Figure 2.

NLK phosphorylates A-Myb and inhibits A-Myb-dependent trans-activation without A-Myb degradation. (A) In vivo phosphorylation of A-Myb. CV-1 cells were transfected with the FLAG-A-Myb expression vector, with or without the expression vector for FLAG-tagged wild-type (WT) or kinase-negative (KN) NLK. Anti-FLAG antibody was used to detect FLAG-A-Myb and FLAG-NLK in whole cell lysates. In lane 3, cell lysates were incubated with λ protein phosphatase (PPase). (B) NLK phosphorylates A-Myb in vitro. 293 cells were transfected with the expression plasmids for FLAG-A-Myb or FLAG-tagged wild-type or kinase-negative NLK, and cell lysates were immunoprecipitated with anti-FLAG antibody. The immunocomplexes were mixed with ³²P-ATP, as indicated above each lane, and analyzed by SDS-PAGE and autoradiography. (C) NLK negatively regulates the trans-activating capacity of A-Myb. CV-1 cells were transfected with a mixture of the luciferase reporter bearing six Myb-binding sites (6MBS-I-luc) or containing the c-myc promoter (c-myc-luc) and either the A-Myb expression plasmid or the control plasmid, with or without increasing amounts of plasmid to express wild-type or kinase-negative NLK. Luciferase activity was then measured. The relative trans-activation capacity of A-Myb (average of 3 experiments) is shown. The degree of activation by A-Myb using 6MBS-I-luc and c-myc-luc was 14.0 ± 0.9- and 25.4 ± 2.1-fold, respectively. (D) Induction of B-Myb degradation by NLK. CV-1 cells were transfected with the FLAG-B-Myb expression vector, with or without the FLAG-NLK expression plasmid. Anti-FLAG antibody was used to detect FLAG-B-Myb and FLAG-NLK in whole cell lysates. In lane 2, transfected cells were treated with the proteasome inhibitor MG132 (50 μM) for 7 h before lysate preparation. (E) Negative regulation of B-Myb-dependent trans-activation by NLK. The effect of NLK on B-Myb-dependent transcription from the 6MBS-I-luc reporter was examined as described in C.