1,043 research outputs found
N-terminal myristoylation is required for membrane localization of cGMP-dependent protein kinase type II
The apical membrane of intestinal epithelial cells harbors a unique
isozyme of cGMP-dependent protein kinase (cGK type II) which acts as a key
regulator of ion transport systems, including the cystic fibrosis
transmembrane conductance regulator (CFTR)-chloride channel. To explore
the mechanism of cGK II membrane-anchoring, recombinant cGK II was
expressed stably in HEK 293 cells or transiently in COS-1 cells. In both
cell lines, cGK II was found predominantly in the particulate fraction.
Immunoprecipitation of solubilized cGK II did not reveal any other tightly
associated proteins, suggesting a membrane binding motif within cGK II
itself. The primary structure of cGK II is devoid of hydrophobic
transmembrane domains; cGK II does, however, contain a penultimate
glycine, a potential acceptor for a myristoyl moiety. Metabolic labeling
showed that cGK II was indeed able to incorporate [3H]myristate. Moreover,
incubation of cGK II-expressing 293 cells with the myristoylation
inhibitor 2-hydroxymyristic acid (1 mM) significantly increased the
proportion of cGK II in the cytosol from 10 +/- 5 to 35 +/- 4%.
Furthermore, a nonmyristoylated cGK II Gly2 --> Ala mutant was localized
predominantly in the cytosol after transient expression in COS-1 cells.
The absence of the myristoyl group did not affect the specific enzyme
activity or the Ka for cGMP and only slightly enhanced the thermal
stability of cGK II. These results indicate that N-terminal myristoylation
fulfills a crucial role in directing cGK II to the membrane
Isotype-specific activation of cystic fibrosis transmembrane conductance regulator-chloride channels by cGMP-dependent protein kinase II
Type II cGMP-dependent protein kinase (cGKII) isolated from pig intestinal
brush borders and type I alpha cGK (cGKI) purified from bovine lung were
compared for their ability to activate the cystic fibrosis transmembrane
conductance regulator (CFTR)-Cl- channel in excised, inside-out membrane
patches from NIH-3T3 fibroblasts and from a rat intestinal cell line
(IEC-CF7) stably expressing recombinant CFTR. In both cell models, in the
presence of cGMP and ATP, cGKII was found to mimic the effect of the
catalytic subunit of cAMP-dependent protein kinase (cAK) on opening
CFTR-Cl-channels, albeit with different kinetics (2-3-min lag time,
reduced rate of activation). By contrast, cGKI or a monomeric cGKI
catalytic fragment was incapable of opening CFTR-Cl- channels and also
failed to potentiate cGKII activation of the channels. The cAK activation
but not the cGKII activation was blocked by a cAK inhibitor peptide. The
slow activation by cGKII could not be ascribed to counteracting protein
phosphatases, since neither calyculin A, a potent inhibitor of phosphatase
1 and 2A, nor ATP gamma S (adenosine 5'-O-(thiotriphosphate)), producing
stable thiophosphorylation, was able to enhance the activation kinetics.
Channels preactivated by cGKII closed instantaneously upon removal of ATP
and kinase but reopened in the presence of ATP alone. Paradoxically,
immunoprecipitated CFTR or CF-2, a cloned R domain fragment of CFTR (amino
acids 645-835) could be phosphorylated to a similar extent with only minor
kinetic differences by both isotypes of cGK. Phosphopeptide maps of CF-2
and CFTR, however, revealed very subtle differences in site-specificity
between the cGK isoforms. These results indicate that cGKII, in contrast
to cGKI alpha, is a potential activator of chloride transport in
CFTR-expressing cell types
Isotype-specific activation of cystic fibrosis transmembrane conductance regulator-chloride channels by cGMP-dependent protein kinase II
Type II cGMP-dependent protein kinase (cGKII) isolated from pig intestinal brush borders and type Iα cGK (cGKI) purified from bovine lung were compared for their ability to activate the cystic fibrosis transmembrane conductance regulator (CFTR)-Cl- channel in excised, inside-out membrane patches from NIH-3T3 fibroblasts and from a rat intestinal cell line (IEC-CF7) stably expressing recombinant CFTR. In both cell models, in the presence of cGMP and ATP, cGKII was found to mimic the effect of the catalytic subunit of cAMP- dependent protein kinase (cAK) on opening CFTR-Cl-channels, albeit with different kinetics (2-3-min lag time, reduced rate of activation). By contrast, cGKI or a monomeric cGKI catalytic fragment was incapable of opening CFTR-Cl- channels and also failed to potentiate cGKII activation of the channels. The cAK activation but not the cGKII activation was blocked by a cAK inhibitor peptide. The slow activation by cGKII could not be ascribed to counteracting protein phosphatases, since neither calyculin A, a potent inhibitor of phosphatase 1 and 2A, nor ATPγS (adenosine 5'-O- (thiotriphosphate)), producing stable thiophosphorylation, was able to enhance the activation kinetics. Channels preactivated by cGKII closed instantaneously upon removal of ATP and kinase but reopened in the presence of ATP alone. Paradoxically, immunoprecipitated CFTR or CF-2, a cloned R domain fragment of CFTR (amino acids 645-835) could be phosphorylated to a similar extent with only minor kinetic differences by both isotypes of cGK. Phosphopeptide maps of CF-2 and CFTR, however, revealed very subtle differences in site-specificity between the cGK isoforms. These results indicate that cGKII, in contrast to cGKIα, is a potential activator of chloride transport in CFTR-expressing cell types.</p
cGMP stimulation of cystic fibrosis transmembrane conductance regulator Cl- channels co-expressed with cGMP-dependent protein kinase type II but not type Ibeta
In order to investigate the involvement of cGMP-dependent protein kinase
(cGK) type II in cGMP-provoked intestinal Cl- secretion, cGMP-dependent
activation and phosphorylation of cystic fibrosis transmembrane
conductance regulator (CFTR) Cl- channels was analyzed after expression of
cGK II or cGK Ibeta in intact cells. An intestinal cell line which stably
expresses CFTR (IEC-CF7) but contains no detectable endogenous cGK II was
infected with a recombinant adenoviral vector containing the cGK II coding
region (Ad-cGK II) resulting in co-expression of active cGK II. In these
cells, CFTR was activated by membrane-permeant analogs of cGMP or by the
cGMP-elevating hormone atrial natriuretic peptide as measured by 125I-
efflux assays and whole-cell patch clamp analysis. In contrast, infection
with recombinant adenoviruses expressing cGK Ibeta or luciferase did not
convey cGMP sensitivity to CFTR in IEC-CF7 cells. Concordant with the
activation of CFTR by only cGK II, infection with Ad-cGK II but not Ad-cGK
Ibeta enabled cGMP analogs to increase CFTR phosphorylation in intact
cells. These and other data provide evidence that endogenous cGK II is a
key mediator of cGMP-provoked activation of CFTR in cells where both
proteins are co-localized, e. g. intestinal epithelial cells. Furthermore,
they demonstrate that neither the soluble cGK Ibeta nor cAMP-dependent
protein kinase are able to substitute for cGK II in this cGMP-regulated
function
EIF4G1 in familial Parkinson's disease: pathogenic mutations or rare benign variants?
International audienceMutations in the eukaryotic translation initiation factor 4-gamma (EIF4G1) gene, encoding a component of the eIF4F translation initiation complex, were recently reported as a possible cause for the autosomal dominant form of Parkinson's disease (PD). Here, we describe the screening of all 31 EIF4G1 coding exons in a series of 251 index cases with autosomal dominant PD, mostly of French origin and in 236 European control subjects. We identified 12 rare coding variants (either nonsynonymous amino acid substitutions or in frame deletions/insertions), including 6 variants present only in cases and 3 in controls. Segregation was possible only for 1 variant (p.E462delInsGK) that was found in 2 affected siblings. In addition, we found 2 previously reported pathogenic variants in 2 isolated patients (p.G686C) and in a control subject (p.R1197W). These data do not support the pathogenicity of several EIF4G1 variants in PD, at least in the French population
Embracing Monogenic Parkinson's Disease: The MJFF Global Genetic PD Cohort
Background As gene-targeted therapies are increasingly being developed for Parkinson's disease (PD), identifying and characterizing carriers of specific genetic pathogenic variants is imperative. Only a small fraction of the estimated number of subjects with monogenic PD worldwide are currently represented in the literature and availability of clinical data and clinical trial-ready cohorts is limited. Objective The objectives are to (1) establish an international cohort of affected and unaffected individuals with PD-linked variants; (2) provide harmonized and quality-controlled clinical characterization data for each included individual; and (3) further promote collaboration of researchers in the field of monogenic PD. Results We collected 3888 variant carriers for our analyses, reported by 92 centers (42 countries) worldwide. Of the included individuals, 3185 had a diagnosis of PD (ie, 1306 LRRK2, 115 SNCA, 23 VPS35, 429 PRKN, 75 PINK1, 13 DJ-1, and 1224 GBA) and 703 were unaffected (ie, 328 LRRK2, 32 SNCA, 3 VPS35, 1 PRKN, 1 PINK1, and 338 GBA). In total, we identified 269 different pathogenic variants; 1322 individuals in our cohort (34%) were indicated as not previously published. Conclusions Within the MJFF Global Genetic PD Study Group, we (1) established the largest international cohort of affected and unaffected individuals carrying PD-linked variants; (2) provide harmonized and quality-controlled clinical and genetic data for each included individual; (3) promote collaboration in the field of genetic PD with a view toward clinical and genetic stratification of patients for gene-targeted clinical trials. © 2023 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society
PTPA variants and impaired PP2A activity in early-onset parkinsonism with intellectual disability
The protein phosphatase 2A complex (PP2A), the major Ser/Thr phosphatase in the brain, is involved in a number of signalling pathways and functions, including the regulation of crucial proteins for neurodegeneration, such as alpha-synuclein, tau and LRRK2. Here, we report the identification of variants in the PTPA/PPP2R4 gene, encoding a major PP2A activator, in two families with early-onset parkinsonism and intellectual disability. We carried out clinical studies and genetic analyses, including genome-wide linkage analysis, whole-exome sequencing, and Sanger sequencing of candidate variants. We next performed functional studies on the disease-associated variants in cultured cells and knock-down of ptpa in Drosophila melanogaster. We first identified a homozygous PTPA variant, c.893T>G (p.Met298Arg), in patients from a South African family with early-onset parkinsonism and intellectual disability. Screening of a large series of additional families yielded a second homozygous variant, c.512C>A (p.Ala171Asp), in a Libyan family with a similar phenotype. Both variants co-segregate with disease in the respective families. The affected subjects display juvenile-onset parkinsonism and intellectual disability. The motor symptoms were responsive to treatment with levodopa and deep brain stimulation of the subthalamic nucleus. In overexpression studies, both the PTPA p.Ala171Asp and p.Met298Arg variants were associated with decreased PTPA RNA stability and decreased PTPA protein levels; the p.Ala171Asp variant additionally displayed decreased PTPA protein stability. Crucially, expression of both variants was associated with decreased PP2A complex levels and impaired PP2A phosphatase activation. PTPA orthologue knock-down in Drosophila neurons induced a significant impairment of locomotion in the climbing test. This defect was age-dependent and fully reversed by L-DOPA treatment. We conclude that bi-allelic missense PTPA variants associated with impaired activation of the PP2A phosphatase cause autosomal recessive early-onset parkinsonism with intellectual disability. Our findings might also provide new insights for understanding the role of the PP2A complex in the pathogenesis of more common forms of neurodegeneration.</p
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