Figure 5: Inhibition of CHK2 activity increases ERK1/2 phosphorylation.

(a) SUDHL6 and Farage cells were treated with CHK2 inhibitor II in RPMI 1640 complete medium for 4 h. The levels of pCHK2, CHK2, pERK1/2 or ERK1/2 in the total cell lysates were monitored by immunoblotting with the indicated antibodies. (M, media; D, DMSO). (b) HeLa and HEK 293 cells were transiently transfected with the expression plasmids as indicated. At 24 h post-transfection, cell lysates were subjected to immunoblotting with anti-pERK1/2, anti-ERK1/2, anti-pCHK2, anti-CHK2 or anti-Myc antibody. (c) Knocking down of CHK2 increases ERK1/2 phosphorylation. Farage cells were transfected with control siRNA or CHK2 siRNA. At 72 h after transfection, the expression of pCHK2, CHK2, pERK1/2, ERK1/2, or Actin was monitored by immunoblotting. (d) Farage and SUDHL6 cells were treated with 5 μM Etoposide in RPMI 1640 complete medium for 15 or 30 min. Cell lysates were subjected to immunoblotting with anti-pERK1/2, anti-ERK1/2, anti-pCHK2 or anti-CHK2 antibody. (e) Knocking down of CHK2 attenuates the regulation of pERK by etoposide. Farage cells were transfected with control siRNA or CHK2 siRNA. At 48 h after transfection, Farage cells were treated with 5 μM Etoposide in RPMI 1640 complete medium for 30 min. Cell lysates were subjected to immunblotting with anti-pERK1/2, anti-ERK1/2, anti-pCHK2 or anti-CHK2 antibody. M, media; D, DMSO. (f) CHK2 directly inhibits ERK2-mediated phosphorylation through a kinase-independent mechanism. Purified wild-type (WT) or inactive (KD) GST–CHK2 and MBP were incubated in the absence or presence of ERK2, and protein phosphorylation was determined. Top panels: autoradiography of CHK2 and ERK2 autophosphorylation and phosphorylation of MBP. CHK2 does not inhibit ERK2 autophosphorylation but does inhibit ERK2-mediated phosphorylation of MBP. Bottom panels: Coomassie stain of CHK2, ERK2, and MBP in the kinase assays. GST was used as a control.