Consequences of lysine 72 mutation on the phosphorylation and activation state of cAMP-dependent kinase.
General strategies to obtain inactive kinases have utilized mutation of key conserved residues in the kinase core, and the equivalent Lys72 in cAMP-dependent kinase has often been used to generate a "dead" kinase. Here, we have analyzed the consequences of this mutation on kinase structure and function. Mutation of Lys72 to histidine (K72H) generated an inactive enzyme, which was unphosphorylated. Treatment with an exogenous kinase (PDK-1) resulted in a mutant that was phosphorylated only at Thr197 and remained inactive but nevertheless capable of binding ATP. Ser338 in K72H cannot be autophosphorylated, nor can it be phosphorylated in an intermolecular process by active wild type C-subunit. The Lys72 mutant, once phosphorylated on Thr197, can bind with high affinity to the RIalpha subunits. Thus a dead kinase can still act as a scaffold for binding substrates and inhibitors; it is only phosphoryl transfer that is defective. Using a potent inhibitor of C-subunit activity, H-89, Escherichia coli-expressed C-subunit was also obtained in its unphosphorylated state. This protein is able to mature into its active form in the presence of PDK-1 and is able to undergo secondary autophosphorylation on Ser338. Unlike the H-89-treated wild type protein, the mutant protein (K72H) cannot undergo the subsequent cis autophosphorylation following phosphorylation at Thr197. Using these two substrates and mammalian-expressed PDK-1, we can elucidate a possible two-step process for the activation of the C-subunit: initial phosphorylation on the activation loop at Thr197 by PDK-1, or a PDK-1-like enzyme, followed by second cis autophosphorylation step at Ser338.
Iyer
Ganesh H
GH
Howard Hughes Medical Institute and Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, California 92093-0654, USA.
Moore
Michael J
MJ
Taylor
Susan S
SS
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry* / Q000378:metabolism*; D004789:Enzyme Activation; D008239:Lysine; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism; D011487:Protein Conformation; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism
D010446:Peptide Fragments; D021122:Protein Subunits; D010768:Phosphoserine; D017868:Cyclic AMP-Dependent Protein Kinases; D008239:Lysine