|
|
|
|
|
|
|
|
|||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | |||||
|
Fig. 5. Identification and functional analysis of the ERK1/2 target sites within FOXM1c. (A) Sequence analysis of FOXM1c identifies three putative ERK1/2 phosphorylation sites (PXS/TP) at amino acids 329-332, 618-621 and 702-705. DBD, DNA binding domain; TAD, transactivating domain. (B) The 331 and 704 motifs are conserved in the FOXM1c coding sequences from multiple species. (C) Activated Raf/MEK/MAPK signaling stimulates FOXM1 phosphorylation. Asynchronized BJ1 cells were incubated with 200 µM ATA or 200 nM TPA for 1 hour. Cells with or without drug treatment were lysed and endogenous FOXM1 immunoprecipitated with anti-FOXM1 antibody or control rabbit antiserum. The immunoprecipitates were immunoblotted with anti-FOXM1 and anti-phosphoserine antibodies. ATA and to a lesser extent TPA, enhanced the phosphorylation of FOXM1. Note that anti-phosphoserine antibody selectively detected the upper band of the FOXM1 doublet. (D) Both the 331 and 704 motifs are important for mediating the caMEK1 enhancing effect. The wild type (WT) and various substitutive mutants (S331A, S704A, SASA) were co-transfected with caMEK1 in transient reporter assays as described in Fig. 3. caMEK1 enhancement is shown as percentage of activation compared to the wild-type control (100%). Immunoblot analysis revealed expression of the various HA-tagged, mutant FOXM1c proteins.
|
