The cAMP-dependent protein kinase as a model system for interaction studies

The cAMP-dependent protein kinase (PKA) is a heterotetramer consisting of two monomeric catalytic (C-) and a dimer of regulatory (R-) subunits. The activation of the inactive holo enzyme complex occurs upon binding of the second messenger cyclic AMP (cAMP) to the cAMP binding sites A and B in the regulatory subunits thus inducing a conformational change and a release of active catalytic subunits which are able to phosphorylate substrate proteins in the cell.


The heat stable protein kinase inhibitor PKI is a thermostable pseudo subtrate inhibitor and binds to the catalytic subunit of cAMP-dependent protein kinase PKA-C(alpha). The interaction between PKI and the catalytic subunit PKA-C(alpha) causes the export from the nucleus to the cytoplasm thus affecting transcriptional activity in the cell.

Figure: Interaction analysis of the heatstable protein kinase inhibitor PKI immobilised on a Biacore sensor surface with the catalytic subunit of cAMP-dependent protein kinase PKA-C(alpha) as analyte in solution. Assuming a 1:1 binding model the non linear data analysis after subtraction of the reference signal yielded the following rate constants [Zimmermann et al., J. Biol. Chem. (1999), 274, 5370-5378]:
kass = 1.5 x106 M-1 s-1; kdiss = 7.6 x 10-4 s-1; KD = 0.5 nM

In the absence of ATP/Mg in a running buffer containing ETDA, PKA changes its conformation from a closed to an open state, resulting in a completely different binding kinetics for PKI. Here the binding affinity is lowered by a factor of 720 to KD = 360 nM.

MAP kinase p38 / SAPK2 alpha

Figure: Interaction analysis of the MAP kinase p38 / SAPK2 alpha immobilised on a Biacore S51 sensor surface with the MAP kinase inhibitor SB203580 as analyte in solution.  Assuming a 1:1 binding model the non linear data analysis after reference subtraction yielded the following rate constants:
kass  = (1,9 ± 1,5) 106 M-1 s-1; kdiss  = (5,1 ± 2,2) 10-2 s-1; KD   = (32,1 ± 10,5) nM

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