ACTIVATION OF (HIS)(6)-RAF-1 IN-VITRO BY PARTIALLY PURIFIED PLASMA-MEMBRANES FROM V-RAS-TRANSFORMED AND SERUM-STIMULATED FIBROBLASTS

The serine-threonine protein kinase Raf-1 is an important signal transducer in mitogenesis, phosphorylating and activating mitogen-activated protein (MAP) kinase kinase. Raf-1 activation in vivo is dependent on Ras, but the mechanism of Raf activation is unknown. The ability of preparations of plasma membranes to activate exogenous (His)(6)-Raf-1 was studied. Plasma membranes of v-Ras-transformed NIH 3T3 cells, but not parental cells, enhanced MAP kinase kinase kinase (MAPKKK) activity dependent on addition of (His)(6)-Raf-1 and ATP/Mg. Treatment of membranes with concentrations of Bacillus cereus phosphatidylcholine-specific phospholipase C that activated Raf-1 in vivo failed to enhance MAPKKK activity in vitro. Activation of (His)(6)Raf-1 in vitro by membranes was dependent on binding to Ras. Membranes from v-Src transformed cells also activated (His)(6)Raf-1 and synergized with v-Ras membranes. Serum-treatment of NIH 3T3 cells stimulated the ability of membranes to activate (His)(6)-Raf-1. Activated (His)(6)-Raf-1 could be recovered on Ni2+-agarose, and this methodology was used to demonstrate that activation by membranes was ATP dependent. These findings demonstrate Ras- and ATP-dependent step(s) for Raf-1 activation by plasma membranes in vitro.