We report a strategy for regulating the activity of a cytoplasmic signaling molecule, the protein kinase encoded by raf-1. Retroviruses encoding a gene fusion between an oncogenic form of human p74`'1 and the hormone-binding domain of the human estrogen receptor (hrafER) were constructed. The fusion protein was nontransforming in the absence of estradiol but could be reversibly activated by the addition or removal of estradiol from the growth media. Activation of hrafER was accompanied in C7 3T3 cells by the rapid, protein synthesis-independent activation of both mitogen-activated protein (MAP) kinase kinase and p42/p44 MAP kinase and by phosphorylation of the resident p74"If-protein as demonstrated by decreased electrophoretic mobility. The phosphorylation of p74`1" had no effect on the kinase activity of the protein, indicating that mobility shift is an unreliable indicator of p74f-' enzymatic activity. Removal of estradiol from the growth media led to a rapid inactivation of the MAP kinase cascade. These results demonstrate that Raf-1 can activate the MAP kinase cascade in vivo, independent of other "upstream" signaling components. Parallel experiments performed with ratla cells conditionally transformed by hrafER demonstrated activation of MAP kinase kinase in response to estradiol but no subsequent activation of p42/p44 MAP kinases or phosphorylation of p74"'. This result suggests that in ratla cells, p42/p44 MAP kinase activation is not required for Raf-l-mediated oncogenic transformation. Estradiol-dependent activation of p42/p44 MAP kinases and phosphorylation of p74

In unfertilized eggs from vertebrates, the cell cycle is arrested in metaphase of the second meiotic division (metaphase II) until fertilization or activation. Maintenance of the long‐term meiotic metaphase arrest requires mechanisms preventing the destruction of the maturation promoting factor (MPF) and the migration of the chromosomes. In frog oocytes, arrest in metaphase II (M II) is achieved by cytostatic factor (CSF) that stabilizes MPF, a heterodimer formed of cdc2 kinase and cyclin. At the metaphase/anaphase transition, a rapid proteolysis of cyclin is associated with MPF inactivation. In Drosophila, oocytes are arrested in metaphase I (M I); however, only mechanical forces generated by the chiasmata seem to prevent chromosome separation. Thus, entirely different mechanisms may be involved in the meiotic arrests in various species. We report here that in mouse oocytes a CSF‐like activity is involved in the M II arrest (as observed in hybrids composed of fragments of metaphase II‐arrested oocytes and activated mitotic mouse oocytes) and that the high activity of MPF is maintained through a continuous equilibrium between cyclin B synthesis and degradation. In addition, the presence of an intact metaphase spindle is required for cyclin B degradation. Finally, MPF activity is preferentially associated with the spindle after bisection of the oocyte. Taken together, these observations suggest that the mechanism maintaining the metaphase arrest in mouse oocytes involves an equilibrium between cyclin synthesis and degradation, probably controlled by CSF, and which is also dependent upon the three‐dimensional organization of the spindle.

Recent studies have demonstrated the existence of a physical complex containing p2lras (RAS), p74raf-4 (RAF-1), and MEK-1. Although it is clear that formation of this complex depends on the activation state of RAS, it is not known whether this complex is regulated by the activation state of the cell and whether MEK-2 is also present in the complex. To analyze the regulation and specificity of this complex, we utilized immobilized RAS to probe lysates of cultured NIH 3T3 fibroblasts and analyzed the proteins complexing with RAS following serum starvation or stimulation. Complex formation among RAS, RAF-1, and MEK-1 was dependent only on RAS:GMP-PNP and not on cell stimulation. Incubations of lysates with immobilized RAS depleted all RAF-1 from the lysate but bound only a small fraction of cytosolic MEK-1, and further MEK-1 could bind immobilized RAS only if exogenous RAF-1 was added to the lysate. This indicates that binding of MEK-1 to RAS depends on the presence of RAF-1 or an equivalent protein. In contrast to MEK-1, MEK-2 was not detected in the RAS signalling complex. A proline-rich region of MEK-1 containing a phosphorylation site appears to be essential for signalling complex formation. Consistent with the preferential binding of MEK-1 to RAS:RAF-1, the basal activity of MEK-1 in v-ras-transformed cells was found to be elevated sixfold, whereas MEK-2 was elevated only twofold, suggesting that the RAS signalling pathway favors MEK-1 activation. p2lras (RAS) is a membrane-associated guanine nucleotidebinding protein which is active when bound to GTP and inactive when bound to GDP (31). Activation of RAS occurs in response to numerous agonists associated with growth and differentiation, reflecting the importance of RAS as a molecular switch regulating diverse cellular responses (22). The ratio of GTP to GDP can be regulated either by controlling the rate of nucleotide exchange or by regulating the rate of GTP hydrolysis (10, 31). Activation of RAS is sufficient to stimulate a kinase cascade leading to the activation of mitogen-activated protein (MAP) kinases (40, 41); in most cases, activation of RAS is also necessary for activation of MAP kinases (9,26,36).Recent studies have clarified some of the components which lie downstream from RAS in the regulation of MAP kinases. The MAP kinases are dually phosphorylated on threonine and tyrosine by MAP kinase kinases (MKKs or MEKs) (1, 2, 44), and at least two MEKs, MEK-1 and MEK-2, are known to be able to catalyze this phosphorylation in vitro (3,7,16,27,33,42,43,46). MEK-1 is a phosphoprotein, regulated by serine/ threonine phosphorylation, and a substantial body of evidence suggests that p74 ra-(RAF-1) is capable of catalyzing this activating phosphorylation (8,14,17,21,25,29). Although v-RAF can phosphorylate MEK-2 in vitro (42), it is as yet unclear how MEK-2 is regulated in vivo and whether there are functional or regulatory differences between MEK-1 and MEK-2.The role of RAS in the regulation of the MAP kinase cascade is thought to involve direct physical ass...