Oncogenic ras triggers the activation of 42-kDa mitogen-activated protein kinase in extracts of quiescent Xenopus oocytes.

Abstract

Quiescent, full-grown Xenopus oocytes, which are arrested at the G2/M border of meiosis, contain an inactive 42-kDa mitogen-activated protein kinase (p42MAPK) that is activated when oocytes are stimulated to resume the meiotic cell cycle. MATERIALS AND METHODSConstruction of Escherichia coli Expression Vectors. PCRs (Taq polymerase, Perkin-Elmer) (25) were performed by Vincent Jung to generate Ha-ras genes with the desired mutations and BamHI as the cloning site (underlined). The 5' oligonucleotide used was 5'-CCCTGAGGjATCCATGAC-GAATATAAG-3' which created a BamHI site (underlined) upstream of the initiation codon (in bold); the 3' oligonucleotide was 5'-CCTCAGGATCCTCAGGAGAGCACA-CACTT-3'. The 3' oligonucleotide used to mutate the membrane localization site was 5'-CCTCAGGATCCTCAG-GAGAGCACACGCCTT-3' with a point mutation (in bold) to change Cys'86 (CAC) to Arg (CGC). The PCR templates were derived from [Gly12]Ha-ras (wild type) and [Vall2]Ha-ras cDNAs (26). These PCR products were first cloned into ADHI promoter-driven expression vectors for Saccharomyces cerevisiae (27) to confirm their biological activity (28). Expression of [Gly12]Ha-ras in yeast suppressed the temperature sensitivity of a strain containing the temperaturesensitive mutation ras2ts. Expression of [Val'2]Ha-ras conferred heat shock sensitivity on a wild-type strain and suppressed the lethality of a strain containing cdc25ts. Finally, expression of [Val"2,Arg186]Ha-ras suppressed the heat shock sensitivity of a strain containing [Val19]RAS2 (29). The fragments were cloned into pTrcHis (Invitrogen, San Diego) and expressed as fusion proteins with N-terminal oligohistidine (30,31).Purification of Intact and Histidine-Tagged Fusion Proteins. Ha-Ras proteins were purified as described (32). His-tagged proteins were prepared according to ref. 31 and manufacturer's instructions (Invitrogen). The His6-Ras proteins were solubilized in sonication buffer [10 mM Tris HCI, pH 7.5/10 mM MgCl2/50 mM NaCI/1 ,M phenylmethanesulfonyl fluoride/20 AM leupeptin/aprotinin (2 ,tg/ml)/2 AM pepstatin/ 0.5% (wt/vol) dodecyl f3-D-maltoside (Boehringer Mannheim)]. The protein was bound to Pro-Bond resin (Invitrogen) overnight at 4°C and eluted with increasing imidazole concentrations. Fractions containing His-tagged proteins, identified by immunoblotting, were dialyzed against microinjection buffer (20 mM Hepes, pH 7.4/20 mM NaCl/4 mM MgCl2/0.5 mM 2-mercaptoethanol). His6-Ras preparations (at least 70% pure) were concentrated using Centriprep-10 concentrators (Amicon) and stored at -70°C in microinjecAbbreviations: MAP kinase, mitogen-activated protein kinase; p42MAPK, 42-kDa MAP kinase; MPF, maturation-promoting factor; GVBD, germinal vesicle breakdown; MBP, myelin basic protein; CHX, cycloheximide. 9831The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Initiation of Xenopus Oocyte Maturation by Activation of the Mitogen-activated Protein Kinase Cascade

“…SESE-MAPKK was moderately active as compared to wild-type MAPKK. 2 To produce a more active mutant of MAPKK, a deletion (32)(33)(34)(35)(36)(37)(38)(39)(40)(41)(42)(43)(44)(45)(46)(47)(48)(49)(50)(51) near the N-terminal was introduced into Xenopus SESE-MAPKK according to the method of Mansour et al (43) using primers (5Ј-AGCCTCGAGGTTTGTCTCGGCTGTAGGG-3Ј and 5Ј-CGTCTCGAGGCTTTTCTCACCCAGAAGC-3Ј) and was ligated at the XhoI site, yielding dSESE-MAPKK. This Xenopus dSESE-MAPKK can function as a strongly active mutant in Xenopus oocytes (this study) and in COS cells.…”

“…MAPK even in the presence of cycloheximide (49). This suggests that some MAPKK kinase other than Mos could be activated in vitro, downstream of MPF.…”

“…There may exist a Mos-independent pathway in activation of the MAPK cascade by MPF, since the addition of nondegradable cyclin to oocyte extracts activates the MAPK cascade in the presence of cycloheximide (49). However, the contribution of this pathway in vivo may be minor, since no activation of MAPK was observed by injecting immature oocytes with cyclin A in the presence of cycloheximide.…”

The Ras-related Protein Rheb Is Farnesylated and Antagonizes Ras Signaling and Transformation

“…Xenopus frog oocyte lysates have been shown to contain all the necessary cellular components to mediate oncogenic Ras activation of p42/p44 MAPKs in vitro (59). Therefore, we determined the ability of Rheb to modulate this activity.…”

Activation of the p42 Mitogen-activated Protein Kinase Pathway Inhibits Cdc2 Activation and Entry into M-Phase in CyclingXenopusEgg Extracts

“…Recently, the involvement of p42MAPK in this checkpoint was demonstrated in vivo by using Xenopus tadpole cells (Wang et al, 1997). Ras and Mos both activate the p42MAPK signaling pathway after microinjection into Xenopus oocytes (Hattori et al, 1992;Shibuya et al, 1992b;Nebreda and Hunt, 1993;Posada et al, 1993) and arrest the cell cycle of early embryonic cells at M-phase (Sagata et al, 1989;Daar et al, 1991). Furthermore, constitutively activated p42MAPK has the same effects (Haccard et al, 1993(Haccard et al, , 1995.…”