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Soluble extracts from uninfected murine cells supplemented with purified viral E1 and E2 proteins support the replication of exogenously added papilloma virus DNA. The E2 transactivator stimulates the binding of the E1 replication protein to the minimal origin of replication and activates DNA replication. These results support the concept that transcription factors have a direct role in the initiation of DNA replication in eukaryotes by participating in the assembly of a complex at the origin of replication.

The mechanism by which transcription factors stimulate DNA replication in eukaryotes is unknown. Bovine papillomavirus DNA synthesis requires the products of the viral E1 gene and the transcriptional activator protein encoded by the E2 gene. Experimental data showed that the 68-kilodalton (kD) E1 protein formed a complex with the 48-kD E2 transcription factor. This complex bound specifically to the viral origin of replication, which contains multiple binding sites for E2. Repressor proteins encoded by the E2 open reading frame failed to complex with E1 suggesting that the 162-amino acid region of E2 that participates in transactivation contained critical determinants for interaction with E1. The physical association between a replication protein and a transcription factor suggests that transcriptional activator proteins may function in targeting replication initiator proteins to their respective origins of replication.

The GTPase-activating protein (GAP) stimulates the GTPase reaction of p21 by 5 orders of magnitude such that the kcat of the reaction is increased to 19 s-'. Mutations of residues in loop Li (Gly-12 and Gly-13), in loop L2 , and in loop LA (Gln-61 and Glu-63) influence the reaction in different ways, but all of these mutant p21 proteins still form complexes with GAP. The C-terminal domain of the human GAP gene product, GAP334, which comprises residues 714 to 1047, is 20 times less active than full-length GAP on a molar basis and has a fourfold lower affinity. This finding indicates that the N terminus of GAP containing the SH2 domains modifies the interaction between the catalytic domain and p21.p21 proteins are the products of the N-, K-, and H-ras genes. They are small guanine nucleotide-binding proteins that are believed to function as signal switch molecules: in the active GTP-bound state, they are switched on; in the GDP-bound state, they are switched off. p21 has an intrinsic GTPase activity, the rate of which determines the lifetime of the active state (5, 10).Mutated forms of the ras genes have been identified in ca. 30% of human tumors. These ras oncogenes code for p21 proteins with point mutations in only three residues: Gly-12, Gly-13, and Gln-61. The mutated proteins have a reduced GTPase activity (5, 8, 9). The GTPase-activating protein (GAP) has been identified by its property of stimulating the conversion of normal p21-GTP to p21-GDP in Xenopus oocytes (32, 41). It has been cloned and sequenced and has a molecular mass of 120 kDa. The fact that it does not stimulate the GTPase activity of oncogenic mutants can explain the transforming properties of those mutants (18,41,42,44).The p21-GAP interaction has not been investigated in detail, and the stimulation of the GTPase by GAP was estimated to be 200-fold (41). By mutational analysis, it has been established that loop L2 of p21 is involved in the interaction with GAP (1,13,18,32). From the threedimensional structure, it would appear that loop L4 could also be involved in this interaction (33,36,37,40). It has also been assumed that the C-terminal domain of GAP is sufficient for the GTPase stimulatory activity (44). We have reevaluated the properties of GAP and show that the maximal stimulation of the GTPase activity by GAP is 105-fold and that mutations in loops Ll, L2, and LA affect the p21-GAP interaction in different ways. We also show that the C-terminal domain of GAP is not sufficient for full activating activity and is apparently modulated by the SH2 (src homologous) domains of GAP.

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Raf-1 is a serine/threonine kinase which is essential in cell growth and differentiation. Tyrosine kinase oncogenes and receptors and p2l' can activate Raf-1, and recent studies have suggested that Raf-1 functions upstream of MEK (MAP/ERK kinase), which phosphorylates and activates ERK. To determine whether or not Raf-1 directly activates MEK, we developed an in vitro assay with purified recombinant proteins. Epitopetagged versions of Raf-1 and MEK and kinase-inactive mutants of each protein were expressed in Sf9 cells, and ERK1 was purified as a glutathione S-transferase fusion protein from bacteria. Raf-1 purified from Sf9 cells which had been coinfected with v-src or v-ras was able to phosphorylate kinase-active and kinase-inactive MEK. A kinase-inactive version of Raf-l purified from cells that had been coinfected with v-src or v-ras was not able to phosphorylate MEK. Raf-1 phosphorylation of MEK activated it, as judged by its ability to stimulate the phosphorylation of myelin basic protein by glutathione S-transferase-ERK1. We conclude that MEK is a direct substrate of Raf-1 and that the activation of MEK by Raf-1 is due to phosphorylation by Raf-1, which is sufficient for MEK activation. We also tested the ability of protein kinase C to activate Raf-1 and found that, although protein kinase C phosphorylation of Raf-1 was able to stimulate its autokinase activity, it did not stimulate its ability to phosphorylate MEK.The serine/threonine kinase Raf-1 is activated by many growth factors, including epidermal growth factor, plateletderived growth factor, erythropoietin, and insulin (1,3,4,6,10,12,13,16,22,23) and is believed to play a central role in cell growth. Several studies have shown that Raf-1 functions downstream of and is required for signalling by receptor and nonreceptor tyrosine kinases and Ras in mammalian cells, in Drosophila melanogaster, and in Caenorhabditis elegans. That Raf-1 acts downstream of Ras is indicated by experiments in which raf-1 antisense RNA and kinase-defective Raf-1 mutants were able to block cell proliferation and transformation by activated Ras (15). The same study also used the expression of raf-1 antisense RNA and kinasedefective Raf-1 mutants to show that Raf-1 is required for the normal growth of the cells. Similarly, the involvement of Ras in the mitogen-activated protein (MAP) kinase pathway is well established (20,24,26,27,31). However, the mechanism by which Src and Ras activate Raf-1 is not known. It has been suggested that the activation of Raf-1 is mediated by yet another kinase whose activity is stimulated by Ras and Src. The hypothesis that Raf-1 is positively regulated by phosphorylation of serine and/or threonine residues is supported by observations that stimulation of growth factor receptors and expression of membrane-bound oncogene products result in the hyperphosphorylation of Raf-1 and an increase in its kinase activity (3,12,13,15,16,22,23). A more recent study showed protein kinase Cot (PKC)-induced phosphorylation of Raf-1 led to increased autokinase a...