Changes In Phosphorylation State Research Articles

Simultaneous Detection of Seven Phosphoproteins of Various Signal Transduction Pathways in a Single Sample During Oocyte Maturation Process

The physiological events during oocyte maturation are still poorly understood, and the molecular approach such as discovery of cellular signaling pathway is very important. To address this issue, we analyzed the change in phosphorylation of seven target proteins in isolated GV, GVBD, MI, and MII (in vitro and vivo) mouse oocytes by using Bio-Plex phosphoprotein assay system (Bio-Rad). Bead-based multiplex phosphorylation assays that detect the phosphorylation of 7 proteins to maximize the information obtained from a single sample at a time in lysates of isolated stage-specific mouse oocytes. Fully grown, GV-intact oocytes were collected in the presence of 0.2 mM IBMX in M2 medium and cultured in M16 medium for 2, 8, and 16 hours to obtain GVBD, MI, and MII (in vitro) oocytes, respectively. Superovulated MII (in vivo) oocytes were obtained after 16 hours of hCG injection, and cumulus cells were removed by treatment with hyaluronidase. Seven target proteins are Akt, GSK-3α/β, IκBα, STAT3 including three MAP kinases, namely ERK1/2, JNK, p38 MAPK. Lysates were obtained from 100 oocytes at each stage, and the experiment was repeated three times. The fold change in phosphorylation state was calculated in comparison to that of GV oocyte as a control. There were two groups, major and minor, of molecules in changing phosphorylation amounts. The phosphorylation of three MAP kinases and that of STAT3 was changed over 3 folds up to 20 folds, while phosphorylation of the other three signal molecules, Akt, GSK-3α/β, and IκBαwas less than 3 folds. Phosphorylation started from GVBD stages and maximized at MI-MII. All of changes except Akt were statistically significant (p<0.05). We succeeded in measuring phosphorylation of seven different important proteins at once in a single lysate sample. We firstly found that the three MAP kinases, ERK1/2, p38, and JNK, are involved in regulating the mouse oocyte maturation. Results of the present study initiate the study of several signaling pathways at once during the oocyte maturation.

Reverse transcription-associated dephosphorylation of hepadnavirus nucleocapsids

Hepatitis B viruses are pararetroviruses that contain a partially dsDNA genome and replicate this DNA through an RNA intermediate (the pregenomic RNA, pgRNA) by reverse transcription. Viral assembly begins with the packaging of the pgRNA into nucleocapsids (NCs), with subsequent reverse transcription within NCs converting the pgRNA into the characteristic dsDNA genome. Only NCs containing this dsDNA (the so-called "mature" NCs) are enveloped by the viral envelope proteins and secreted as virions; "immature" NCs, i.e., those containing pgRNA or immature reverse transcription intermediates, are excluded from virion formation. This phenomenon is thought to be caused by the emergence of an intrinsic maturation signal only on the mature NCs. To define the maturation signal, we have devised a method to separate mature from immature duck hepatitis B virus NCs and have compared them to NCs derived from secreted virions. Detailed mass spectrometric analyses revealed that the core protein from immature NCs was phosphorylated on at least six sites, whereas the core protein from mature NCs and that from secreted virions was entirely dephosphorylated. These results, together with the known requirement of core phosphorylation for pgRNA packaging and DNA synthesis, suggest that the NC undergoes a dynamic change in phosphorylation state to fulfill its multiple roles at different stages of viral replication. Although phosphorylation of the NCs is required for efficient RNA packaging and DNA synthesis by the immature NCs, dephosphorylation of the mature NCs may trigger envelopment and secretion.

Ceramide Promotes Apoptosis in Lung Cancer-Derived A549 Cells by a Mechanism Involving c-Jun NH2-Terminal Kinase

Ceramide regulates diverse signaling pathways involving cell senescence, the cell cycle, and apoptosis. Ceramide is known to potently activate a number of stress-regulated enzymes, including the c-Jun NH(2)-terminal kinase (JNK). Although ceramide promotes apoptosis in human lung cancer-derived A549 cells, a role for JNK in this process is unknown. Here, we report that ceramide promotes apoptosis in A549 cells by a mechanism involving JNK. The JNK inhibitor SP600125 proved effective at protecting cells from the lethal effects of ceramide. To understand which JNK-mediated pathway may be involved, a number of JNK target proteins were examined, including the transcription factor, c-Jun, and the apoptotic regulatory proteins Bcl-X(L) and Bim. A549 cells exhibited basal levels of phosphorylated c-Jun in nuclear fractions, revealing that active c-Jun is present in these cells. Ceramide was found to inhibit c-Jun phosphorylation, suggesting that JNK-mediated phosphorylation of c-Jun is not likely involved in ceramide-induced apoptosis. Ceramide did not promote Bcl-X(L) phosphorylation. On the other hand, ceramide promoted phosphorylation of Bim and induced translocation of active JNK from the nucleus to the cytoplasm and mitochondrial fraction. Ceramide-mediated changes in localization of JNK were consistent with the observed changes in phosphorylation status of c-Jun and Bim. Furthermore, ceramide promoted Bim translocation to the mitochondria. Mitochondrial localization of Bim has been shown recently to promote apoptosis. These results suggest that JNK may participate in ceramide-induced apoptosis in A549 cells by a mechanism involving Bim.

Tau, tangles, and Alzheimer's disease

Neurofibrillary tangles (NFT) are comprised of the microtubule-associated protein tau, in the form of filamentous aggregates. In addition to the well-known changes in phosphorylation state, tau undergoes multiple truncations and shifts in conformation as it transforms from an unfolded monomer to the structured polymer characteristic of NFT. Truncations at both the amino- and carboxy-termini directly influence the conformation into which the molecule folds, and hence the ability of tau to polymerize into fibrils. Certain of these truncations may be due to cleavage by caspases as part of the apoptotic cascade. In this review, we discuss evidence that strongly suggests that these truncations occur in an orderly pattern and directly influence the ability of tau to polymerize into filaments.

Proteomic analysis of hyperdynamic mouse hearts with enhanced sarcoplasmic reticulum calcium cycling.

Depressed sarcoplasmic reticulum (SR) Ca-cycling is a hallmark of human and experimental heart failure. Strategies to improve this impairment by either increasing SERCA2a levels or decreasing phospholamban (PLN) activity have been suggested as promising therapeutic targets. Indeed, ablation of PLN gene in mice was associated with greatly enhanced cardiac Ca-cycling and performance. Intriguingly, this hyperdynamic cardiac function was maintained throughout the lifetime of the mouse without observable pathological consequences. To determine the cellular alterations in the expression or modification of myocardial proteins, which are associated with the enhanced cardiac contractility, we performed a proteomics-based analysis of PLN knockout (PLN-KO) hearts in comparison to isogenic wild-types. By use of 2-dimensional gel electrophoresis (2-DE), approximately 3300 distinct protein spots were detected in either wild-type or PLN-KO ventricles. Protein spots observed to be altered between PLN-KO and wild-type hearts were subjected to tryptic peptide mass fingerprinting for identification by MALDI-TOF mass spectrometry in combination with LC/MS/MS analysis. In addition, two-dimensional 32P-autoradiography was performed to analyze the phosphorylation profiles of PLN-KO cardiomyocytes. We identified alterations in the expression level of more than 100 ventricular proteins, along with changes in phosphorylation status of important regulatory proteins in the PLN-KO. These protein changes were observed mainly in two subcellular compartments: the cardiac contractile apparatus, and metabolism/energetics. Our findings suggest that numerous alterations in protein expression and phosphorylation state occurred upon ablation of PLN and that a complex functional relationship among proteins involved in calcium handling, myofibrils, and energy production may exist to coordinately maintain the hyperdynamic cardiac contractile performance of the PLN-KO mouse in the long term.

Revealing Signaling Network Heterogeneity

Irish et al . set out to determine whether the variation in the genetics and clinical phenotypes of human cancer correlated with altered responsiveness of signaling networks. They used multiparameter flow cytometry to quantify the amounts of various phosphorylated proteins in acute myeloid leukemia (AML) cells from multiple patients, normal CD33 + cells, and two cell lines, HL-60 AML cells and U937 lymphoma cells. They found substantial variation among the 30 AML patient samples in the basal phosphorylation state of the transcriptional regulators STAT1, STAT3, STAT5, and STAT6, extracellular signal-regulated protein kinases 1 and 2 (ERK1/2), and the stress-activated protein kinase p38. Furthermore, the AML samples showed differential responsiveness to various cytokines in terms of the change in phosphorylation state of the measured proteins. Using an unsupervised clustering algorithm, the authors classified the AML patient samples into four groups on the basis of their basal phosphorylation status and change in response to cytokine stimulation. The frequency with which a particular cytogenetic profile, Flt3 mutation (Flt3 is a receptor tyrosine kinase, and aberrant signaling through this receptor occurs in 30% of AML patients), the abundance of CD15, and response to chemotherapy occurred in each of the four groups was analyzed. From this analysis, particular alterations in phosphoprotein biosignature were statistically correlated with clinical and genetic phenotypes. This single cell analysis may allow the mechanisms for cancer cell heterogeneity to be matched to particular aberrant signaling networks and thus mechanism-based therapies can be developed. J. M. Irish, R. Hovland, P. O. Krutzik, O. D. Perez, Ø. Bruserud, B. T. Gjertsen, G. P. Nolan, Single cell profiling of potentiated phospho-protein networks in cancer cells. Cell 118 , 217-288 (2004). [Online Journal]

Phase I and correlative science trial of UCN-01 plus cisplatin (CDDP) in advanced solid tumors: A California Cancer Consortium study

9591 Background: UCN-01 (7-hydroxystaurosporine), a novel kinase inhibitor, promotes sequence-specific abrogation of DNA damage-induced G2 arrest by inhibition of checkpoint regulators Chk1 and Chk2, potentiating the activity of CDDP, particularly in cells with a dysfunctional p53. We previously reported the preliminary results of an NCI-sponsored phase I trial designed to determine the safety, maximum tolerated dose (MTD) and pharmacokinetics (PK) of escalating doses of CDDP over 5 dose levels as a 1 hour infusion prior to fixed-dose UCN-01 (45 mg/m2/d), delivered as a 72-hour continuous infusion in cycle 1, then over 36 hours in subsequent cycles. Cycle length = 3 weeks (Gandara, ASCO 2003). Here we report the results of the correlative science and PK studies, and the final clinical results of the phase I trial. Methods: PK was assessed using high performance liquid chromatography (HPLC). Molecular analysis by Western blotting evaluated the effects of UCN-01 on key cell cycle-associated targets in serial patient tumor specimens. Results: Patient characteristics - male/female: 3/7, performance status 0–1/2: 8/2, prior chemotherapy (yes/no): 9/1. Accrual was halted at dose level 2 (cisplatin 30 mg/m2) due to dose limiting toxicities (DLT) in 2 of 3 pts. One partial response was seen. PK parameters for plasma and salivary UCN-01 were similar to those seen with single-agent UCN-01. Terminal UCN-01 half-life was > 300 hours. Western blot analysis of pre- and post-treatment tumor biopsies in 3 patients showed mobility shifts of Chk1 and Cdc25C consistent with changes in phosphorylation status. In one patient who progressed rapidly, dramatically increased levels of cyclin B and moderately increased Cdk1 phosphorylation in the post-treatment sample were indicative of a CDDP-induced G2 arrest and failure of UCN-01-induced G2 abrogation. Conclusions: These findings are consistent with predicted cell cycle regulatory effects of UCN-01 in modulating the molecular target (Chk1). Further studies of UCN-01 and platinum compounds using alternative dose schedules are warranted, and are ongoing. (CA62505, ACS-CRTG 19701-CCE) No significant financial relationships to disclose.

Phase I and correlative science trial of UCN-01 plus cisplatin (CDDP) in advanced solid tumors: A California Cancer Consortium study

9591 Background: UCN-01 (7-hydroxystaurosporine), a novel kinase inhibitor, promotes sequence-specific abrogation of DNA damage-induced G2 arrest by inhibition of checkpoint regulators Chk1 and Chk2, potentiating the activity of CDDP, particularly in cells with a dysfunctional p53. We previously reported the preliminary results of an NCI-sponsored phase I trial designed to determine the safety, maximum tolerated dose (MTD) and pharmacokinetics (PK) of escalating doses of CDDP over 5 dose levels as a 1 hour infusion prior to fixed-dose UCN-01 (45 mg/m2/d), delivered as a 72-hour continuous infusion in cycle 1, then over 36 hours in subsequent cycles. Cycle length = 3 weeks (Gandara, ASCO 2003). Here we report the results of the correlative science and PK studies, and the final clinical results of the phase I trial. Methods: PK was assessed using high performance liquid chromatography (HPLC). Molecular analysis by Western blotting evaluated the effects of UCN-01 on key cell cycle-associated targets in serial patient tumor specimens. Results: Patient characteristics - male/female: 3/7, performance status 0–1/2: 8/2, prior chemotherapy (yes/no): 9/1. Accrual was halted at dose level 2 (cisplatin 30 mg/m2) due to dose limiting toxicities (DLT) in 2 of 3 pts. One partial response was seen. PK parameters for plasma and salivary UCN-01 were similar to those seen with single-agent UCN-01. Terminal UCN-01 half-life was > 300 hours. Western blot analysis of pre- and post-treatment tumor biopsies in 3 patients showed mobility shifts of Chk1 and Cdc25C consistent with changes in phosphorylation status. In one patient who progressed rapidly, dramatically increased levels of cyclin B and moderately increased Cdk1 phosphorylation in the post-treatment sample were indicative of a CDDP-induced G2 arrest and failure of UCN-01-induced G2 abrogation. Conclusions: These findings are consistent with predicted cell cycle regulatory effects of UCN-01 in modulating the molecular target (Chk1). Further studies of UCN-01 and platinum compounds using alternative dose schedules are warranted, and are ongoing. (CA62505, ACS-CRTG 19701-CCE) No significant financial relationships to disclose.

Human homolog of disc-large is required for adherens junction assembly and differentiation of human intestinal epithelial cells.

We and others have shown that phosphatidylinositol 3-kinase (PI3K) is recruited to and activated by E-cadherin engagement. This PI3K activation is essential for adherens junction integrity and intestinal epithelial cell differentiation. Here we provide evidence that hDlg, the homolog of disc-large tumor suppressor, is another key regulator of adherens junction integrity and differentiation in mammalian epithelial cells. We report the following. 1) hDlg co-localizes with E-cadherin, but not with ZO-1, at the sites of cell-cell contact in intestinal epithelial cells. 2) Reduction of hDlg expression levels by RNA(i) in intestinal cells not only severely alters adherens junction integrity but also prevents the recruitment of p85/PI3K to E-cadherin-mediated cell-cell contact and inhibits sucrase-isomaltase gene expression. 3) PI3K and hDlg are associated with E-cadherin in a common macromolecular complex in living differentiating intestinal cells. 4) This interaction requires the association of hDlg with E-cadherin and with Src homology domain 2 domains of the p85/PI3K subunit. 5) Phosphorylation of hDlg on serine and threonine residues prevents its interaction with the p85 Src homology domain 2 in subconfluent cells, whereas phosphorylation of hDlg on tyrosine residues is essential. We conclude that hDlg may be a determinant in E-cadherin-mediated adhesion and signaling in mammalian epithelial cells.

The transcription factor Eyes absent is a protein tyrosine phosphatase.

Post-translational modifications provide sensitive and flexible mechanisms to dynamically modulate protein function in response to specific signalling inputs. In the case of transcription factors, changes in phosphorylation state can influence protein stability, conformation, subcellular localization, cofactor interactions, transactivation potential and transcriptional output. Here we show that the evolutionarily conserved transcription factor Eyes absent (Eya) belongs to the phosphatase subgroup of the haloacid dehalogenase (HAD) superfamily, and propose a function for it as a non-thiol-based protein tyrosine phosphatase. Experiments performed in cultured Drosophila cells and in vitro indicate that Eyes absent has intrinsic protein tyrosine phosphatase activity and can autocatalytically dephosphorylate itself. Confirming the biological significance of this function, mutations that disrupt the phosphatase active site severely compromise the ability of Eyes absent to promote eye specification and development in Drosophila. Given the functional importance of phosphorylation-dependent modulation of transcription factor activity, this evidence for a nuclear transcriptional coactivator with intrinsic phosphatase activity suggests an unanticipated method of fine-tuning transcriptional regulation.

Marked modulation by phosphate of phosphoenolpyruvate carboxylase in leaves of Amaranthus hypochondriacus, a NAD-ME type C4 plant: decrease in malate sensitivity but no change in the phosphorylation status.

The effect of Pi on the properties of phosphoenolpyruvate carboxylase (PEPC) from Amaranthus hypochondriacus, a NAD-ME type C4 plant, was studied in leaf extracts as well as with purified protein. Efforts were also made to modulate the Pi status of the leaf by feeding leaves with either Pi or mannose. Inclusion of 30 mM Pi during the assay enhanced the enzyme activity in leaf extracts or of purified protein by >2-fold. The effect of Pi on the enzyme purified from dark-adapted leaves was more pronounced than that from light-adapted ones. The Ki for malate increased >2.3-fold and >1.9-fold by Pi in the enzyme purified from dark-adapted leaves and light-adapted leaves, respectively. Pi also induced an almost 50-60% increase in Km for PEP or Ka for glucose-6-phosphate. Feeding the leaves with Pi also increased the activity of PEPC in leaf extracts, while decreasing the malate sensitivity of the enzyme. On the other hand, Pi sequestering by mannose marginally decreased the activity, while markedly suppressing the light activation, of PEPC. There was no change in phosphorylation of PEPC in leaves of A. hypochondriacus due to the feeding of 30 mM Pi. However, feeding with mannose decreased the light-enhanced phosphorylation of PEPC. The marked decrease in malate sensitivity of PEPC with no change in phosphorylation state indicates that the changes induced by Pi are independent of the phosphorylation of PEPC. It is suggested here that Pi is an important factor in regulating PEPC in vivo and could also be used as a tool to analyse the properties of PEPC.

Kennedy's Disease: PHOSPHORYLATION OF THE POLYGLUTAMINE-EXPANDED FORM OF ANDROGEN RECEPTOR REGULATES ITS CLEVAGE BY CASPASE-3 AND ENHANCES CELL DEATH

X-linked spinal and bulbar muscular atrophy is a degenerative disease affecting motor neurons that is caused by polyglutamine (polyQ) expansion within the androgen receptor (AR). The polyQ-expanded form of AR is cytotoxic to cells, and proteolytic cleavage enhances cell death. The intracellular signaling pathways activated and/or required for cell death induced by the expanded form of AR (AR112) are unknown. We found that AR regulates mitogen-activated protein kinase (MAP kinase) pathways and, therefore, hypothesized that these pathway(s) may be required for AR112-induced cell death. The polyQ expansion in AR activates three MAP kinase pathways, causing increasing levels of phosphorylation of p44/42, p38, and SAPK/JNK MAP kinase. Inhibitors of either the JNK or p38 pathways had no effect on AR112-induced cell death, suggesting they are not required for polyQ-induced cell death. Strikingly, the MEK1/2 inhibitor, U0126, which selectively inhibits the p44/42 MAP kinase pathway, reduces AR112-stimulated cell death. The inhibition of the MEK1/2 pathway correlates directly with a change in phosphorylation state of the androgen receptor. Mutation of the MAP kinase consensus phosphorylation site in AR at serine 514 blocked AR-induced cell death and the generation of caspase-3-derived cleavage products. We propose a mechanism by which phosphorylation at serine 514 of AR enhances the ability of caspase-3 to cleave AR and generate cytotoxic polyQ fragments.

Physiological and Pharmacological Regulation of Hepatic 3-Hydroxy-3- Methylglutaryl Coenzyme A Reductase

The conversion of 3-hydroxy-3-methylglutaryl coenzyme A to mevalonate, catalyzed by 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), is considered to be the ratelimiting step in the overall pathway of cholesterol biosynthesis. Although expressed in virtually every tissue, HMGR is normally expressed at the highest level in liver. Also the magnitude of regulation of HMGR in liver exceeds that in any other tissue. Thus, this review focuses on regulation of hepatic HMGR. Feedback regulation by the end product of the pathway, cholesterol, has been, perhaps, the most extensively studied of the physiological and pharmacological factors that act to regulate HMGR expression. This constitutes an important homeostatic mechanism to maintain cholesterol levels within rather narrow limits. A series of recent studies have now demonstrated that this regulation occurs at the level of mRNA translation - not at the level of transcription as has been widely accepted. The rapid stimulation of hepatic HMGR activity by insulin is due to increased transcription rather than a change in phosphorylation status. Glucagon opposes this effect. Insulins effect is mediated through phosphorylation of cAMP Response Element Binding Protein. Thyroid hormone acts, after a lag period of about 36 hrs, to markedly increase hepatic HMGR expression as a result of an increased rate of transcription as well as stabilization of the mRNA. Glucocorticoids oppose the mRNA stabilization. Statins act to increase hepatic HMGR protein levels as much as 700-fold through a combination of increased transcription, increased translation and stabilization of HMGR protein. Thyromimetics act to increase HMGR mRNA levels and also hepatic LDL receptor mRNA and protein levels. Oxylanosterols act to profoundly lower hepatic HMGR protein levels by acting to decrease the rate of translation. The physiological and pharmacological factors that modulate hepatic HMGR expression appear to employ a plethora of regulatory strategies. The molecular details of these regulatory mechanisms are the subjects of several current investigations. Keywords: hepatic 3-hydroxy-3-methylglutaryl, coenzyme a reductase, cholesterol biosynthesis, hmgr expression, hmgr protein levels

Dimerization of the Polycomb-group protein Mel-18 is regulated by PKC phosphorylation

The Polycomb-group (Pc-G) gene products form complexes via protein–protein interactions and maintain the transcriptional repression of genes involved in embryogenesis, cell cycle, and tumorigenesis. Previously, we have shown that mouse Mel-18, a Pc-G protein, has tumor suppressor gene-like activity and negatively regulates transcription. Here, we show in vitro by pull-down assays and in vivo in transiently transfected COS-7 cells that Mel-18 forms homodimers. Deletion analysis revealed that the N-terminal RING-finger and α-helix domains are required for homodimer formation. In addition, we demonstrated that Mel-18 homo-dimerization is regulated by protein kinase C (PKC) and protein phosphatases, such that dephosphorylated Mel-18 is able to homo-dimerize. These results suggest that the stoichiometry and/or equilibrium of subunits of the class II Polycomb complex containing Mel-18 might be regulated by changes in phosphorylation status via the PKC signaling pathway.

Temporal profiles of the subcellular localization of Bim, a BH3-only protein, during middle cerebral artery occlusion in mice.

BH3-only proteins are a subfamily of proapoptotic Bcl-2 proteins that act upstream of the mitochondrially mediated cell death pathway, and their association with the pathogenesis of brain ischemia remains largely unknown. The authors explored the temporal profiles of the expression levels and subcellular localization of BH3-only proteins in permanent middle cerebral artery occlusion (MCAO) by Western blot analysis. They observed an increased mitochondrial distribution of Bim at 3 to 6 hours of MCAO that appeared unrelated to transcriptional upregulation, as assessed by semiquantitative reverse transcription-polymerase chain reaction. At 3 to 6 hours of MCAO, Bim immunoreactivity was enhanced in neurons and oligodendrocytes in the ischemic regions. The increased mitochondrial localization of Bim coincided with a marked cytochrome c release and preceded the peak of caspase-9 activation. The authors observed an association of Bim with the dynein intermediate chain, a major component of the dynein motor complex, in the brain using a coimmunoprecipitation assay. Cerebral ischemia induced a time-dependent significant decrease in dynein expression, which started at 3 hours of MCAO. The authors deduced that the liberation of Bim from the dynein motor complex is a likely mechanism for the increased mitochondrial localization of Bim. During MCAO, Bad did not show any change in phosphorylation state or subcellular localization.

Adrenocorticotropin/Cyclic Adenosine 3′,5′-Monophosphate-Mediated Transcription of the Human CYP17 Gene in the Adrenal Cortex Is Dependent on Phosphatase Activity

cAMP-dependent transcription of steroid hydroxylase genes involves activation of cAMP-dependent protein kinase (PKA) and subsequent phosphorylation of downstream target proteins. Although the requirement for the activation of PKA is well established, none of the transcription factors required for steroid hydroxylase gene transcription have been found to be PKA phosphoproteins. In this study we examined the role of changes in phosphorylation state on the expression and transcriptional activity of the human CYP17 gene (hCYP17). Using inhibitors of serine/threonine phosphatase activity (okadaic acid) and phosphotyrosine phosphatase activity (peroxyvanadate), we can inhibit the cAMP-inducible binding of the steroidogenic factor-1 (SF-1), p54(nrb)/NonO, and polypyrimidine tract-binding protein-associated splicing factor (PSF) complex required for regulation of transcription to the promoter of hCYP17. Further, both okadaic acid and peroxyvanadate attenuate cAMP-stimulated increases in endogenous hCYP17 mRNA expression and in hCYP17 promoter-reporter construct luciferase activity. In vivo phosphorylation and immunoprecipitation of SF-1 show a cAMP-stimulated decrease in (32)P-labeled SF-1. Our findings demonstrate that activation of protein phosphatase(s) is essential for cAMP-dependent transcription of hCYP17 in H295R cells and suggest a role for PKA in phosphatase activation, which leads to dephosphorylation of SF-1 and increased gene transcription.

Pyruvate,orthophosphate dikinase in leaves and chloroplasts of C(3) plants undergoes light-/dark-induced reversible phosphorylation.

Pyruvate,orthophosphate (Pi) dikinase (PPDK) is best recognized as a chloroplastic C(4) cycle enzyme. As one of the key regulatory foci for controlling flux through this photosynthetic pathway, it is strictly and reversibly regulated by light. This light/dark modulation is mediated by reversible phosphorylation of a conserved threonine residue in the active-site domain by the PPDK regulatory protein (RP), a bifunctional protein kinase/phosphatase. PPDK is also present in C(3) plants, although it has no known photosynthetic function. Nevertheless, in this report we show that C(3) PPDK in leaves of several angiosperms and in isolated intact spinach (Spinacia oleracea) chloroplasts undergoes light-/dark-induced changes in phosphorylation state in a manner similar to C(4) dikinase. In addition, the kinetics of this process closely resemble the reversible C(4) process, with light-induced dephosphorylation occurring rapidly (< or =15 min) and dark-induced phosphorylation occurring much more slowly (> or =30-60 min). In intact spinach chloroplasts, light-induced dephosphorylation of C(3) PPDK was shown to be dependent on exogenous Pi and photosystem II activity but independent of electron transfer from photosystem I. These in organello results implicate a role for stromal pools of Pi and adenylates in regulating the reversible phosphorylation of C(3)-PPDK. Last, we used an in vitro RP assay to directly demonstrate ADP-dependent PPDK phosphorylation in desalted leaf extracts of the C(3) plants Vicia faba and rice (Oryza sativa). We conclude that an RP-like activity mediates the light/dark modulation of PPDK phosphorylation state in C(3) leaves and chloroplasts and likely represents the ancestral isoform of this unusual and key C(4) pathway regulatory "converter" enzyme.

Electrostatic properties of the structure of the docking and dimerization domain of protein kinase A IIalpha.

The structure of the N-terminal docking and dimerization domain of the type IIalpha regulatory subunit (RIIalpha D/D) of protein kinase A (PKA) forms a noncovalent stand-alone X-type four-helix bundle structural motif, consisting of two helix-loop-helix monomers. RIIalpha D/D possesses a strong hydrophobic core and two distinct, exposed faces. A hydrophobic face with a groove is the site of protein-protein interactions necessary for subcellular localization. A highly charged face, opposite to the former, may be involved in regulation of protein-protein interactions as a result of changes in phosphorylation state of the regulatory subunit. Although recent studies have addressed the hydrophobic character of packing of RIIalpha D/D and revealed the function of the hydrophobic face as the binding site to A-kinase anchoring proteins (AKAPs), little attention has been paid to the charges involved in structure and function. To examine the electrostatic character of the structure of RIIalpha D/D we have predicted mean apparent pKa values, based on Poisson-Boltzmann electrostatic calculations, using an ensemble of calculated dimer structures. We propose that the helix promoting sequence Glu34-X-X-X-Arg38 stabilizes the second helix of each monomer, through the formation of a (i, i +4) side chain salt bridge. We show that a weak inter-helical hydrogen bond between Tyr35-Glu19 of each monomer contributes to tertiary packing and may be responsible for discriminating from alternative quaternary packing of the two monomers. We also show that an inter-monomer hydrogen bond between Asp30-Arg40 contributes to quaternary packing. We propose that the charged face comprising of Asp27-Asp30-Glu34-Arg38-Arg40-Glu41-Arg43-Arg44 may be necessary to provide flexibility or stability in the region between the C-terminus and the interdomain/autoinhibitory sequence of RIIalpha, depending on the activation state of PKA. We also discuss the structural requirements necessary for the formation of a stacked (rather than intertwined) dimer, which has consequences for the orientation of the functionally important and distinct faces.

Gabapentin-mediated inhibition of voltage-activated Ca 2+ channel currents in cultured sensory neurones is dependent on culture conditions and channel subunit expression

We have used the whole cell patch clamp method and fura-2 fluorescence imaging to study the actions of gabapentin (1-(aminoethyl) cyclohexane acetic acid) on voltage-activated Ca 2+ entry into neonatal cultured dorsal root ganglion (DRG) neurones and differentiated F-11 (embryonic rat DRG×neuroblastoma hybrid) cells. Gabapentin (2.5 μM) in contrast to GABA (10 μM) did not influence resting membrane potential or input resistance. In current clamp mode gabapentin failed to influence the properties of evoked single action potentials but did reduce the duration of action potentials prolonged by Ba 2+. Gabapentin attenuated high voltage-activated Ca 2+ channel currents in a dose- and voltage- dependent manner in DRG neurones and reduced Ca 2+ influx evoked by K + depolarisation in differentiated F-11 cells loaded with fura-2. The sensitivity of DRG neurones to gabapentin was not changed by the GABA B receptor antagonist saclofen but pertussis toxin pre-treatment reduced the inhibitory effects of gabapentin. Experiments following pre-treatment of DRG neurones with a PKA-activator and a PKA-inhibitor implicated change in phosphorylation state as a mechanism, which influenced gabapentin action. Sp- and Rp-analogues of cAMP significantly increased or decreased gabapentin-mediated inhibition of voltage-activated Ca 2+ channel currents. Culture conditions used to maintain DRG neurones and passage number of differentiated F-11 cells also influenced the sensitivity of Ca 2+ channels to gabapentin. We analysed the Ca 2+ channel subunits expressed in populations of DRG neurones and F-11 cells that responded to gabapentin had low sensitivity to gabapentin or were insensitive to gabapentin, by Quantitative TaqMan PCR. The data obtained from this analysis suggested that the relative abundance of the Ca 2+ channel β 2 and α 2δ subunit expressed was a key determinant of gabapentin sensitivity of both cultured DRG neurones and differentiated F-11 cells. In conclusion, gabapentin inhibited part of the high voltage-activated Ca 2+ current in neonatal rat cultured DRG neurones via a mechanism that was independent of GABA receptor activation, but was sensitive to pertussis toxin. Gabapentin responses identified in this study implicated Ca 2+ channel β 2 subunit type as critically important to drug sensitivity and interactions with α 1 and α 2δ subunits may be implicated in antihyperalgesic therapeutic action for this compound.

Modulation of cyclic-nucleotide-gated channels and regulation of vertebrate phototransduction.

Cyclic-nucleotide-gated (CNG) channels are crucial for sensory transduction in the photoreceptors (rods and cones) of the vertebrate retina. Light triggers a decrease in the cytoplasmic concentration of cyclic GMP in the outer segments of these cells, leading to closure of CNG channels and hyperpolarization of the membrane potential. Hence, CNG channels translate a chemical change in cyclic nucleotide concentration into an electrical signal that can spread through the photoreceptor cell and be transmitted to the rest of the visual system. The sensitivity of phototransduction can be altered by exposing the cells to light, through adaptation processes intrinsic to photoreceptors. Intracellular Ca(2+) is a major signal in light adaptation and, in conjunction with Ca(2+)-binding proteins, one of its targets for modulation is the CNG channel itself. However, other intracellular signals may be involved in the fine-tuning of light sensitivity in response to cues internal to organisms. Several intracellular signals are candidates for mediating changes in cyclic GMP sensitivity including transition metals, such as Ni(2+) and Zn(2+), and lipid metabolites, such as diacylglycerol. Moreover, CNG channels are associated with protein kinases and phosphatases that catalyze changes in phosphorylation state and allosterically modulate channel activity. Recent studies suggest that the effects of circadian rhythms and retinal transmitters on CNG channels may be mediated by such changes in phosphorylation. The goal of this paper is to review the molecular mechanisms underlying modulation of CNG channels and to relate these forms of modulation to the regulation of light sensitivity.