Figure 2.

Osmotic stress-induced, MK2-dependent phosphorylation of CNOT2 at Ser101.

(A) Alignment of CNOT2 amino acid sequences in various organisms and the consensus sequence for MK2, MAPK or CDK substrates. Asterisks indicate putative phosphorylation sites. (B) HEK293T cells transfected with vectors expressing the indicated CNOT2 constructs were treated with (+) or without sorbitol (-). Cell lysates were immunoprecipitated using anti-FLAG antibody. The Anti-FLAG immunoprecipitates were analysed by Phos-tag SDS-PAGE, followed by immunoblotting using anti-FLAG antibody. A schematic representation of immunoblot images in CNOT2 WT treated with (+) or without sorbitol (-) is shown at the bottom. (C) HEK293T cells transfected with vectors expressing the indicated CNOT2 constructs were treated with (+) or without sorbitol (-). Cell lysates were immunoprecipitated with anti-FLAG antibody. Immunoprecipitates (IP) and lysates were analysed by immunoblot. Anti-FLAG antibody detects exogenously expressed CNOT2. Immunoblots for phospho-p38MAPK was used to monitor the presence of osmotic stress-induced response (bottom). (D) HEK293T cells were transfected with the indicated constructs. Cell lysates were immunoprecipitated using anti-FLAG antibody. IPs and lysates were analysed by immunoblot. Anti-FLAG antibody and anti-Myc antibody detect exogenously expressed FLAG-CNOT2 and Myc-p38MAPK-AS (constitutively active p38MAPK), respectively. (E) HeLa cells were pretreated with inhibitors for 30 min and then treated with sorbitol for an additional 30 min. Cell lysates were analysed by immunoblot. SP: SP600125 (JNK inhibitor), SB: SB203580 (p38MAPK inhibitor), MK2i: (MK2 inhibitor). (F) An in vitro kinase assay was performed by incubating recombinant MK2 and indicated CNOT2 fragments in the absence (-) or presence (+) of ATP. Reaction products were analysed by immunoblot using phospho-CNOT2 S101 antibody (upper) and CBB staining (lower). A schematic representation of CNOT2 fragments is shown on the right.