Figure 3.

MPK3 Phosphorylates AZI1—In Vitro Kinase Assay.

Recombinant proteins of AZI1 (proline-rich domain; the five putative phosphorylation sites are indicated) and GST–MPK3 were isolated from E. coli and incubated in kinase reaction buffer containing [γ-³²P]-ATP. Myelin basic protein (MBP), a known MAPK substrate, served as positive control. SDS–PAGE followed by autoradiography revealed incorporation of ³²P into AZI1 peptide and MBP (right). Left: Coomassie Blue staining of the corresponding gel section. (During protein purification, the fusion tag (intein–chitin binding domain) partially co-elutes with AZI1 peptides.)