Single Turnover Autophosphorylation Cycle of the PKA RIIβ Holoenzyme
Fig 3
Single turnover and pre-steady-state kinetics for RIIβ holoenzyme phosphorylation in the presence of Mg2+ versus Ca2+.
(A) Holoenzyme phosphorylation can be followed by gel electrophoresis in which the pRIIβ is detected by antibodies. The C-subunit phosphorylates RIIβ using MgATP or CaATP in 30 min. Shown is a representative western blot in which the C-subunit was incubated with RIIβ alone, with RIIβ and MgATP, or with RIIβ and CaATP. The starting RIIβ sample was loaded on the last lane showing no phosphorylation before incubation with C-subunit and nucleotide. Also, no phosphorylation was observed when the C-subunit and RIIβ were incubated without nucleotide, suggesting that it is not ATP contamination of either protein sample that contributes to RIIβ phosphorylation. (B) Shown is a western blot in which the C-subunit was incubated with RIIβ alone, with RIIβ and AMP-PNP, or with RIIβ and ATP. Mg2+ or Ca2+ were added to the reaction buffer, which contains nucleotides. The phosphorylation status of RIIβ was monitored at 30 min, 1 d, and 2 d. The sample with AMP-PNP shows phosphorylation of RIIβ that increases over time. (C) Pre-steady-state kinetics of RIIβ holoenzyme in the presence of Mg2+ and Ca2+. Holoenzyme phosphorylation as followed by rapid quench analysis.