Phosphorylation of raf-1 by kinase suppressor of ras is inhibited by "MEK-specific" inhibitors PD 098059 and U0126 in differentiating HL60 cells

Determination of the involvement of MAP kinase cascades in signaling cell g rowth or differentiation is aided by the use of the inhibitors PD 098059 [2 -(2'amino-3'-methoxyphenyl)oxananphthalen-4-one] and U0126 [1,4-diamino-2,3 -dicyano-1,4-bis(2-aminophenylthio)butadiene], believed to be MEK-specific kinase inhibitors. We report here that the activity of kinase suppressor of ras (KSR-1), a kinase upstream of raf-1, is inhibited by both these compou nds at concentrations similar to those that inhibit MEK-1. Further, in HL60 cells induced to differentiate with 1,25-dihydroxyvitamin D-3 raf-1 and p9 0RSK, but not ERK1/2, are coregulated, and their expression as well as mono cytic differentiation is inhibited in parallel by PD 098059. Thus, in this system raf-1 is phosphorylated by KSR-1, and PD 098059 as well as U0126 inh ibits this phosphorylation. This suggests great caution in the interpretati on of experiments that utilize these pharmacological inhibitors of kinase a ctivity as evidence for a role for the MEK-ERK module in ras or raf-1 signa ling. (C) 2001 Academic Press.