Association of catalytic and regulatory subunits of cyclic AMP-dependent protein kinase requires a negatively charged side group at a conserved threonine.
In Saccharomyces cerevisiae, as in higher eucaryotes, cyclic AMP (cAMP)-dependent protein kinase is a tetramer composed of two catalytic (C) subunits and two regulatory (R) subunits. In the absence of cAMP, the phosphotransferase activity of the C subunit is inhibited by the tight association with R. Mutation of Thr-241 to Ala in the C1 subunit of S. cerevisiae reduces the affinity of this subunit for the R subunit approximately 30-fold and results in a monomeric cAMP-independent C subunit. The analogous residue in the mammalian C subunit is known to be phosphorylated. Peptide maps of in vivo 32P-labeled wild-type C1 and mutant C1(Ala241) suggest that Thr-241 is phosphorylated in yeast cells. Substituting Thr-241 with either aspartate or glutamate partially restored affinity for the R subunit. Uncharged and positively charged residues substituted at this site resulted in C subunits that failed to associate with the R subunit. Replacement with the phosphorylatable residue serine resulted in a C subunit with wild-type affinity for the R subunit. Analysis of this protein revealed that it appears to be phosphorylated on Ser-241 in vivo. These data suggest that the interaction between R and C involves a negatively charged phosphothreonine at position 241 of yeast C1, which can be mimicked by either aspartate, glutamate, or phosphoserine.
Levin
L R
LR
Cold Spring Harbor Laboratory, New York 11724.
Zoller
M J
MJ
D000595:Amino Acid Sequence; D004252:DNA Mutational Analysis; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D010766:Phosphorylation; D011485:Protein Binding; D011494:Protein Kinases / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D012694:Serine; D013329:Structure-Activity Relationship; D013912:Threonine
D046911:Macromolecular Substances; D013912:Threonine; D012694:Serine; D011494:Protein Kinases