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.
Highlights
Cyclic guanosine monophosphate acts as a second messenger in various cell types and tissues of eukaryotes [1,2]
The intracellular concentration of Cyclic guanosine monophosphate (cGMP) depends on the rate of its synthesis and degradation. cGMP is generated by cytosolic soluble guanylyl cyclases in response to NO or by membrane-bound particulate guanylyl cyclases that are activated by natriuretic peptides and some bacterial toxins. cGMP is hydrolyzed to GMP by phosphodiesterases, whose catalytic activity is often regulated by binding of cGMP or cAMP
Two additional non-purified antisera with good specificities for phosphorylated over non-phosphorylated sites were identified by Enzyme-linked immunosorbent assay (ELISA): polyclonal serum 6 (PS6) detects phospho-Thr84 of cGKIa (Fig. 2B), and polyclonal serum 7 (PS7) detects phosphoThr56, phospho-Ser63, and phospho-Ser79 of cGKIb (Fig. 2C)
Summary
Cyclic guanosine monophosphate (cGMP) acts as a second messenger in various cell types and tissues of eukaryotes [1,2]. The intracellular concentration of cGMP depends on the rate of its synthesis and degradation. CGMP is hydrolyzed to GMP by phosphodiesterases, whose catalytic activity is often regulated by binding of cGMP or cAMP. At least three classes of cGMP effector proteins are known: cyclic nucleotide-gated cation channels, which transduce changes in cGMP concentrations into changes of membrane potential; cGMP-regulated cAMP-hydrolyzing phosphodiesterases, which mediate a cross-talk of cGMP and cAMP signaling; and cGMP-dependent protein kinases, which upon binding of cGMP phosphorylate a variety of target proteins at Ser/Thr residues. The cGMP-dependent protein kinase type I (cGKI, known as PKG-I or PRKG1) is considered a major mediator of cGMP signaling in mammals. The development of such drugs has been hampered, in part, because the in vivo-biochemistry of cGKI is not well understood
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