Volume 14 Supplement 1
Phospho-specific antisera to monitor N-terminal autophosphorylation of cGMP-dependent protein kinase type I
© Vallur et al; licensee BioMed Central Ltd. 2013
Published: 29 August 2013
Although the cGMP-dependent protein kinase type I (cGKI) is an important mediator of cGMP signaling in many cells and tissues, its’ in vivo-biochemistry is not well understood. It has been shown that the purified enzyme can autophosphorylate multiple sites in its N-terminal region in the presence of ATP and cyclic nucleotides (cGMP and/or cAMP). N-terminal autophosphorylation might be involved in the activation of the kinase by cGMP in vitro, but it is not clear whether or not this also happens in intact cells .
To detect autophosphorylated cGKI in cells and tissues, we have generated polyclonal rabbit antisera against the major in vitro autophosphorylation sites of murine cGKI-alpha (Ser-50; Thr-58, Ser-72, and Thr-84) and cGKI-beta (Thr-56, Ser-63, and Ser-79). ELISAs with peptides containing the respective amino acids in their non-phosphorylated or phosphorylated form as well as Western blots with purified cGKI-alpha and cGKI-beta indicated that the antisera specifically recognized the autophosphorylated N-termini of these isoforms. The sensitivity of detection was comparable to a highly sensitive pan-cGKI antiserum. Interestingly, the addition of ATP (100 µM) alone was sufficient to induce autophosphorylation of the purified isozymes in vitro. Surprisingly, we were not able to detect phospho-cGKI species in intact fibroblasts and vascular smooth muscle cells, both under basal conditions as well as after induction of cGKI kinase activity (monitored as VASP phosphorylation) with cGMP-elevating compounds.
We have generated phospho-specific antisera against the N-terminal regions of cGKI-alpha and cGKI-beta and could confirm the previously reported autophosphorylation of these isozymes in vitro. However, our results question the relevance of N-terminal autophosphorylation of cGKI in intact cells.
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.