Figure 3.
Expression of ERK3 stimulates activity of MK5. (A, B) Coexpression of MK5 and ERK3 in HEK293 cells. (A) Autoradiograph of MK5 IP kinase assay. GFP-MK5 was precipitated by anti-GFP antibodies from unstimulated cells (C), HEK293 cells stimulated by 150 μM arsenite treatment (A) and from arsenite-stimulated cells pretreated for 1 h with 10 μM SB203580 (AS) expressing GFP-MK5 alone (Ctr), or coexpressing Flag-p38 or His-ERK3. MK5 kinase activity in the IP was determined by incorporation of phosphate from [γ-33P]ATP into the in vitro substrate Hsp25. In the IP, ERK3 and GFP-MK5 were also phosphorylated. (B) Quantification of Hsp25 phosphorylation by phospho-imaging of two independent experiments, each with double determinations. (C) Autoradiograph of IP kinase assay using MK5 antibodies for WT and MK5-deficient (−/−) MEFs transfected with control plasmid (C) or expressing His-ERK3.