The Biochemistry department is located at the Heinrich Plett Strasse 40 in Kassel and is part of the Institute of Biology within the Faculty of Mathematics and Natural Sciences at Kassel University. The head of department, Prof. Friedrich W. Herberg currently leads a group of about 20 people working on protein kinases. A main focus is cyclic nucleotide signaling, analyzing both, cAMP dependent protein kinase (PKA) (see figure 1) and the cGMP dependent protein kinase (PfPKG) in a structure / function approach. PKA is used as a model system to understand basic mechanisms of protein kinase function. This PKA-model system is also used to implement and validate biochemical / biophysical techniques summarized under Biomolecular Interaction Analysis (BIA) as well as for the development of novel pharmaceutical tools. BIA is further developed in the section biosensing of the Center for Interdisciplinary Nanostructure Science and Technology (CINSaT). The technology platform is used for the detailed characterization of molecular interactions within cellular protein networks also employed the University Kassel´s graduate program clocks.
More recently, work has been extended to larger multi domain protein kinases, here in particular the Leucine-Rich Repeat Kinase 2 (LRRK2) with research funded primarily by the Michael J. Fox foundation for Parkinson’s research, and the Microtubule Associated Kinase (MAST-kinase) funded by the University Kassel Zukunftsprojekt Phosmorg.
Figure 1: cAMP-signaling
Extracellular stimuli i.e. hormones (ligand) bind to a G-Protein coupled receptor (GPCR) and activate via a trimeric G-protein an Adenylate Cyclase (AC) generating the second messenger cAMP whereas Phosphodiesterases (PDEs) degrade cAMP. Primary target of cAMP are the Regulatory subunits (R) of PKA releasing the Catalytic subunits (C) after cAMP-binding. Further targets of cAMP are cyclic nucleotide gated channels (CNG) and hyperpolarization-activated cyclic nucleotide - gated cation channel (HCN) as well as the exchange protein activated by cAMP (Epac). A-Kinase Anchoring Proteins (AKAPs) mediate the spatial/temporal organization of PKA-signalling. The heat stable Protein Kinase Inhibitor (PKI) controls PKA-activity in the nucleus. Figure from ref. [D. Bertinetti, 2007]
We were among the first to quantify the binding affinities of various effectors in cAMP-signaling (PKA subunits, cyclic nucleotides, A-Kinase Anchoring Proteins (AKAPs, Phosphodiesterases, Epac). We study the effects of nucleotides, metal ions, substrates and cofactors on binding kinetics between the catalytic (C)-subunit and physiological inhibitors, i.e. the regulatory subunits (R) and the heat stable protein kinase inhibitor (PKI). We investigate the role of components of the cAMP-signaling system in the context of neuropathic pain. Recently we have expanded our work to structure / function studies on the cGMP-dependent protein kinase (PKG) in humans and in Plasmodium falciparum.
Figure 2: Architecture of the conserve kinase core exemplified in the PKA C-subunit
The core of eukaryotic protein kinases is defined by a bilobal structure with a C-terminal lobe (C-lobe) built up by helices, and a smaller N-lobe mainly consisting of β-strands. (A) The catalytic center structurally arranges the correct positioning of various motifs involved in substrate binding and phosphoryl transfer. As depicted here for the catalytic subunit of PKA, the activation loop phosphorylation (T197) anchors the α-C Helix (H87) and the Mg binding loop and thereby renders the kinase active. The invariant β3-Lys (K72) binds the α- and β-phosphates of ATP and stabilizes ATP binding through interaction with another invariant residue in the α-C Helix (E91). (B) The Gly-rich loop is the most flexible part of the kinase domain and serves as lid, which closes the active upon nucleotide and substrate binding. (C) Correct positioning of the polyphosphate chain is accomplished by two functionally non-redundant divalent metal ions (Me1, N171 and Me2, D184), which are also critically involved in the binding but also release of nucleotides and substrates. (D) Protein kinases of the AGC family possess a special regulatory motif directly following the kinase domain, the C-tail. Phosphorylation’s within this motif are important for stability as well as activation of AGC kinases. Modified from ref. [Taylor SS et al., Philos. Trans. R. Soc. B Biol. Sci. 2012]. The structure (PKA, PDB 1ATP) was visualized using PyMOL v1.3 (Schrödinger LLC).