Figure 7.

Autophosphorylation and phosphoamino acid analysis of recombinant BRI1-KD. A) Affinity purified calmodulin-binding peptide-BRI1 kinase domain recombinant protein (CBP-BRI1-KD, Lane 1) or the mutant CBP-BRI1-K911E (Lane 2) was incubated with 20 μCi [γ-32P]-ATP in kinase buffer for 1 h at ambient temperature, followed by PAGE. Lanes 3 and 4 represent the Coomassie Blue stained gel corresponding to the autoradiograph in Lanes 1 and 2. Molecular mass of CBP-BRI1-KD was determined by MALDI/MS. B) CBP-BRI1-KD was autophosphorylated and transferred to PVDF membrane as described above. The membrane was digested with HCl and subjected to phosphoamino acid analysis by TLE with pSer, pThr and pTyr standards. CBP-BRI1-KD autophosphorylated primarily on Ser residues with a minor Thr component and no detectable phospho-Tyr residues. Adapted from Oh et al., (2000).