Cell-Free Assay System for Ras- and Rap1-Dependent Activation of MAP-Kinase Cascade

It is well-established that Ras activates the mitogen-activated protein (MAP) kinase cascade consisting of MAP kinase, extracellular signal-regulated kinase (ERK), ERK kinase (MEK), and MEK kinase in mammals (for reviews, see refs. 1 –4 ). MAP kinase is phosphorylated at both serme/threonme and tyrosine residues by MEK and this phosphorylation causes the MAP-kinase activation (1 –3 ). MEK is also phosphorylated at serine/threonme residues by MEK kinase, and this phosphorylation causes the MEK activation (5 –7 ). Many MEK kinases have been identified: these include c-Raf-I (8 –10 ), B-Raf (11 –15 ), Mos (16 –17 ), and mStel 1 (11 –18 ). There are several lines of evidence that Ras is an upstream regulator of c-Raf-1:

1	The antisense c-Raf-1 RNA and dominant negative c-Raf-1 inhibit the Rasinduced DNA synthesis and growth (19 );
2.	Ras genetically posmons upstream of c-Raf-1 in Drosophila (20 ) and Caenorhabditis elegans (21 );
3	Dominant negative c-Raf-1 inhibits the Ras-induced MAP kinase activation in intact cells (22 ), and
4.	GTP-Ras directly interacts with c-Raf-1 (23 –28 )