Student's t test in Progenesis Discovery to identify spots whose expression was significantly altered (p < 0.05) due to treatment.
Protein Digestion and MALDI Analysis. Protein spots were automatically detected and excised using the Xcise apparatus (Shimadzu Biotech). Gel pieces were washed twice with 150 µl of 25 mM ammonium bicarbonate, pH 8.2, and 50% v/v acetonitrile and then dehydrated by the addition of 100% ACN and air-dried. Next 30 ng of trypsin (Promega, Madison, WI) in 25 mM ammonium bicarbonate (20 µg/µl) was added to each gel piece and incubated at 30°C for 16 h. The peptides were extracted by sonication. The peptide solution was automatically desalted and concentrated using ZipTips from Millipore Corporation (Bedford, MA) on the Xcise apparatus and spotted onto the Axima (Kratos, Manchester, UK) MALDI target plate. Peptide mass fingerprints of and postsource decay tryptic peptides were generated by MALDI-TOF-MS using an Axima-CFR mass spectrometer (Kratos).
MS Database Search. All spectra were automatically analyzed by the BioinformatIQ integrated suite of bioinformatics tools from Proteome Systems, using a combination of Ioniq and Mascot searching algorithms. Protein identifications were assigned by comparing peak lists to a database containing theoretical tryptic digests of NCBI and Swiss Prot sequence databases. Protein identification was evaluated based on percent coverage, Mowse score, number of peptide matches, peak intensity, and match of pI and molecular weight with the location of the protein on the 2D gel. Postsource decay experiments were also acquired to confirm protein identifications.
|
Results |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
|
|
|
Pro-Q Diamond from Invitrogen is a sensitive new noncovalent fluorescent dye staining technology for the detection of phosphoserine-, phosphothreonine- and phosphotyrosine-containing proteins displayed on SDS-polyacrylamide gels and 2D gels. Gels were fixed, washed with water, and then stained by incubation in a single solution. The stain can quantify protein loads ranging from 1.0 ng to 1.0 µg on a single electrophoresis gel. The stain was fully compatible with matrix-assisted laser desorption time-of-flight mass spectrometry, thus facilitating protein identification after gel electrophoresis.
Subsequent to Pro-Q Diamond staining, SYPRO Ruby was used to restain the same gel to reveal total protein level. This protein gel stain is a noncovalent stain that does not require aldehyde fixation. As little as 75 fmol of stained protein can be recovered from the gel and accurately identified using MALDI-TOF mass spectrometry. The stain has a broad linear quantitation range, extending over 3 orders of magnitude. These features made it possible to obtain accurate protein quantitation for both highly expressed and minimally expressed proteins in the gel. The specificity of the Pro-Q Diamond stain was also confirmed by analyzing the liver flow-through and eluent from the QIAGEN column by SDS-polyacrylamide gel electrophoresis. Flow-through and eluent from the QIAGEN column were separated by SDS-polyacrylamide gel electrophoresis, and gels were stained with Pro-Q Diamond. The eluent, consisting of a concentrated fraction of phosphorylated protein, stained heavily with Pro-Q Diamond compared with the light staining of the flow-through samples, which contained mostly nonphosphorylated protein (data not shown).
After QIAGEN column enrichment, each sample was resolved on 2D gel in two pH ranges, 3 to 10 and 4 to 7, in triplicate, and stained with Pro-Q diamond and SYPRO Ruby dyes. Protein separation and gel staining was highly reproducible (Fig. 3, A and B). Proteins were much better resolved on the narrow-range pH 4 to 7 gels, which is important to note because changes in phosphorylation status may result in a relatively small change in the pI of a protein. The combined use of Pro-Q Diamond and the total protein dye, SYPRO Ruby, enabled the detection and quantitation of low abundance, but highly phosphorylated, proteins.
|
|
|
|
Discussion |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
; Rakus et al., 2003) and Rakus et al. (2003) proposed that phosphorylation of FBPase may regulate its activity. PKA phosphorylation of the liver isoform of fructose 2,6-bisphosphatase (an inhibitor of fructose 1,6-bisphosphatase) at serine 32 has been reported (Kurland et al., 2000). Serines 388, 341, and 356 of FBPase have also been reported to be phosphorylation sites for PKA (Ekman and Dahlqvist-Edberg, 1981; Ekdahl, 1987). These data are consistent with the fact that cAMP plays a major, if not primary, role in the regulation of hepatic gluconeogenesis. The relationship between the observed difference in fructose 2,6-bisphosphatase phosphorylation and hepatic metabolism was not determined in this study, but increased expression of hepatic fructose 2,6-bisphosphate resulted in lowered blood glucose levels in normal mice accompanied by increased plasma lactate, triglycerides, and free fatty acids (Wu et al., 2001). Fructose 1,6-bisphosphatase was also found to be very sensitive for assessing cadmium-induced nephrotoxicity (Rajanna et al., 1984). Additional work is underway to determine whether the phosphorylation state of this protein family can serve as a specific biomarker of cAMP/PKA pathway modulation in liver and the relationship to hepatic glucose metabolism.
We also observed that heterogeneous nuclear ribonucleoprotein (hnRNP) was differentially phosphorylated in H89-treated liver protein extracts. hnRNP proteins play important roles in mRNA processing. Xie et al. (2003) reported that nucleocytoplasmic transport of hnRNP1 was regulated by cAMP-dependent PKA. They also showed direct phosphorylation of hnRNP on Ser-16 via PKA activation and demonstrated that phosphorylation of Ser-16 modulates the nucleocytoplasmic distribution of hnRNP in PC12 cells. An interesting follow-up to these studies will be the effect of H89 on hepatic mRNA processing.
N-Ethylmaleimide-sensitive fusion protein cofactor p47 (p97 cofactor 47) is an accessory protein for the p97-mediated fusion pathway, one of the distinct pathways of membrane fusion involved in Golgi reassembly during mitosis (Kondo et al., 1997). It is essential for the p97-mediated regrowth of Golgi cisternae from mitotic Golgi fragments, a process restricted to animal cells. Phosphorylation of p47 on serine 140 by cdc2 appears to be important for Golgi disassembly-assembly during the cell cycle (Uchiyama et al., 2003). Recently, Kano et al. reported that maintenance of the endoplasmic reticulum network requires a process mediated by p97/p47, and cell cycle-dependent morphological changes of the endoplasmic reticulum network are regulated through phosphorylation/dephosphorylation of p47 (Kano et al., 2005). It is widely recognized that cAMP and PKA are required to maintain G2 arrest and that a drop in PKA activity is required for M phase-promoting factor activation. There have been reports of cdc25 being a substrate of PKA (Duckworth et al., 2002). Identification of the specific residue modified by H89 treatment may provide additional evidence of associating camp/PKA, cdc2, and regulation of p47. Thioredoxin domain-containing protein 4 precursor and eukaryotic translation initiation factor 4E were also identified as differential phosphorylated proteins in protein extracts from H89-treated liver. Confirmation of regulation of these two proteins by H89 in liver will require additional study.
The reported specificity of H89 is an important factor in interpreting these data. In a standard in vitro cell-free assay conducted at 0.1 mM ATP, H89 (at 10 µM) inhibited eight protein kinases by 80 to 100% (Davies et al., 2000). IC50 values were determined for the protein kinases that were inhibited most strongly, and these studies revealed that three (MSK1, S6K1, and ROCK-II) were inhibited with potency similar to or greater than that for PKA (Davies et al., 2000). Of the proteins identified in our in vivo study, none of them appeared to have predicted MSK1, S6K1, or ROCK-II phosphorylation sites, but there were other protein spots that remain to be identified. It is remarkable that the proteins with the most robust changes in phosphorylation in samples from H89-treated animals all have a potential regulatory connection to cAMP/PKA. The results presented support the idea that key information about kinase inhibitor activity can be derived from whole animal studies via differential profiling of phosphoproteins isolated from tissues. Furthermore, this method shows promise for identification of substrate biomarkers that can be used to establish kinase pathway modulation in vivo.
|
Acknowledgements |
|---|
|
Footnotes |
|---|
ABBREVIATIONS: H89, N-(2-[p-bromocinnamylamino-]ethyl)-5-isoquinolinesulfonamide; PKA, protein kinase A; 2D gel, two-dimensional gel electrophoresis; MALDI, matrix-assisted laser desorption/ionization; TOF, time-of-flight; MS, mass spectrometry; DMSO, dimethyl sulfoxide; LC, liquid chromatography; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; IEF, isoelectric focusing; PCA, principle component analysis; FBPase, fructose 1,6-bisphosphatase; hnRNP, heterogeneous nuclear ribonucleoprotein.
Address correspondence to: Dr. Myrtle Davis, Toxicology and Drug Disposition, Lilly Research Laboratories, Eli Lilly and Company, Greenfield, IN 46140. E-mail: davisma{at}lilly.com
|
References |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Choi J, Choi BH, Hahn SJ, Yoon SH, Min DS, Jo Y, and Kim M (2001) Inhibition of Kv1.3 channels by H-89 (N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide) independent of protein kinase A. Biochem Pharmacol 61: 1029-1032.[CrossRef][Medline]
Davies SP, Reddy H, Caivano M, and Cohen P (2000) Specificity and mechanism of action of some commonly used protein kinase inhibitors. Biochem J 351: 95-105.[CrossRef][Medline]
Dolan S and Nolan AM (2001) Biphasic modulation of nociceptive processing by the cyclic AMP-protein kinase A signalling pathway in sheep spinal cord. Neurosci Lett 309: 157-160.[CrossRef][Medline]
Duckworth BC, Weaver JS, and Ruderman JV (2002) G2 arrest in Xenopus oocytes depends on phosphorylation of cdc25 by protein kinase A. Proc Natl Acad Sci USA 99: 16794-16799.
Ekdahl KN (1987) Rat liver fructose-1,6-bisphosphatase: identification of serine 338 as a third major phosphorylation site for cyclic AMP-dependent protein kinase and activity changes associated with multisite phosphorylation in vitro. J Biol Chem 262: 16699-16703.
Ekman P and Dahlqvist-Edberg U (1981) The kinetics of unphosphorylated, phosphorylated and proteolytically modified fructose bisphosphatase from fat liver. Biochim Biophys Acta 662: 265-270.[Medline]
Fang L, Wu J, Lin Q, and Willis WD (2003) Protein kinases regulate the phosphorylation of the GluR1 subunit of AMPA receptors of spinal cord in rats following noxious stimulation. Brain Res Mol Brain Res 118: 160-165.[Medline]
Hidaka H, Inagaki M, Kawamoto S, and Sasaki Y (1984) Isoquinolinesulfonamides, novel and potent inhibitors of cyclic nucleotide dependent protein kinase and protein kinase C. Biochemistry 23: 5036-5041.[CrossRef][Medline]
Hua XY, Chen P, and Yaksh TL (1999) Inhibition of spinal protein kinase C reduces nerve injury-induced tactile allodynia in neuropathic rats. Neurosci Lett 276: 99-102.[CrossRef][Medline]
Kano F, Kondo H, Yamamoto A, Tanaka AR, Hosokawa N, Nagata K, and Murata M (2005) The maintenance of the endoplasmic reticulum network is regulated by p47, a cofactor of p97, through phosphorylation by cdc2 kinase. Genes Cells 10: 333-344.
Kondo H, Rabouille C, Newman R, Levine TP, Pappin D, Freemont P, and Warren G (1997) p47 is a cofactor for p97-mediated membrane fusion. Nature (Lond) 388: 75-78.[CrossRef][Medline]
Kurland IJ, Chapman B, and El-Maghrabi MR (2000) N and C termini modulate the effects of pH and phosphorylation on hepatic 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. Biochem J 347: 459-467.[CrossRef][Medline]
Li KC and Chen J (2003) Differential roles of spinal protein kinases C and A in development of primary heat and mechanical hypersensitivity induced by subcutaneous bee venom chemical injury in the rat. Neurosignals 12: 292-301.[CrossRef][Medline]
Lim G, Wang S, and Mao J (2005) cAMP and protein kinase A contribute to the downregulation of spinal glutamate transporters after chronic morphine. Neurosci Lett 376: 9-13.[CrossRef][Medline]
Makaula S, Lochner A, Genade S, Sack MN, Awan MM, and Opie LH (2005) H-89, a non-specific inhibitor of protein kinase A, promotes post-ischemic cardiac contractile recovery and reduces infarct size. J Cardiovasc Pharmacol 45: 341-347.[CrossRef][Medline]
Makrantoni V, Antrobus R, Botting CH, and Coote PJ (2005) Rapid enrichment and analysis of yeast phosphoproteins using affinity chromatography, 2D-PAGE and peptide mass fingerprinting. Yeast 22: 401-414.[CrossRef][Medline]
Murray KJ, El-Maghrabi MR, Kountz PD, Lukas TJ, Soderling TR, and Pilkis SJ (1984) Amino acid sequence of the phosphorylation site of rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. J Biol Chem 259: 7673-7681.
Rajanna B, Hobson M, Reese J, Sample E, and Chapatwala KD (1984) Chronic hepatic and renal toxicity by cadmium in rats. Drug Chem Toxicol 7: 229-241.[Medline]
Rakus D, Zarzycki M, and Dzugaj A (2003) Rabbit muscle fructose-1,6-bisphosphatase is phosphorylated in vivo. Acta Biochim Pol 50: 115-121.[Medline]
Schulenberg B, Goodman TN, Aggeler R, Capaldi RA, and Patton WF (2004) Characterization of dynamic and steady-state protein phosphorylation using a fluorescent phosphoprotein gel stain and mass spectrometry. Electrophoresis 25: 2526-2532.[CrossRef][Medline]
Sun RQ, Tu YJ, Lawand NB, Yan JY, Lin Q, and Willis WD (2004) Calcitonin gene-related peptide receptor activation produces PKA- and PKC-dependent mechanical hyperalgesia and central sensitization. J Neurophysiol 92: 2859-2866.
Uchiyama K, Jokitalo E, Lindman M, Jackman M, Kano F, Murata M, Zhang X, and Kondo H (2003) The localization and phosphorylation of p47 are important for Golgi disassembly-assembly during the cell cycle. J Cell Biol 161: 1067-1079.
Vargas ML, Abella C, and Hernandez J (2001) Diazepam increases the hypothalamic-pituitary-adrenocortical (HPA) axis activity by a cyclic AMP-dependent mechanism. Br J Pharmacol 133: 1355-1361.[CrossRef]
Wu C, Okar DA, Newgard CB, and Lange AJ (2001) Overexpression of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase in mouse liver lowers blood glucose by suppressing hepatic glucose production. J Clin Investig 107: 91-98.[Medline]
Wu J, Lenchik NJ, Pabst MJ, Solomon SS, Shull J, and Gerling IC (2005) Functional characterization of two-dimensional gel-separated proteins using sequential staining. Electrophoresis 26: 225-237.[CrossRef][Medline]
Xie J, Lee JA, Kress TL, Mowry KL, and Black DL (2003) Protein kinase A phosphorylation modulates transport of the polypyrimidine tract-binding protein. Proc Natl Acad Sci USA 100: 8776-8781.
This article has been cited by other articles:
|
|
 |
|
  N. R. Zearfoss, J. M. Alarcon, P. Trifilieff, E. Kandel, and J. D. Richter A Molecular Circuit Composed of CPEB-1 and c-Jun Controls Growth Hormone-Mediated Synaptic Plasticity in the Mouse Hippocampus J. Neurosci., August 20, 2008; 28(34): 8502 - 8509. [Abstract] [Full Text] [PDF]
|
|
Read all eLetters
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|
 |
 |
 |
