Raf is a mitogen-stimulated protein kinase that functions as a component of the signaling cascade that leads to the stimulation of mitogen-activated protein kinase. Here we show that the native structure of Raf is a large multi-subunit protein complex with an apparent mass of 300-500 kDa that interacts with Ras and the mitogen-activated protein kinase kinase Mek. Analysis of the structure of the Raf complex demonstrates that it contains a single Raf protein kinase together with the molecular chaperones hsp90 and p50. The Raf-hsp90-p50 complex was observed in starved cells and in cells activated with serum or phorbol ester. Thus, changes in complex formation with hsp90 and p50 are not required for activation of the Raf protein kinase. However, Raf activation caused by Ras was associated with the translocation of the cytoplasmic Raf-hsp90-p50 complex to the cell membrane. Significantly, it is only the membrane-bound complex that exhibits increased protein kinase activity. Thus, the Ras-activated Raf protein kinase functions as a membrane-bound multi-subunit complex.

Treatment of cells with agents that stimulate the release of arachidonic acid causes increased serine phosphorylation and activation of cytosolic phospholipase A2 (cPLA2). Here we report that cPLA2 is a substrate for mitogen-activated protein (MAP) kinase. Moreover, phosphorylation by MAP kinase increases the enzymatic activity of cPLA2. The site of cPLA2 phosphorylation by MAP kinase, Ser-505, is identical to the major site of cPLA2 phosphorylation observed in phorbol ester-treated cells. Replacement of Ser-505 with Ala resulted in a mutant cPLA2 that is not a substrate for MAP kinase and causes little or no enhanced agonist-stimulated arachidonate release from intact cells. Taken together, these data indicate that MAP kinase mediates, at least in part, the agonist-induced activation of cPLA2.

A growth factor-stimulated protein kinase activity that phosphorylates the epidermal growth factor (EGF) receptor at Thr669 has been described (Countaway, J. L., Northwood, I. C., and Davis, R. J. (1989) J. Biol. Chem. 264, 10828-10835). Anion-exchange chromatography demonstrated that this protein kinase activity was accounted for by two enzymes. The first peak of activity eluted from the column corresponded to the microtubule-associated protein 2 (MAP2) kinase. However, the second peak of activity was found to be a distinct enzyme. We present here the purification of this enzyme from human tumor KB cells by sequential ion-exchange chromatography. The isolated protein kinase was identified as a 46-kDa protein by polyacrylamide gel electrophoresis and silver staining. Gel filtration chromatography demonstrated that the enzyme was functional in a monomeric state. A kinetic analysis of the purified enzyme was performed at 22 degrees C using a synthetic peptide substrate based on the primary sequence of the EGF receptor (KREL VEPLT669PSGEAPNQALLR). The Km(app) for ATP was 40 +/- 5 microM (mean +/- S.D., n = 3). GTP was not found to be a substrate for the purified enzyme. The Km(app) for the synthetic peptide substrate was 260 +/- 40 microM (mean +/- S.D., n = 3). The Vmax(app) for the isolated protein kinase was determined to be 400-900 nmol/mg/min. The purified enzyme was designated EGF receptor Thr669 (ERT) kinase. It is likely that the MAP2 and ERT kinases account for the phosphorylation of the EGF receptor at Thr669 observed in cultured cells. The marked stimulation of protein kinase activity caused by growth factors indicates that these enzymes may have an important function during signal transduction.