Microinjection of pp60v-src into Xenopus oocytes increases phosphorylation of ribosomal protein S6 and accelerates the rate of progesterone-induced meiotic maturation.

Microinjection of purified pp60v-src into Xenopus oocytes caused the phosphorylation of ribosomal protein S6 on serine residues and also increased total protein phosphorylation, with almost a two-fold increase in the percentage of phosphotyrosine present. In addition, pp60v-src accelerated the time course of progesterone-induced oocyte maturation, suggesting that the biochemical pathway influenced by pp60v-src is related to that induced by progesterone.     

Tyrosine phosphorylation of the gap junction protein connexin43 is required for the pp60v-src-induced inhibition of communication.

Gap junction communication in some cells has been shown to be inhibited by pp60v-src, a protein tyrosine kinase encoded by the viral oncogene v-src. The gap junction protein connexin43 (Cx43) has been shown to be phosphorylated on serine in the absence of pp60v-src and on both serine and tyrosine in cells expressing pp60v-src. However, it is not known if the effect of v-src expression on communication results directly from tyrosine phosphorylation of the Cx43 or indirectly, for example, by activation of other second-messenger systems. In addition, the effect of v-src expression on communication based on other connexins has not been examined. We have used a functional expression system consisting of paired Xenopus oocytes to examine the effect of v-src expression on the regulation of communication by gap junctions comprised of different connexins. Expression of pp60v-src completely blocked the communication induced by Cx43 but had only a modest effect on communication induced by connexin32 (Cx32). Phosphoamino acid analysis showed that pp60v-src induced tyrosine phosphorylation of Cx43, but not Cx32. A mutation replacing tyrosine 265 of Cx43 with phenylalanine abolished both the inhibition of communication and the tyrosine phosphorylation induced by pp60v-src without affecting the ability of this protein to form gap junctions. These data show that the effect of pp60v-src on gap junctional communication is connexin specific and that the inhibition of Cx43-mediated junctional communication by pp60v-src requires tyrosine phosphorylation of Cx43.     

A 90,000-dalton binding protein common to both steroid receptors and the Rous sarcoma virus transforming protein, pp60v-src

Previous studies have shown that the avian progesterone receptor, when in the nontransformed 8 S state, is complexed to another cellular protein having a molecular weight of 90,000. In this report, we show that this receptor-binding protein is indistinguishable from the 90,000-dalton protein which associates in a complex with the Rous sarcoma virus transforming protein, pp60v-src. This identity was established by the following criteria. 1) Monoclonal antibodies directed against the pp60v-src-associated 90-kDa protein recognized the 90-kDa progesterone receptor binding protein in an immunoblot assay. Conversely, monoclonal antibodies that recognize the progesterone receptor binding protein bind to the 90-kDa protein which complexes with pp60v-src. 2) Peptide maps prepared from the 90-kDa proteins immunoprecipitated from chicken cells with monoclonal antibodies directed against either the 90-kDa receptor binding protein or the 90-kDa pp60v-src-associated protein were indistinguishable. 3) Preincubation of the progesterone receptor complex with monoclonal antibodies prepared against the pp60v-src-associated protein caused a shift in the sedimentation of the progesterone receptor. Previous studies have established that the pp60v-src-associated protein is indistinguishable from one of the major heat shock proteins which are induced under a variety of stress conditions in eukaryotic cells. These present studies implicate a new role for this 90-kDa protein in the action of steroid hormones.     

The development of competence for meiotic maturation during oogenesis in Xenopus laevis

Meiotic maturation of large, 1.2-1.4 mm in diameter, stage VI oocytes of Xenopus laevis can be induced to mature in vitro by exposure to progesterone or by microinjection of maturation-promoting factor (MPF). Small, 0.95 mm in diameter, stage IV oocytes do not respond to progesterone but do undergo germinal vesicle breakdown (GVBD) in response to microinjection of MPF. The possibility that small oocytes are nonresponsive to progesterone due to a specific defect in an event known to occur with large oocytes is investigated. Both large and small oocytes possess a plasma membrane steroid receptor (Mr = 110,000) as measured by photoaffinity labeling with [3H]R5020, but the density of receptors in small oocytes is only 20% of that in large oocytes. Adenylate cyclase activity stimulated by guanyl-5'-yl-imidodiphosphate is equally inhibited by steroid (50%) in plasma membranes from both large and small oocytes with an apparent IC50 of 2 X 10(-7) M progesterone. Microinjection of the heat-stable inhibitor protein of cAMP-dependent protein kinase induces GVBD in large but not in small oocytes. These results indicate that the nonresponsiveness of small, stage IV oocytes to progesterone is due to a deficiency in an event(s) subsequent to cAMP fluctuations but prior to MPF action.     

Oncogenic ras protein induces meiotic maturation of amphibian oocytes in the presence of protein synthesis inhibitors

Microinjection of the activated ras oncogenic protein can induce the meiotic maturation of Xenopus laevis oocytes, a process that can also be triggered by progesterone or high concentrations of insulin. Cycloheximide and puromycin, well-known inhibitors of protein synthesis, block the maturation process induced by progesterone and insulin but do not affect the maturation caused by H-raslys12 protein microinjection. Theophylline, an inhibitor of cAMP phosphodiesterase that also affects oocyte protein synthesis, does cause a partial inhibition of ras protein-induced maturation. These findings indicate that ras protein acts on the oocyte maturation process at a point that is downstream of the protein synthesis requirement, a characteristic shared with the maturation promoting factor, an activity that appears in oocytes and mitotic cells at the onset of cell division.     

Requirement of phospholipase C-catalyzed hydrolysis of phosphatidylcholine for maturation of Xenopus laevis oocytes in response to insulin and ras p21

Recent studies have demonstrated the activation of phospholipase C-mediated hydrolysis of phosphatidylcholine both by growth factors and by the product of ras oncogene, ras p21. Also, evidence has been presented indicating that the stimulation of this phospholipid-degradative pathway is sufficient to activate mitogenesis in fibroblasts. In Xenopus laevis oocytes, microinjection of transforming ras p21 is a potent inducer of maturation, whereas microinjection of a neutralizing anti-ras p21 antibody specifically inhibits maturation induced by insulin but not by progesterone. The results presented here demonstrated that microinjection of phosphatidylcholine-hydrolyzing phospholipase C is sufficient to induce maturation of Xenopus laevis oocytes. Furthermore, microinjection of a neutralizing anti-phosphatidylcholine-hydrolyzing phospholipase C specifically blocks the maturation program induced by ras p21/insulin but not by progesterone.     

Identification of protein phosphatases 1 and 2B as ribosomal protein S6 phosphatases in vitro and in vivo

Protein phosphatases 1 and 2B from rabbit skeletal muscle were found to catalyze the dephosphorylation of ribosomal protein S6 in vitro. Phosphorylation of protein phosphatase-1 by the transforming protein of Rous sarcoma virus, pp60v-src, abolished S6 dephosphorylation by the purified enzyme. Analysis of the dephosphorylation of phosphorylase a and phosphorylase kinase in Xenopus oocyte extracts and after microinjection indicated the presence of oocyte enzymes similar to protein phosphatases-1 and -2B. Studies with 32P-labeled 40 S ribosomal subunits suggested that these enzymes were functioning as S6 phosphatases in oocytes. These findings support the hypothesis that regulation of protein phosphatase activity may be involved in the increase in S6 phosphorylation observed after mitogenic stimulation.     

The incorporation of myo-inositol into phosphatidylinositol derivatives is stimulated during hormone-induced meiotic maturation of amphibian oocytes

The incorporation of myo-[3H]inositol into phosphatidylinositol and its phosphorylated derivatives was studied by microinjection of the radioactive precursor into Xenopus laevis oocytes. Induction of meiotic maturation of the oocytes by treatment with either progesterone one or insulin resulted in a significant increase in the incorporation of myo-[3H]inositol into the phospholipid fraction. This increase occurred 3-6 h after hormonal treatment, a time coincident with the start of the breakdown of the nuclear envelope, and requires protein synthesis. The effect of progesterone and insulin contrasts with the effect of acetylcholine, which acts through a muscarinic receptor causing the activation of phospholipase C, since the latter effector causes an increase in myo-[3H]inositol incorporation, which is more rapid and does not require protein synthesis. These results suggest that the meiotic maturation process is connected with changes in inositol metabolism in the amphibian oocyte.     

A mutation at the ATP-binding site of pp60v-src abolishes kinase activity, transformation, and tumorigenicity.

We constructed a mutant, called RSV-SF2, at the ATP-binding site of pp60v-src. In this mutant, lysine-295 is replaced with methionine. SF2 pp60v-src was found to have a half-life similar to that of wild-type pp60v-src and was localized in the membranous fraction of the cell. Rat cells expressing SF2 pp60v-src were morphologically untransformed and do not form tumors. The SF2 pp60v-src isolated from these cells lacked kinase activity with either specific immunoglobulin or other substrates, and expression of SF2 pp60v-src failed to cause an increase of total phosphotyrosine in the proteins of infected cells. Wild-type pp60v-src was phosphorylated on serine and tyrosine in infected cells, and the analogous phosphorylations could also be carried out in vitro. Phosphorylation of serine was catalyzed by a cyclic AMP-dependent protein kinase, and phosphorylation of tyrosine was perhaps catalyzed by pp60v-src itself. By contrast, SF2 pp60v-src could not be phosphorylated on serine or tyrosine either in infected cells or in vitro. These findings strengthen the belief that the phosphotransferase activity of pp60v-src is required for neoplastic transformation by the protein and suggest that the binding of ATP to pp60v-src elicits an allosteric change required for phosphorylation of serine in the protein.     

Two Distinct Mechanisms Control the Accumulation of Cyclin B1 and Mos in Xenopus Oocytes in Response to Progesterone

Progesterone-induced meiotic maturation of Xenopus oocytes requires the synthesis of new proteins, such as Mos and cyclin B. Synthesis of Mos is thought to be necessary and sufficient for meiotic maturation; however, it has recently been proposed that newly synthesized proteins binding to p34(cdc2) could be involved in a signaling pathway that triggers the activation of maturation-promoting factor. We focused our attention on cyclin B proteins because they are synthesized in response to progesterone, they bind to p34(cdc2), and their microinjection into resting oocytes induces meiotic maturation. We investigated cyclin B accumulation in response to progesterone in the absence of maturation-promoting factor-induced feedback. We report here that the cdk inhibitor p21(cip1), when microinjected into immature Xenopus oocytes, blocks germinal vesicle breakdown induced by progesterone, by maturation-promoting factor transfer, or by injection of okadaic acid. After microinjection of p21(cip1), progesterone fails to induce the activation of MAPK or p34(cdc2), and Mos does not accumulate. In contrast, the level of cyclin B1 increases normally in a manner dependent on down-regulation of cAMP-dependent protein kinase but independent of cap-ribose methylation of mRNA.     

Inhibition of v-src-induced transformation by a GTPase-activating protein.

Previous work has shown that microinjection into cells of antibodies against p21ras blocks transformation by src, suggesting that oncogenic transformation by pp60v-src is dependent on p21ras. The activity of p21ras itself is regulated by its cyclic association with GDP-GTP, where p21ras-GTP is the active form and p21ras-GDP is the inactive form. A GTPase-activating protein (GAP) mediates the inactivation of p21ras by facilitating the conversion of the active p21ras-GTP to the inactive p21ras-GDP. This predicts that overexpression of GAP would inactivate p21ras and block transformation of cells by src. In this paper, we confirm this prediction. We report that overexpression of GAP in NIH 3T3 cells blocks transformation by pp60v-src but not by v-ras. Susceptibility to transformation by v-src is restored when GAP expression is lowered to levels comparable to that in control cells. These results support the suggestion that p21ras plays a central role in the signalling pathway used by pp60v-src.     

ATP-gamma-S (adenosine 5'-0(3-thiotriphosphate)) blocks progesterone-induced maturation of the Xenopus oocyte

ATP-gamma-S microinjection into Xenopus oocyte prevents progesterone induced maturation. Inhibition is time and dose dependent; 50% inhibition occurs when 50 nl of 0.5 mM ATP-gamma-S solution are microinjected/oocyte 1 hr prior to the hormonal trigger. ATP-gamma-S inhibited oocytes can be induced to mature (100%) following microinjection of extracts containing maturation promoting factor (MPF). Our results suggest that the maturation protein(s) has been stabilized in ovo by ATP-gamma-S microinjection, in its phosphorylated inhibitory form.     

Forms of pp60v-src isolated from Rous sarcoma virus-transformed cells.

It has previously been shown that an electrophoretic variant form of the Rous sarcoma virus transforming protein, pp60v-src, exists in src-transformed cells. This variant, which was readily observed in vanadate-treated cells, was characterized as possessing extensive amino-terminal domain phosphotyrosine modification. Its appearance was further correlated with increased src-specific protein kinase activity. In this study, we used a src-specific monoclonal antibody (MAb) to resolve immunologic forms of pp60v-src. The MAb was able to distinguish between two populations of typical lower-band pp60v-src and was unreactive with the electrophoretic variant upper-band pp60v-src species. Using serial immunoprecipitations, we resolved four populations of pp60v-src: src protein either immunoreactive or unreactive with the MAb from both untreated and vanadate-treated transformed cells. The pp60v-src in each fraction displayed a distinct phosphoamino acid composition and tryptic phosphopeptide profile. However, analysis of their tyrosyl kinase specific activities showed that the immunologically resolved populations of pp60v-src from a given culture did not differ. Both pp60v-src fractions from vanadate-treated cells exhibited similar kinase specific activities, which were greatly enhanced over those of enzyme preparations from untreated cells. Since the MAb-reactive pp60v-src fraction from vanadate-treated cells lacked the electrophoretic variant upper-band pp60v-src species yet still possessed enhanced enzymatic specific activity, the initially stated correlation between the appearance of the electrophoretic variant src form and increased src kinase activity breaks down. These results suggest that yet to be defined modifications of the src protein may be involved in its functional regulation.     

Restriction of the in vitro and in vivo tyrosine protein kinase activities of pp60c-src relative to pp60v-src.

The tyrosine protein kinase activities of pp60c-src and pp60v-src were compared. The activities were qualitatively similar in vitro when the src proteins were bound in an immune complex with monoclonal antibody; both proteins utilized either ATP or GTP as phosphate donors, preferred Mn2+ to Mg2+, and had similar exogenous substrate specificities. The specific activity of pp60c-src was about 10-fold lower than that of pp60v-src for exogenous substrate phosphorylation but was only 1.1- to 2-fold lower than that of pp60v-src for autophosphorylation. Six glycolytic enzymes, including three not previously identified as substrates for pp60src phosphorylation, were phosphorylated by both pp60c-src and pp60v-src. Levels of pp60c-src fourfold higher than the amount of pp60v-src in src-plasmid-transformed cells did not detectably alter the level of phosphotyrosine in cellular proteins, but increasing the expression of pp60c-src another twofold (which induces cells to form foci in monolayer culture (P.J. Johnson, P.M. Coussens, A.V. Danko, and D. Shalloway, Mol. Cell. Biol. 5:1073-1083, 1985) resulted in a threefold increase in the level of cellular protein phosphotyrosine. Immunoprecipitation and analysis of the alkali-stable phosphoproteins by two-dimensional electrophoresis showed that, in contrast to pp60v-src-transformed cells, pp36 and enolase are only weakly phosphorylated in these high-level pp60c-src overexpresser cells. Even allowing for the in vitro differences in specific activities of phosphorylation, these results suggest that the pp60c-src tyrosine protein phosphorylating activity may be restricted relative to that of pp60v-src by additional in vivo mechanisms.     

Intracellular localization and processing of pp60v-src proteins expressed by two distinct temperature-sensitive mutants of Rous sarcoma virus.

The transforming protein of Rous sarcoma virus, pp60v-src, is known to be a tyrosine protein kinase, but the mechanism of cell transformation remains unclear. In further investigating pp60v-src structure and function, we have analyzed two temperature-sensitive (ts) Rous sarcoma virus src gene mutants, tsLA29 and tsLA32. The mutations in tsLA29 and tsLA32 map in the carboxy-terminal region and the amino-terminal half of pp60v-src, respectively, and encode mutant proteins with either temperature-labile (tsLA29) or -stable (tsLA32) kinase activities. Here we examined the intracellular processing and localization of these pp60v-src mutants and extended our characterization of transformation parameters expressed by cells infected by the Rous sarcoma virus variants. No obvious defects in functional integrity of the tsLA32 pp60v-src could yet be demonstrated, whereas the tsLA29 pp60v-src was perturbed not only in kinase activity, but also in aspects of protein processing and localization. Analysis of transformation parameters expressed by infected cells demonstrated the complete temperature lability of both mutants.     

Boron deficiency disables Xenopus laevis oocyte maturation events

Processes of oocyte maturation that may be affected by boron (B) deficiency were studied to potentially determine a possible biochemical role of B in the Xenopus laevis oocyte. More specifically, the Xenopus oocyte membrane progesterone receptor (OMPR) in B-deficient oocytes was characterized by evaluating progesterone affinity for the OMPR and OMPR responsiveness to progesterone stimulation. The responsiveness of B-deficient oocytes to microinjection of a purified oocyte cytoplasmic fraction (OCF) from B-adequate oocytes was also studied to evaluate which aspects of the maturation process were affected by B deficiency. Results suggested that B deficiency resulted in incomplete oocyte maturation and that maturation could not be induced by the administration of exogenous progesterone. Progesterone successfully induced germinal vesicle breakdown (GVBD) in oocytes from females fed a B-supplemented diet (+B) and females administered a traditional diet of beef liver and lung (B adequate). Addition of exogenous B to the -B oocytes increased the rate of progesterone-induced GVBD slightly. The B-deficient X. laevis oocytes were capable of undergoing GVBD when endogenously stimulated by microinjected purified B-adequate OCF. These results indicated that the inability of the B-deficient oocytes to undergo GVBD was not associated with the cytoplasmic induction process specifically, but possibly in the progesterone receptor or signal transduction pathways. Radio-binding studies found that progesterone binding to the B-deficient OPMR was greatly reduced compared to B-adequate or B-supplemented OMPR. Moreover, washout studies determined that progesterone binding to the OMPR in B-deficient oocytes was more transient than the B adequate or +B oocytes.     

Progesterone and cAMP-dependent protein kinase regulate in vivo the level of phosphorylation of two proteins (Mr 20,000 and Mr 32,000) in Xenopus oocytes

The [32P]phosphoproteins and [35S]thiophosphoproteins were analyzed by electrophoresis and autoradiography after microinjection of [gamma-32P]ATP or of [35S]ATP-gamma-S into living Xenopus oocytes. The level of 32P incorporation into a 20-kDA protein was decreased following progesterone treatment (between 1 and 2 hr). This 20-kDa protein was partially thiophosphorylated in vivo by [35S]ATP-gamma-S. Furthermore it was found that this phosphoprotein was partially purified by TCA (1%) extraction and heat treatment. Microinjection of the C-subunit of cAMP-dependent protein kinase (0.6 to 1.2 pmole) inhibited maturation and provoked an increase in the level of phosphorylation of the 20-kDa protein and of a 32-kDa protein, indicating that both proteins were in vivo substrates (directly or indirectly) for cAMP-dependent protein kinase. When inhibitor-1 of protein phosphatase-1 was microinjected (5 to 10 pmole per oocyte) meiotic maturation was inhibited and the level of phosphorylation of the 32-kDa protein was increased; the same result was obtained following ATP-gamma-S (1 mM) microinjection. Altogether these results suggest that a 20-kDa phosphoprotein, whose level of phosphorylation is decreased by progesterone, could be involved in the regulation of maturation by lowering the level phosphorylation of a 32-kDa phosphoprotein. An attractive hypothesis would be that the 20-kDa phosphoprotein is an inhibitor of protein phosphatase-1.