cAMP‐dependent protein kinase: prototype for a family of enzymes

Abstract

Protein kinases represent a diverse family of enzymes that play critical roles in regulation. The simplest and best-understood biochemically is the catalytic (C) subunit of cAMP-dependent protein kinase, which can serve as a framework for the entire family. The amino-terminal portion of the C subunit constitutes a nucleotide binding site based on affinity labeling, labeling of lysines, and a conserved triad of glycines. The region beyond this nucleotide fold also contains essential residues. Modification of Asp 184 with a hydrophobic carbodiimide leads to inactivation, and this residue may function as a general base in catalysis. Despite the diversity of the kinase family, all share a homologous catalytic core, and the residues essential for nucleotide binding or catalysis in the C subunit are invariant in every protein kinase. Affinity labeling and intersubunit cross-linking have localized a portion of the peptide binding site, and this region is variable in the kinase family. The crystal structure of the C subunit also is being solved. The C subunit is maintained in its inactive state by forming a holoenzyme complex with an inhibitory regulatory (R) subunit. This R subunit has a well-defined domain structure that includes two tandem cAMP binding domains at the carboxy-terminus, each of which is homologous to the catabolite gene activator protein in Escherichia coli. Affinity labeling with 8N3 cAMP has identified residues that are in close proximity to the cAMP binding sites and is consistent with models of the cAMP binding sites based on the coordinates of the CAP crystal structure. An expression vector was constructed for the RI subunit and several mutations have been introduced. These mutations address 1) the major site of photoaffinity labeling, 2) a conserved arginine in the cAMP binding site, and 3) the consequences of deleting the entire second cAMP binding domain.

Mutation of a kinase allosteric node uncouples dynamics linked to phosphotransfer

“…The conserved kinase core, first defined by the structure of PKA (26)(27)(28), is a bilobal structure that brings together two substrates (nucleotide and peptide/protein) at its active-site cleft to generate a phosphorylated product. Our mechanistic understanding of these enzymes is limited by simplification of these structures as rigid bodies that do not allow for molecular explanations of subtleties in kinase function.…”

Protein kinase A isozyme switching: eliciting differential cAMP signaling and tumor reversion

“…Functional characterization of cAMP-binding mutation in PKA type I has been extensively studied (McKnight et al, 1988), whereas functional analysis of the RIa subunit pseudophosphorylation site (Taylor et al, 1988) has scarcely been studied (Durgerian and Taylor, 1989;Lee et al, 1999). Here, we used the RIa mutant, RIa-P, which gains an autophosphorylation site (ala-ser) by point mutation of G-T (Durgerian and Taylor, 1989;Lee et al, 1999) to functionally mimic RII.…”

Regulation of Human Endothelial Cell Focal Adhesion Sites and Migration by cGMP-dependent Protein Kinase I

“…VASP phosphorylation by 8-pCPTcGMP-stimulated endogenous cGK I was completely inhibited by 5 ϫ 10 10 particles of mutant cGK I␤-containing adenoviral vector/ml, which produced a severalfold overexpression of the mutant kinase compared with the endogenous kinase. The mutated Lys 405 is the homolog of the essential Lys 72 in the ATP-binding site of cAK that is conserved in all protein kinases (39). The mutant's inhibitory effect on wild-type cGK I function could be abolished by raising the concentration of 8-pCPTcGMP from 100 to 500 M (data not shown), suggesting that the inactive mutant competed with wild-type cGK I for the cGMP analog.…”