Analysis of the functional role of cGMP-dependent protein kinase in intact human platelets using a specific activator 8-para-chlorophenylthio-cGMP

Butt, Elke; Nolte, Christiané; Schulz, Susanne; Beltman, Jerlyn; Beavo, Joseph A.; Jastorff, Bernd; Walter, Ulrich

doi:10.1016/0006-2952(92)90148-c

Cited by 141 publications

(96 citation statements)

References 24 publications

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“…The latter issue was addressed more systematically in VASP phosphorylation studies with intact human platelets. Established conditions (27,28) also confirmed by experiments with cGPK-deficient human platelets (29) for potent and specific activation of either platelet cAPK (incubation with 1 M prostacyclin or 0.5 mM 5, 6-DCl-cBIMPS) or cGPK (10 M sodium nitroprusside or 1.0 mM 8-pCPT-cGMP) were chosen. In extracts of washed, unstimulated human platelets the M4 VASP antiserum detected only the 46-kDa VASP species, whereas the 16C2 antibody detected no specific signal at all (Fig.…”

Section: Resultsmentioning

confidence: 99%

Analysis and Regulation of Vasodilator-stimulated Phosphoprotein Serine 239 Phosphorylation in Vitro and in Intact Cells Using a Phosphospecific Monoclonal Antibody

Smolenski

Bachmann

Reinhard

et al. 1998

Journal of Biological Chemistry

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314

322

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Although cGMP-dependent protein kinases (cGPKs) 1 have been recognized as important components of major signal transduction pathways (1-3), quantitative analysis of cGPK activation in intact cells has been very difficult (1-4). This is because of the relatively low expression of cGPK in most cell types compared with the relatively high expression of its closest functional homolog, the cAMP-dependent protein kinase (cAPK), and the scarcity of specific cGPK substrates. Unfortunately, the mediating role of cGPK for a given effect/function is often implied or excluded by the use of cGPK activators and/or inhibitors alone, which is clearly insufficient to establish or rule out functional roles of cGPKs (1-4). One of the few established cGPK substrates is the 46-kDa/50-kDa vasodilator-stimulated phosphoprotein (VASP), which was initially discovered and characterized as a substrate of both cAPK and cGPK in human platelets (5-8). VASP phosphorylation in response to cyclic nucleotide-regulating vasodilators (i.e. cAMP-elevating prostaglandins and cGMP-elevating nitric oxide donors) closely correlates with platelet inhibition and in particular with the inhibition of fibrinogen binding to the integrin ␣ IIb ␤ 3 of human platelets (9 -11). Molecular cloning of human, canine, and mouse VASP predicted highly homologous proteins and revealed a proline-rich protein that is organized into three structural segments of different sequence complexity (12,13). VASP is the founding member of a new family of proline-rich proteins, which includes Enabled (Ena), a dose-dependent suppressor of Drosophila Abl-and Disabled-dependent phenotypes, its mammalian homolog Mena, and the Ena-VASP-like protein Evl (14 -16). These proteins all share an overall domain organization consisting of highly homologous NH 2 -terminal and COOHterminal domains (Ena-VASP homology domains 1 and 2, EVH1 and EVH2), which are separated by a proline-rich central domain of low complexity (12-16). In platelets and many other cells including vascular smooth muscle cells, endothelial cells, and fibroblasts, VASP has been found to be associated with stress fibers, focal adhesions, cell-cell contacts, and highly dynamic membrane regions (16,17). VASP colocalizes with profilins and binds directly to their poly(L-proline) binding site (18), binds to and colocalizes with zyxin and vinculin (16,19), and also directly binds to Listeria monocytogenes surface protein ActA, which is essential for the actin polymerization-based intracellular motility of this pathogen (20). Functional evidence indicates that VASP is a crucial factor involved in the enhancement of spatially confined actin filament formation (16,20,21).Three distinct phosphorylation sites were biochemically identified in VASP (serine 157, serine 239, and threonine 278) which are used in vitro and in intact human platelets by both cAPK and cGPK and by the serine/threonine protein phosphatases 2A and 2B with overlapping selectivity (8,22). Phosphorylation of serine 157, the site preferred by the cAPK, leads to a marked...

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Section: Resultsmentioning

confidence: 99%

Analysis and Regulation of Vasodilator-stimulated Phosphoprotein Serine 239 Phosphorylation in Vitro and in Intact Cells Using a Phosphospecific Monoclonal Antibody

Smolenski

Bachmann

Reinhard

et al. 1998

Journal of Biological Chemistry

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314

322

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“…However, it remains unknown which conformer of cAMP and cGMP is the genuine substrate for other PDE families. Early studies suggested that the preferable substrates are: syn cAMP and cGMP for PDE1 and PDE2, anti cAMP for PDE3 and PDE4, and anti cGMP for PDE3 and PDE5 (37,38). Because the substrates and products have different nucleotide configurations: syn for cAMP and cGMP, and anti for AMP and GMP, it would be interesting to know whether the enzyme takes an additional step to perform the syn to anti conversion during catalysis.…”

Section: Discussionmentioning

confidence: 99%

Structural insight into substrate specificity of phosphodiesterase 10

Wang

Liu

Hou

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

104

139

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Phosphodiesterases (PDEs) hydrolyze the second messengers cAMP and cGMP. It remains unknown how individual PDE families selectively recognize cAMP and cGMP. This work reports structural studies on substrate specificity. The crystal structures of the catalytic domains of the D674A and D564N mutants of PDE10A2 in complex with cAMP and cGMP reveal that two substrates bind to the active site with the same syn configuration but different orientations and interactions. The products AMP and GMP bind PDE10A2 with the anti configuration and interact with both divalent metals, in contrast to no direct contact of the substrates. The structures suggest that the syn configurations of cAMP and cGMP are the genuine substrates for PDE10 and the specificity is achieved through the different interactions and conformations of the substrates. The PDE10A2 structures also show that the conformation of the invariant glutamine is locked by two hydrogen bonds and is unlikely to switch for substrate recognition. Sequence alignment shows a potential pocket, in which variation of amino acids across PDE families defines the size and shape of the pocket and thus determines the substrate specificity.crystal structure ͉ cyclic nucleotides cAMP and cGMP C yclic nucleotide phosphodiesterases (PDEs) are enzymes hydrolyzing the second messengers adenosine and guanosine 3Ј,5Ј-cyclic monophosphates (cAMP and cGMP). The human genome encodes 21 PDE genes that are categorized into 11 families (1, 2). Selective inhibitors against individual PDE families have been developed as therapeutics for treatment of various human diseases (3-8). The best known examples are the PDE5 inhibitors sildenafil (Viagra), vardenafil (Levitra), and tadalafil (Cialis) that have been used for treatment of male erectile dysfunction (5). Sildenafil (Revatio) has also been approved for treatment of pulmonary hypertension (9). PDE10 was independently identified by three groups in 1999 and shows a dual activity on hydrolysis of both cAMP and cGMP (10-12). PDE10 is highly expressed in brain striatum (13-16). Reduction of PDE10A mRNA and protein levels in striatum of transgenic mice implies a role of PDE10A in Huntington's disease (17,18). Knockout mice experiments suggest that PDE10A is involved in regulating striatal output, possibly by reducing the sensitivity of medium spiny neurons to glutamatergic excitation (19). The PDE10 inhibitor papaverine is effective in improving executive function deficits associated with schizophrenia, and therefore inhibition of PDE10 may represent an approach to treatment of psychosis (20,21).PDE families contain a variable N-terminal regulatory domain and a conserved C-terminal catalytic domain. Individual PDE families show different substrate preferences. Crystal structures have been reported for the catalytic domains of seven PDE families in the unliganded form or in complex with inhibitors or products: PDE1B, PDE2A, PDE3B, PDE4B/4D, PDE5A, PDE7A, and PDE9A (22-34). However, it remains a puzzle how the conserved catalytic pocket of the PDE famili...

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“…To determine whether potential diffusional limitations associated with arterial remodeling limit access of 8-BrcGMP to VSM in lungs from CH rats, vasodilatory responses to the more membrane-permeable analog the Rp diastereomer of 8-(4-chlorophenylthio)-guanosine 3',5'-cyclic monophosphate (8-pCPT-cGMP) (6) were assessed in control and CH rats. After attainment of a stable vasoconstrictor response to U-46619, 8-cCPT-cGMP (1 M) was administered.…”

Section: Isolated Lung Experimentsmentioning

confidence: 99%

Pulmonary PKG-1 is upregulated following chronic hypoxia

Jernigan

Walker

Resta

2003

American Journal of Physiology-Lung Cellular and Molecular Phys

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. Pulmonary PKG-1 is upregulated following chronic hypoxia. Am J Physiol Lung Cell Mol Physiol 285: L634-L642, 2003. First published May 23, 2003 10.1152/ ajplung.00328.2002.-Recent studies from our laboratory indicate that pulmonary vasodilatory responses to exogenous nitric oxide (NO) are attenuated following chronic hypoxia (CH) and that this NO-dependent vasodilation is mediated by cGMP. Similarly, we have demonstrated that CH attenuates vasodilatory responses to the cGMP analog 8-bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP). We hypothesized that attenuated pulmonary vasodilation to 8-BrcGMP following CH is mediated by decreased protein kinase G-1 (PKG-1) expression/activity. Therefore, we examined vasodilatory responses to 8-BrcGMP (1 M) in isolated, saline-perfused lungs from control and CH (4 wk at barometric pressure of 380 mmHg) rats in the presence of the competitive PKG inhibitor Rp-␤-phenyl-1, N2-etheno-8-bromoguanosine 3',5'-cyclic monophosphorothionate (30 M) or the highly specific PKG inhibitor KT-5823 (10 M). PKG-1 expression and activity were determined in whole lung homogenates from each group, and vascular PKG-1 levels were assessed by quantitative immunohistochemistry. PKG inhibition with either Rp-8-Br-PET-cGMPS or KT-5823 diminished vasodilatory responses to 8-BrcGMP in lungs from both control and CH rats, thus indicating a role for PKG in mediating reactivity to 8-BrcGMP in each group. However, in contrast to our hypothesis, PKG-1 levels were approximately twofold greater in lungs from CH rats vs. controls, and furthermore, this upregulation was localized to the vasculature. This correlates with an increase in PKG activity following CH. We conclude that PKG-1 is involved in 8-BrcGMPmediated vasodilation; however, attenuated pulmonary vasodilation following CH is not associated with decreased expression/activity of PKG-1. isolated rat lung; nitric oxide; pulmonary hypertension; 8-bromoguanosine 3',5'-cyclic monophosphate; KT-5823; Rp-␤-phenyl-1, N2-etheno-8-bromoguanosine 3',5'-cyclic monophosphorothioate CHRONIC EXPOSURE TO HYPOXIA has been shown to augment pulmonary vasodilatory responses to endothelium-derived nitric oxide (EDNO)-dependent vasodilators (18, 26, 28, 32-34, 36, 37). EDNO is synthesized in the vasculature by endothelial nitric oxide synthase (eNOS), and several studies have demonstrated that chronic hypoxia (CH) is associated with increased pulmonary eNOS levels, gene expression, and activity (18,21,26,33,34,39,46). Consistent with elevated eNOS levels, nitric oxide (NO) synthesis appears to be greater in lungs isolated from CH rats compared with controls (18,26,44). In contrast to enhanced reactivity to our laboratory (19,34) and others (35) have demonstrated impaired responsiveness to exogenous NO following CH. However, the mechanism(s) responsible for this diminished reactivity is not fully understood.NO leads to pulmonary vascular smooth muscle (VSM) relaxation by stimulating soluble guanylyl cyclase (sGC) (7). The subsequent elevation in cGMP activates prot...

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Analysis of the functional role of cGMP-dependent protein kinase in intact human platelets using a specific activator 8-para-chlorophenylthio-cGMP

Cited by 141 publications

References 24 publications

Analysis and Regulation of Vasodilator-stimulated Phosphoprotein Serine 239 Phosphorylation in Vitro and in Intact Cells Using a Phosphospecific Monoclonal Antibody

Analysis and Regulation of Vasodilator-stimulated Phosphoprotein Serine 239 Phosphorylation in Vitro and in Intact Cells Using a Phosphospecific Monoclonal Antibody

Structural insight into substrate specificity of phosphodiesterase 10

Pulmonary PKG-1 is upregulated following chronic hypoxia

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