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@ARTICLE{Vallur:137446,
author = {Vallur, Raghavan and Kalbacher, Hubert and Feil, Robert},
title = {{C}atalytic activity of c{GMP}-dependent protein kinase
type {I} in intact cells is independent of {N}-terminal
autophosphorylation.},
journal = {PLOS ONE},
volume = {9},
number = {6},
issn = {1932-6203},
address = {San Francisco, California, US},
publisher = {PLOS},
reportid = {DZNE-2020-03768},
pages = {e98946},
year = {2014},
abstract = {Although cGMP-dependent protein kinase type I (cGKI) is an
important mediator of cGMP signaling and upcoming drug
target, its in vivo-biochemistry is not well understood.
Many studies showed that purified cGKI autophosphorylates
multiple sites at its N-terminus. Autophosphorylation might
be involved in kinase activation, but it is unclear whether
this happens also in intact cells. To study cGKI
autophosphorylation in vitro and in vivo, we have generated
phospho-specific antisera against major in
vitro-autophosphorylation sites of the cGKI isoforms, cGKIα
and cGKIβ. These antisera detected specifically and with
high sensitivity phospho-cGKIα (Thr58), phospho-cGKIα
(Thr84), or phospho-cGKIβ (Thr56/Ser63/Ser79). Using these
antisera, we show that ATP-induced autophosphorylation of
cGKI in purified preparations and cell extracts did neither
require nor induce an enzyme conformation capable of
substrate heterophosphorylation; it was even inhibited by
pre-incubation with cGMP. Interestingly, phospho-cGKI
species were not detectable in intact murine cells and
tissues, both under basal conditions and after induction of
cGKI catalytic activity. We conclude that N-terminal
phosphorylation, although readily induced in vitro, is not
required for the catalytic activity of cGKIα and cGKIβ in
vivo. These results will also inform screening strategies to
identify novel cGKI modulators.},
keywords = {Animals / Catalysis / Cattle / Cyclic GMP-Dependent Protein
Kinase Type I: chemistry / Cyclic GMP-Dependent Protein
Kinase Type I: immunology / Cyclic GMP-Dependent Protein
Kinase Type I: metabolism / Immune Sera: immunology / Mice /
Phosphorylation / Protein Interaction Domains and Motifs /
Protein Isoforms / Recombinant Proteins: chemistry /
Recombinant Proteins: metabolism / Substrate Specificity /
Immune Sera (NLM Chemicals) / Protein Isoforms (NLM
Chemicals) / Recombinant Proteins (NLM Chemicals) / Cyclic
GMP-Dependent Protein Kinase Type I (NLM Chemicals)},
cin = {AG N.N. 3},
ddc = {610},
cid = {I:(DE-2719)1240015},
pnm = {344 - Clinical and Health Care Research (POF3-344)},
pid = {G:(DE-HGF)POF3-344},
typ = {PUB:(DE-HGF)16},
pubmed = {pmid:24897423},
pmc = {pmc:PMC4045857},
doi = {10.1371/journal.pone.0098946},
url = {https://pub.dzne.de/record/137446},
}