Figure 5

ZmPti1a protein expression and kinase activity. (A) Immunodetection of ZmPti1a protein in various tissues at different developmental stages of maize. Proteins from the sterile flavonol-deficient whp maize line and its corresponding wild type (WT) were size fractionated using PAGE (silks pollen, pollinated silks 6 h after pollination; see legend Fig. 3 for other tissues). Proteins were subjected to Western blot and detected using a polyclonal antibody raised against recombinant ZmPti1a. Ponceau stain of the blot is given as a control for loading of the gel. A strong band of the expected molecular size of the ZmPti1a protein of approximately 41 kDa is visible in extracts from mature pollen. Faint bands could be detected in protein of staminate spikelets at anthesis (0 dba) and pollinated silks after extended exposure times. (B) Autophosphorylation and cross-phosphorylation of ZmPti1a. Wild type ZmPti1a-His fusion protein, wild type MBP-SlPto, and the kinase-deficient mutants ZmPti1a-His(K100N) and GST-SlPti1(K96N) were over-expressed and purified in equal amounts from E. coli, using Ni-NTA magnetic beads, GST-Bind-Resin or amylose resin, respectively. Immobilized proteins were incubated alone or in pairs with [γ-³²P]ATP in kinase buffer, separated by PAGE and exposed to X-ray film. Cross-phosphorylation of GST-SlPti1(K96N) by MBP-SlPto served as positive control. ZmPti1a is capable of autophosphorylation. The K100N mutation completely abolished autophosphorylation of ZmPti1a. ZmPti1a-His(K100N) is moderately phosphorylated by MBP-SlPto whereas ZmPti1a-His cannot phosphorylate GST-SlPti1. (C) Magnetocapture interaction kinase assay. Wild type ZmPti1a-His or mutant ZmPti1a-His(K100N) was immobilized on Ni-NTA magnetic beads and incubated with native protein extracts from pollen, silks or seedlings. ZmPti1 and bound proteins were collected by magnetic force, washed and subjected to kinase assays. Unloaded Ni-NTA magnetic beads were used as control. Proteins were separated by PAGE and exposed to X-ray film. Pollen and silk but not seedling extracts contained kinase activities capable of interaction with and cross-phosphorylating ZmPti1a-His(K100N).