Figure 4. AIR-2 Phosphorylation Increases the Kinase Activity of TLK-1.

(A) Wild-type TLK-1, TLK-1(S634E), and TLK-1(S634A) were incubated with ASF-1 and MYBP in kinase buffer supplemented with γ-³²P-ATP. ³²P incorporation was measured by phosphoimaging, and protein loading was determined by antibody and Ponceau S staining. All three TLK-1 fusion proteins underwent autophosphorylation (P-TLK-1) and phosphorylated ASF-1 (P-ASF-1) and MYBP (P-MYBP). However, the relative levels of ³²P incorporation differed, with TLK-1(S634E) displaying increased kinase activity with respect to wild-type TLK-1 or TLK-1(S634A).

(B) Quantitation of ³²P incorporation into MYBP and ASF-1 in the kinase assays shown in (A). Error bars represent the standard deviation from at least three separate experiments.

(C) Wild-type TLK-1, TLK-1(S634A), and TLK-1(S634E) were incubated with ASF-1 in kinase buffer supplemented with γ-³²P-ATP in the presence and absence of AIR-2/ICP-1. All three TLK-1 proteins underwent autophosphorylation (P-TLK-1) and phosphorylated ASF-1 (P-ASF-1) (lanes 2, 4, and 6). However, ASF-1 phosphorylation by wild-type TLK-1 was increased in the presence of AIR-2/ICP-1 (lane 3). Phosphorylation of ASF-1 by TLK-1(S634A) and TLK-1(S634E) did not change in the presence or absence of AIR-2/ICP-1 (lanes 4–7).

(D) Quantitation of ³²P incorporation into ASF-1 in the kinase assays shown in (C). Error bars represent the standard deviation from at least three separate experiments.