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. 2004 Sep;16(9):2406–2417. doi: 10.1105/tpc.104.023903

Figure 2.

Figure 2.

TTL Is a Putative Substrate for BRI1.

(A) Different bri1 mutations have different effects on the kinase activity of BRI1. In vitro autophosphorylation activity was measured using the E. coli expressed GST fusion proteins of the wild-type BRI1 cytoplasmic kinase domain and its three mutated counterparts containing bri1-101, bri1-104, and bri1-114, respectively. The top panel shows the amount of purified recombinant proteins visualized by Coomassie blue staining, and the bottom panel reveals the autophosphorylation level by autoradiography.

(B) Yeast two-hybrid assay of the interaction between TTL and the three mutated BRI1 cytoplasmic kinases.

(C) In vitro transphosphorylation assay of TTL by BRI1. The wild type and mutated BRI1 cytoplasmic kinase domains were fused to GST, whereas TTL was fused to MBP for recombinant protein expression in E. coli cells. Transphosphorylation assay was performed by mixing MBP-TTL with GST-fused wild type or mutated BRI1CK. Lane 1, GST-BRI1CK alone; lane 2, GST-BRI1 (one-fifth of the amount loaded on lane 1) mixed with MBP-TTL; lane 3, GST-BRI1CK (bri1-101) alone; lane 4, GST-BRI1CK (bri1-101) (one-fifth of the amount loaded on lane 3) mixed with MBP-TTL; lane 5, MBP-TTL alone; lane 6, MBP alone; lane 7, GST-BRI1CK mixed with MBP. The top panel displays the purified recombinant protein by Coomassie blue staining, and the bottom panel shows the level of protein phosphorylation by autoradiography.