86 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
Endogenous type II cGMP-dependent protein kinase exists as a dimer in membranes and can Be functionally distinguished from the type I isoforms
In mammalian tissues two types of cGMP-dependent protein kinase (cGK) have
been identified. In contrast to the dimeric cGK I, cGK II purified from
pig intestine was shown previously to behave as a monomer. However,
recombinant rat cGK II was found to have hydrodynamic parameters
indicative of a homodimer. Chemical cross-linking studies showed that pig
cGK II in intestinal membranes h
Membrane targeting of cGMP-dependent protein kinase is required for cystic fibrosis transmembrane conductance regulator Cl- channel activation
A recently cloned isoform of cGMP-dependent protein kinase (cGK),
designated type II, was implicated as the mediator of cGMP-provoked
intestinal Cl- secretion based on its localization in the apical membrane
of enterocytes and on its capacity to activate cystic fibrosis
transmembrane conductance regulator (CFTR) Cl- channels. In contrast, the
soluble type I cGK was unable to activate CFTR in intact cells, although
both cGK I and cGK II could phosphorylate CFTR in vitro. To investigate
the molecular basis for the cGK II isotype specificity of CFTR channel
gating, we expressed cGK II or cGK I mutants possessing different membrane
binding properties by using adenoviral vectors in a CFTR-transfected
intestinal cell line, and we examined the ability of cGMP to phosphorylate
and activate the Cl- channel. Mutation of the cGK II N-terminal
myristoylation site (Gly2 --> Ala) reduced cGK II membrane binding and
severely impaired cGK II activation of CFTR. Conversely, a chimeric
protein, in which the N-terminal membrane-anchoring domain of cGK II was
fused to the N terminus of cGK Ibeta, acquired the ability to associate
with the membrane and activate the CFTR Cl- channel. The potency order of
cGK constructs for activation of CFTR (cGK II > membrane-bound cGK I
chimer >> nonmyristoylated cGK II > cGK Ibeta) correlated with the extent
of 32P incorporation into CFTR observed in parallel measurements. These
results strongly support the concept that membrane targeting of cGK is a
major determinant of CFTR Cl- channel activation in intact cells
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
A cGMP-dependent protein kinase is implicated in wild-type motility in C. elegans
Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/65790/1/j.1471-4159.2001.00131.x.pd
The Nitric Oxide-Cyclic GMP Pathway Regulates FoxO and Alters Dopaminergic Neuron Survival in Drosophila
Activation of the forkhead box transcription factor FoxO is suggested to be involved in dopaminergic (DA) neurodegeneration in a Drosophila model of Parkinson's disease (PD), in which a PD gene product LRRK2 activates FoxO through phosphorylation. In the current study that combines Drosophila genetics and biochemical analysis, we show that cyclic guanosine monophosphate (cGMP)-dependent kinase II (cGKII) also phosphorylates FoxO at the same residue as LRRK2, and Drosophila orthologues of cGKII and LRRK2, DG2/For and dLRRK, respectively, enhance the neurotoxic activity of FoxO in an additive manner. Biochemical assays using mammalian cGKII and FoxO1 reveal that cGKII enhances the transcriptional activity of FoxO1 through phosphorylation of the FoxO1 S319 site in the same manner as LRRK2. A Drosophila FoxO mutant resistant to phosphorylation by DG2 and dLRRK (dFoxO S259A corresponding to human FoxO1 S319A) suppressed the neurotoxicity and improved motor dysfunction caused by co-expression of FoxO and DG2. Nitric oxide synthase (NOS) and soluble guanylyl cyclase (sGC) also increased FoxO's activity, whereas the administration of a NOS inhibitor L-NAME suppressed the loss of DA neurons in aged flies co-expressing FoxO and DG2. These results strongly suggest that the NO-FoxO axis contributes to DA neurodegeneration in LRRK2-linked PD
Verfahren und Vorrichtung zur Erzeugung eines Wasserabrasivstrahls
DE 10348805 A UPAB: 20050715 NOVELTY - In a process an abrasive medium, water and a supply of solid particles are brought together in a mixer head (1). Water and the abrasive medium are drawn through tubes (7, 12) to the mixer head. The supply of water and abrasive medium is regulated by valves (13). Also claimed is a commensurate mixer and jet assembly. USE - Process and assembly to generate water jet transporting abrasive particulate medium. ADVANTAGE - The abrasive effect is more efficient at high pressure than prior art
Large, Unstable Inserts in the Chromosome Affect Virulence Properties Of Uropathogenic Escherichia coli 06 Strain 536
The hemolytic, uropathogenic Escherichia coli 536 (06:K15:H31) contains two inserts in its chromosome (insert I and insert II), both of which carried hly genes, were rather unstable, and were deleted spontaneously with a frequen~y of 10-3 to 10-4• These inserts were not found in the chromosome of two nonhemolytic E. coli strains, whereas the chromosomal ~equences adjacent to these inserts appeared tobe again homologous in the uropathogenic and two other E. coü strains. Insert I was 75 kilobases in size and was ftanked at both ends by 16 base pairs (bp) (TTCGACTCCTGTGATC) which were arranged in direct orientation. For insert I it was demonstrated that deletion occurred by recombination between the two 16-bp ftanking sequences, since mutants lacking this insert still carried a single copy of the 16-bp sequence in the chromosome. 8oth inserts contained a functional hemolysin determinant. However, the loss of the inserts not only atfected the hemolytic phenotype bot led to a considerable reduction in serum resistance and the loss of mannose-resistant hemagglutination, caused by the presence of S-type funbriae (sja). lt is shown that the Sfa-negative phenotype is due to a block in transcription of the sfa genes. Mutants of strain 536 which lacked both inserts were entirely avirulent when tested in several animal model systems
Large, unstable inserts in the chromosome affect virulence properties of uropathogenic Escherichia coli O6 strain 536.
The hemolytic, uropathogenic Escherichia coli 536 (O6:K15:H31) contains two inserts in its chromosome (insert I and insert II), both of which carried hly genes, were rather unstable, and were deleted spontaneously with a frequency of 10(-3) to 10(-4). These inserts were not found in the chromosome of two nonhemolytic E. coli strains, whereas the chromosomal sequences adjacent to these inserts appeared to be again homologous in the uropathogenic and two other E. coli strains. Insert I was 75 kilobases in size and was flanked at both ends by 16 base pairs (bp) (TTCGACTCCTGTGATC) which were arranged in direct orientation. For insert I it was demonstrated that deletion occurred by recombination between the two 16-bp flanking sequences, since mutants lacking this insert still carried a single copy of the 16-bp sequence in the chromosome. Both inserts contained a functional hemolysin determinant. However, the loss of the inserts not only affected the hemolytic phenotype but led to a considerable reduction in serum resistance and the loss of mannose-resistant hemagglutination, caused by the presence of S-type fimbriae (sfa). It is shown that the Sfa-negative phenotype is due to a block in transcription of the sfa genes. Mutants of strain 536 which lacked both inserts were entirely avirulent when tested in several animal model systems
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