TY  - JOUR
AU  - Vallur, Raghavan
AU  - Kalbacher, Hubert
AU  - Feil, Robert
TI  - Catalytic activity of cGMP-dependent protein kinase type I in intact cells is independent of N-terminal autophosphorylation.
JO  - PLOS ONE
VL  - 9
IS  - 6
SN  - 1932-6203
CY  - San Francisco, California, US
PB  - PLOS
M1  - DZNE-2020-03768
SP  - e98946
PY  - 2014
AB  - 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.
KW  - Animals
KW  - Catalysis
KW  - Cattle
KW  - Cyclic GMP-Dependent Protein Kinase Type I: chemistry
KW  - Cyclic GMP-Dependent Protein Kinase Type I: immunology
KW  - Cyclic GMP-Dependent Protein Kinase Type I: metabolism
KW  - Immune Sera: immunology
KW  - Mice
KW  - Phosphorylation
KW  - Protein Interaction Domains and Motifs
KW  - Protein Isoforms
KW  - Recombinant Proteins: chemistry
KW  - Recombinant Proteins: metabolism
KW  - Substrate Specificity
KW  - Immune Sera (NLM Chemicals)
KW  - Protein Isoforms (NLM Chemicals)
KW  - Recombinant Proteins (NLM Chemicals)
KW  - Cyclic GMP-Dependent Protein Kinase Type I (NLM Chemicals)
LB  - PUB:(DE-HGF)16
C6  - pmid:24897423
C2  - pmc:PMC4045857
DO  - DOI:10.1371/journal.pone.0098946
UR  - https://pub.dzne.de/record/137446
ER  -