CAMP-dependent protein kinase: prototype for a family of enzymes.
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.
Taylor
S S
SS
Department of Chemistry, University of California, San Diego, La Jolla 92093.
Bubis
J
J
Toner-Webb
J
J
Saraswat
L D
LD
First
E A
EA
Buechler
J A
JA
Knighton
D R
DR
Sowadski
J
J
D000595:Amino Acid Sequence; D000818:Animals; D046911:Macromolecular Substances; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011494:Protein Kinases / Q000378:metabolism*
D046911:Macromolecular Substances; D011494:Protein Kinases