Activation and inhibition mechanisms of the cGMP-dependent protein kinase studied by mass spectrometry

Abstract

Post translational modification plays an important role in the regulation of cellular functions via the modulation of the structural and functional properties of strategically selected proteins. Through the key mechanism of protein phosphorylation, proteins can be rapidly and reversibly modified, providing and ‘on-off switch’ for a given protein activity. Protein kinases and protein phosphatases are the key regulators in protein phosphorylation and protein dephosphorylation processes. Consequently, protein kinases and phosphatases are together with their corresponding protein substrates intensely studied in an effort to elucidate signaling pathways mediated via reversible protein phosphorylation. In particular protein kinases are intriguing enzymes, since they are endowed with a tremendous precision in recognizing their target. The primary goal of the work described in this thesis was aimed at gathering biochemical insight into structural and functional aspects of the 3’-,5’- cyclic guanosine monophosphate dependent protein kinase (PKG) type Iα using mass spectrometry based approaches. Type Iα PKG mediates essential physiological functions in cGMP-signaling pathways in for example vascular smooth muscle cells. Although there is significant progress in understanding the specific functional role of PKG, relative little is known about the overall structure of PKG, the significance of its autophosphorylation reaction, the nature of conformational changes induced by the binding of its various ligands, substrates and or inhibitors. New information about these molecular determinants is of importance in the design of molecular tools to delineate cGMP-signaling networks and eventually for future therapeutics that may target PKG-mediated signaling pathways. Non-covalent interactions between PKG and its natural activator cGMP, ATP and ATP analogs, as well as potent peptide inhibitors such as DT2 were studied by nanoflow electrospray mass spectrometry. Insight into binding order and stoichiometry of the various ligands could be directly derived from the acquired mass spectra. An interesting observation made in these studies is the unusual binding behavior of DT2. Mass spectra of PKG, acquired in the presence of cGMP and DT2, showed that preferentially 1 DT2 peptide binds to dimeric PKG. In addition, since high-resolution structural information is currently not available for PKG, binding sites of substrate and inhibitor peptides were localized using an approach combining photoaffinity labeling, stable isotope labeling and mass spectrometry. This approach was first tested on a highly specific substrate peptide. Initial results demonstrated that this peptide binds directly to the active site of PKG, and more importantly, binding is cGMP-dependent. Finally, a new and innovative method for the selective enrichment of phosphopeptides was developed. This method is based on the unique ion-exchange properties of spherical titanium oxide particles. In an on-line configuration phosphorylated peptides could be isolated from the non-phosphorylated peptides. Using this setup the autophosphorylation reaction of PKG was examined in time. Up to 8 phosphorylation sites were identified, of which 2 were previously uncharacterized. These results clearly show that titanium oxide has a high potential in the field of phosphoproteomics.