Folding and activity of cAMP‐dependent protein kinase mutants

Abstract

The catalytic subunit of cAMP-dependent protein kinase (PKA) can easily be expressed in Escherichia coli and is catalytically active. Four phosphorylation sites are known in PKA (S10, S139, T197 and S338), and the isolated recombinant protein is a mixture of different phosphorylated forms. Obtaining uniformly phosphorylated protein requires separation of the protein preparation leading to significant loss in protein yield. It is found that the mutant S10A/S139D/S338D has similar properties as the wild-type protein, whereas additional replacement of T197 with either E or D reduces protein expression yield as well as folding propensity of the protein. Due to its high sequence homology to Akt/PKB, which cannot easily be expressed in E. coli, PKA has been used as a surrogate kinase for drug design. Several mutations within the ATP binding site have been described to make PKA even more similar to Akt/PKB. Two proteins with Akt/PKB-like mutations in the ATP binding site were made (PKAB6 and PKAB8), and in addition S10, S139 and S338 phosphorylation sites have been removed. These proteins can be expressed in high yields but have reduced activity compared to the wild-type. Proper folding of all proteins was analyzed by 2D 1 H, 15 N-TROSY NMR experiments.

Probing Protein-Protein Interactions Using Asymmetric Labeling and Carbonyl-Carbon Selective Heteronuclear NMR Spectroscopy

“…Furthermore, we applied this technique to resolve assignment ambiguities on the 41 kDa catalytic subunit of cAMP-dependent protein kinase A (PKA-C) [ 33 , 34 ]. PKA-C is the prototypical Ser/Thr kinase and, until relatively recently, had remained unexplored by NMR due to its size and presence of conformational exchange effects on the μs-ms timescale [ 35 , 36 , 37 , 38 ]. Advances in pulse sequence design and sample preparation have since made it possible to investigate this system using NMR [ 39 , 40 , 41 , 42 ].…”

Carbonyl carbon label selective (CCLS) 1H–15N HSQC experiment for improved detection of backbone 13C–15N cross peaks in larger proteins

“…Protein kinases are involved in a number of signal transduction cascades, including cell growth, proliferation, and death, making them ideal candidates as a target for drug design (Taylor et al 2005). This family of enzymes has remained relatively unexplored by NMR because of undesirable spectral characteristics related to the size of its members and the presence of conformational exchange effects on the μs-ms timescale (Langer et al 2004(Langer et al , 2005Vogtherr et al 2005Vogtherr et al , 2006. Previous efforts to assign the resonances of PKA-C by TROSY-based 3D experiments yielded assignments to only ~55% of the non-proline residues (Langer et al 2004).…”

A tripartite cooperative mechanism confers resistance of the protein kinase A catalytic subunit to dephosphorylation

“…Biophysical studies showed that PKAc with defective activation loop phosphorylation reduces protein expression, folding, and stability (36,37). Also, alkylation and oxidative agents reduce PKAc activation loop phosphorylation and PKAc stability in cells (9).…”