Figure 4.

STRK1 Phosphorylates CatC Mainly at Tyr-210 and Activates Its Activity.

(A) STRK1 phosphorylates CatC in vitro. His-TF or His-TF-CatC was incubated with GST-STRK1 and [γ-³²P]ATP. Upper panel shows autoradiography and bottom panel Coomassie brilliant blue (CBB) staining of the gel.

(B) Phosphorylation of CatC is indeed dependent on the kinase activity of STRK1. His-TF-CatC was incubated with GST-STRK1, GST-STRK1^K95E and ³²P-γ-ATP. GST-STRK1^K95E is the kinase dead form of STRK1. Upper panel shows autoradiography and bottom panel Coomassie brilliant blue (CBB) staining of the gel.

(C) STRK1 stimulates CatC activity in vitro. Recombinant His-TF-CatC expressed in E. coli was purified, and the CatC activity was measured with GST or GST-STRK1 in the presence of ATP. Data are presented as mean ± sd (n = 3, **P ≤ 0.01, Student’s t test).

(D) An equal amount of His-TF fused CatC, CatC^Y210F, CatC^Y360F, and CatC^Y210,360F was incubated with GST-STRK1 and [γ-³²P]ATP. Upper panel shows autoradiography and bottom panel Coomassie brilliant blue staining of the gel.

(E) Phosphorylation of CatC at Tyr-210 is essential for CatC activity. CatC^Y210Fand CatC^Y360F, recombinant CatC with Tyr-210 and Tyr-360 mutated to Phe, CatC^Y210D and CatC^Y360D, and recombinant CatC with Tyr-210 and Tyr-360 mutated to Asp. Each data point represents the mean ± se (n = 6). Asterisk indicates significant difference relative to CatC activity in the absence of STRK1 and ATP (Student’s t test, **P < 0.01).

(F) and (G) CAT activity (F) and H₂O₂ contents (G) in transgenic seedlings (T0) overexpressing CatC^Y210F and CatC^Y210D in the Ri11 background, a STRK1 knockdown mutant. The Kitaake (WT) and Ri11 seedlings regenerated from the corresponding calli were used as control. Data are presented as mean ± sd (n = 3, *P ≤ 0.05, **P ≤ 0.01, Student’s t test).