Accumulation of free amino acids in growing Xenopus laevis oocytes

The sizes of amino acid pools in growing Xenopus laevis oocytes have been measured. The total free amino acid content per oocyte increases nearly 25-fold during oocyte growth. Together, glutamic acid and aspartic acid account for approximately 59-75% of the total amino acid pool in Xenopus oocytes. On the other hand, methionine and cysteine are the least abundant of the amino acids detected, each accounting for less than 0.7% of the total pool in developing oocytes. It is argued that the acid-extractable amino acid pool represents the precursor pool used in protein synthesis.     

Transport of amino acids and nucleosides in metaphase-arrested unfertilized oocytes of Xenopus laevis

Metaphase-arrested, unfertilized shed oocytes of Xenopus laevis obtained after hormonal stimulation of the female are able to take up nucleosides (U, T) and amino acids (Ala, Gly, Glu, Gln, Tyr). For alanine, tyrosine, and glutamic acid the transport is uphill. The transport of the amino acids studied is activated by Na+, whereas the uptake of the nucleosides is independent of the Na+ concentration. Ouabain does not inhibit the uptake of amino acids significantly. The uptake of alanine and thymidine is not measurably affected by the presence of the jelly coat.     

Localized measurement of kinase activation in oocytes of Xenopus laevis.

We have combined a rapid cytoplasmic sampling technique with capillary electrophoresis to measure the activation of protein kinase C (PKC) in a small region (approximately 60 microm) of a Xenopus oocyte. The phosphorylation of a fluorescent PKC substrate was measured following addition of a pharmacological or physiological stimulus to an oocyte. When substrates for cdc2 kinase (cdc2K), PKC, and protein kinase A (PKA) were comicroinjected into an oocyte, all three substrates could be identified on the electropherogram after cytoplasmic sampling. With this new method, it should be possible to measure simultaneously the activation of multiple different kinases in a single cell, enabling the quantitative dissection of signal transduction pathways.     

S6 phosphorylation is regulated at multiple levels in Xenopus laevis oocytes

It is known that the 40s ribosomal protein S6 undergoes a dramatic increase in its level of phosphorylation during Xenopus oocyte meiotic maturation in response to progesterone stimulation. During prophase arrest, the majority of S6 has 0 moles phosphate per mole protein; this increases to 4-5 moles phosphate per mole protein by the time of germinal vesicle breakdown (GVBD). Our in vitro and in vivo studies indicate that the accumulation of phosphate on S6 is the net result of a 4-5-fold increase in S6 kinase activity and a 30-50% decrease in the rate of dephosphorylation and/or turnover of phosphate groups on S6 in maturing oocytes. In addition, the level of phosphorylation of S6 on 80s monosomes injected into non-hormone-stimulated oocytes was unexpectedly high. This indicates that the S6 kinase/phosphatase ratio in prophase arrested oocytes is higher than anticipated from previous studies. This observation implies that the majority of the oocyte ribosomes may be sequestered from any S6 kinase during meiotic prophase. Furthermore, these observations suggest that a portion of the increased accumulation of phosphate on S6 may be the result of increased accessibility of the ribosomes to S6 kinase during oocyte meiotic maturation.     

Mos stimulates MAP kinase in Xenopus oocytes and activates a MAP kinase kinase in vitro.

Several protein kinases, including Mos, maturation-promoting factor (MPF), mitogen-activated protein (MAP) kinase, and MAP kinase kinase (MAPKK), are activated when Xenopus oocytes enter meiosis. De novo synthesis of the Mos protein is required for progesterone-induced meiotic maturation. Recently, bacterially synthesized maltose-binding protein (MBP)-Mos fusion protein was shown to be sufficient to initiate meiosis I and MPF activation in fully grown oocytes in the absence of protein synthesis. Here we show that MAP kinase is rapidly phosphorylated and activated following injection of wild-type, but not kinase-inactive mutant, MBP-Mos into fully grown oocytes. MAP kinase activation by MBP-Mos occurs within 20 min, much more rapidly than in progesterone-treated oocytes. The MBP-Mos fusion protein also activates MPF, but MPF activation does not occur until approximately 2 h after injection. Extracts from oocytes injected with wild-type but not kinase-inactive MBP-Mos contain an activity that can phosphorylate MAP kinase, suggesting that Mos directly or indirectly activates a MAPKK. Furthermore, activated MBP-Mos fusion protein is able to phosphorylate and activate a purified, phosphatase-treated, rabbit muscle MAPKK in vitro. Thus, in oocytes, Mos is an upstream activator of MAP kinase which may function through direct phosphorylation of MAPKK.     

Nek2B stimulates zygotic centrosome assembly in Xenopus laevis in a kinase-independent manner

Pronuclear migration and formation of the first mitotic spindle depend upon assembly of a functional zygotic centrosome. For most animals, this involves both paternal and maternal contributions as sperm basal bodies are converted into centrosomes competent for microtubule nucleation through recruitment of egg proteins. Nek2B is a vertebrate NIMA-related protein kinase required for centrosome assembly, as its depletion from egg extracts delays microtubule aster formation from sperm basal bodies. Using Xenopus as a model system, we now show that protein expression of Nek2B begins during mid-oogenesis and increases further upon oocyte maturation. This is regulated, at least in part, at the level of protein translation. Nek2B protein is weakly phosphorylated in mitotic egg extracts but its recruitment to the sperm basal body, which occurs independently of its kinase activity, stimulates its phosphorylation, possibly through sequestration from a phosphatase present in mitotic egg cytoplasm. Importantly, although Nek2B is not required to organize acentrosomal microtubule asters, we show that addition of either active or kinase-dead recombinant Nek2B can restore centrosome assembly in a dose-dependent manner to a depleted extract. These results support a model in which maternal Nek2B acts to promote assembly of a functional zygotic centrosome in a kinase-independent manner.     

Activation of a ribosomal protein S6 protein kinase in Xenopus oocytes by insulin and insulin-receptor kinase.

Previous studies in this laboratory have shown that insulin treatment of Xenopus oocytes leads to an increase in phosphorylation of ribosomal protein S6. To investigate the mechanism of this increase, S6 kinase activity was measured in lysates of oocytes exposed to insulin. Insulin caused a rapid 4- to 6-fold increase in S6 kinase activity, which was maximal by 20 min and which could be reversed by removal of insulin prior to homogenization. Dose-response curves showed a detectable increase in specific activity at 1 nM insulin with a maximal effect at 100 nM. Treatment of oocytes with puromycin did not prevent this increase in S6 kinase activity, suggesting activation rather than synthesis of the enzyme. DEAE-Sephacel chromatography of extracts from insulin-treated oocytes revealed two peaks of S6 kinase activity, and the specific activity of the peak eluting at 300 nM NaCl was increased 3-fold in oocytes treated with insulin. The same peak of S6 kinase activity was increased 40% within 10 min in oocytes injected with highly purified insulin-receptor kinase. These results indicate that the insulin-dependent increase in S6 phosphorylation is due, at least in part, to activation of an S6 protein kinase, and this activation may result from the action of the insulin receptor at an intracellular location.     

Ligand modulates VDR-Ser/Thr protein phosphatase interaction and p70S6 kinase phosphorylation in a cell-context-dependent manner

We have recently shown that in colon cancer cells, Vitamin D receptor (VDR) interacts with the catalytic subunit of Ser/Thr protein phosphatases, PP1c and PP2Ac, and induces their enzymatic activity in a ligand-dependent manner. The VDR-PP1c and VDR-PP2Ac interactions were ligand independent in vivo, and 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3))-mediated increase in VDR-associated phosphatase activity resulted in dephosphorylation and inactivation of p70S6 kinase in colon cancer cells. Here, we demonstrate that in myeloid leukemia cells, 1,25(OH)(2)D(3) treatment increased the Thr389 phosphorylation of p70S6 kinase. Accordingly, 1,25(OH)(2)D(3) decreased VDR-associated Ser/Thr protein phosphatase activity by dissociating VDR-PP1c and VDR-PP2Ac interactions. Further, 1,25(OH)(2)D(3) increased the association between VDR and Thr389 phosphorylated p70S6 kinase. Finally, by using non-secosteroidal VDR ligands, we demonstrate a separation between transactivation and p70S6 kinase phosphorylation activities of VDR and show pharmacologically that p70S6 kinase phosphorylation correlates with HL-60 cell differentiation.     

Antibodies to Xenopus egg S6 kinase II recognize S6 kinase from progesterone- and insulin-stimulated Xenopus oocytes and from proliferating chicken embryo fibroblasts.

Ribosomal protein S6 becomes highly phosphorylated during progesterone- or insulin-induced maturation of Xenopus laevis oocytes. We have previously purified an Mr 92,000 protein as one of the major S6 kinases from Xenopus unfertilized eggs. In this paper we confirm by renaturation of activity from a sodium dodecyl sulfate-polyacrylamide gel that this protein is an S6 kinase. This enzyme, termed S6 kinase II (S6 K II), was used for the preparation of polyclonal antiserum. Immunocomplexes formed with the antiserum and purified S6 K II were able to express kinase activity with the same substrate specificity as that of the purified enzyme, including autophosphorylation of S6 K II itself. The antiserum did not react with S6 kinase I, another major S6 kinase present in Xenopus eggs, which is chromatographically distinct from S6 K II. The administration of progesterone to oocytes resulted in a 20- to 25-fold increase in S6 kinase activity in extracts of these cells. Immunocomplex kinase assays done on extracts revealed that anti-S6 K II serum reacted with S6 kinase from progesterone-treated oocytes. This antiserum also reacted with the activated S6 kinase from insulin-stimulated oocytes. In addition, anti-S6 K II serum reacted with activated S6 kinase from chicken embryo fibroblasts stimulated with serum or transformed by Rous sarcoma virus. These results indicate that S6 K II or an antigenically related S6 kinase(s) is subject to regulation by mitogenic stimuli in various cell types.     

Immunopurified mammalian target of rapamycin phosphorylates and activates p70 S6 kinase alpha in vitro

p70 S6 kinase alpha (p70alpha) is activated in vivo through a multisite phosphorylation in response to mitogens if a sufficient supply of amino acids is available or to high concentrations of amino acids per se. The immunosuppressant drug rapamycin inhibits p70alpha activation in a manner that can be overcome by coexpression of p70alpha with a rapamycin-resistant mutant of the mammalian target of rapamycin (mTOR) but only if the mTOR kinase domain is intact. We report here that a mammalian recombinant p70alpha polypeptide, extracted in an inactive form from rapamycin-treated cells, can be directly phosphorylated by the mTOR kinase in vitro predominantly at the rapamycin-sensitive site Thr-412. mTOR-catalyzed p70alpha phosphorylation in vitro is accompanied by a substantial restoration in p70alpha kinase activity toward its physiologic substrate, the 40 S ribosomal protein S6. Moreover, sequential phosphorylation of p70alpha by mTOR and 3-phosphoinositide-dependent protein kinase 1 in vitro resulted in a synergistic stimulation of p70alpha activity to levels similar to that attained by serum stimulation in vivo. These results indicate that mTOR is likely to function as a direct activator of p70 in vivo, although the relative contribution of mTOR-catalyzed p70 phosphorylation in each of the many circumstances that engender p70 activation remains to be defined.     

p70(S6K) controls selective mRNA translation during oocyte maturation and early embryogenesis in Xenopus laevis

In mammalian cells, p70(S6K) plays a key role in translational control of cell proliferation in response to growth factors. Because of the reliance on translational control in early vertebrate development, we cloned a Xenopus homolog of p70(S6K) and investigated the activity profile of p70(S6K) during Xenopus oocyte maturation and early embryogenesis. p70(S6K) activity is high in resting oocytes and decreases to background levels upon stimulation of maturation with progesterone. During embryonic development, three peaks of activity were observed: immediately after fertilization, shortly before the midblastula transition, and during gastrulation. Rapamycin, an inhibitor of p70(S6K) activation, caused oocytes to undergo germinal vesicle breakdown earlier than control oocytes, and sensitivity to progesterone was increased. Injection of a rapamycin-insensitive, constitutively active mutant of p70(S6K) reversed the effects of rapamycin. However, increases in S6 phosphorylation were not significantly affected by rapamycin during maturation. mos mRNA, which does not contain a 5'-terminal oligopyrimidine tract (5'-TOP), was translated earlier, and a larger amount of Mos protein was produced in rapamycin-treated oocytes. In fertilized eggs rapamycin treatment increased the translation of the Cdc25A phosphatase, which lacks a 5'-TOP. Translation assays in vivo using both DNA and RNA reporter constructs with the 5'-TOP from elongation factor 2 showed decreased translational activity with rapamycin, whereas constructs without a 5'-TOP or with an internal ribosome entry site were translated more efficiently upon rapamycin treatment. These results suggest that changes in p70(S6K) activity during oocyte maturation and early embryogenesis selectively alter the translational capacity available for mRNAs lacking a 5'-TOP region.     

Intracellular signals trigger ultrastructural events characteristic of meiotic maturation in oocytes of Xenopus laevis

Summary Oocytes of Xenopus laevis were treated with agents which induce individual intracellular signals normally evoked during the process of meiotic maturation. Ultrastructural analysis of these oocytes allowed identification of specific second messengers that individually trigger single ultrastructural changes characteristic of the meiotic maturation process: Manipulation of intracellular cAMP levels induced changes in cortical granule position. Cytoplasmic alkalinization triggered a disruption of the annulate lamellae, a specialized organelle in the periphery of oocytes. Activation of protein kinase C caused rapid formation of a cortical endoplasmic reticulum and subsequent disruption of cortical granules. Manipulation of transmembrane calcium flux had varied results dependent upon the agent employed. Two of the treatments, Verapamil and zero external calcium, induced a reorganization in the oocyte periphery. The results indicate that these ultrastructural events are under the control of specific intracellular signals known to be elicited during meiotic maturation.     

Evidence for a role of protein kinase C zeta subspecies in maturation of Xenopus laevis oocytes.

A number of studies have demonstrated the activation of phospholipase C-mediated hydrolysis of phosphatidylcholine (PC-PLC) both by growth factors and by the product of the ras oncogene, p21ras. Evidence has been presented indicating that the stimulation of this phospholipid degradative pathway is sufficient to activate mitogenesis in fibroblasts as well as that it is sufficient and necessary for induction of maturation in Xenopus laevis oocytes. However, the mechanism whereby PC-PLC transduces mitogenic signals triggered by growth factors or oncogenes remains to be elucidated. In this study, data are presented that show the involvement of protein kinase C zeta subspecies in the channelling of the mitogenic signal activated by insulin-p21ras-PC-PLC in Xenopus oocytes as well as the lack of a critical role of protein kinase C isotypes alpha, beta, gamma, delta, and epsilon in these pathways.     

Endogenous L-glutamate transport in oocytes of Xenopus laevis.

The existence of an endogenous Na(+)-glutamate cotransporter in the oocytes of Xenopus laevis is demonstrated. The transporter does not accept D-glutamate as substrate. The dependence on substrate displays two saturating components with low (K1/2 = 9 mM) and high (K1/2 = 0.35 microM) affinities for L-glutamate. The dependence on external Na+ exhibits a saturating component with a K1/2 value of about 5 mM and a component that has not saturated up to 110 mM Na+. In voltage-clamped oocytes, it is possible to demonstrate that Na(+)-dependent L-glutamate transport is directly coupled to countertransport of Rb+. The analysis of the voltage dependence of the Na+,K(+)-dependent L-glutamate uptake suggests that positive charges are moved inwardly during the transport cycle.     

Insulin promotes rat retinal neuronal cell survival in a p70S6K-dependent manner

The purpose of this study was to examine the role of the ribosomal protein S6 protein kinase (p70S6K), a protein synthesis regulator, in promoting retinal neuronal cell survival. Differentiated R28 rat retinal neuronal cells were used as an experimental model. Cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% newborn calf serum, and during the period of experimentation were exposed either to the absence or presence of 10 nm insulin. Insulin treatment induced p70S6K, mTOR, and Akt phosphorylation, effects that were completely prevented by the PI3K inhibitor, LY294002. Insulin-induced phosphorylation of p70S6K and mTOR was prevented by the mTOR inhibitor, rapamycin. Apoptosis, induced by serum deprivation and evaluated by Hoechst staining, was inhibited by insulin treatment in R28 cells, but not in L6 muscle cells. This effect of insulin was also largely prevented by rapamycin. Inhibition of p70S6K activity by exogenous expression of a dominant negative mutant of p70S6K prevented insulin-induced cell survival, whereas, overexpression of wild type p70S6K or expression of a rapamycin resistant form of the kinase enhanced the effect of insulin on survival. Enhanced cell survival under the latter condition was accompanied by increased p70S6K activity and phosphorylation. Rapamycin did not inhibit insulin induced p70S6K phosphorylation and activity in cells transfected with the rapamycin-resistant mutant. Together, these results suggest that p70S6K plays a key role in insulin stimulated retinal neuronal cell survival.     

Repair of UV-induced lesions in Xenopus laevis oocytes.

We characterized a DNA repair system in frog oocytes by comicroinjection of UV-irradiated pBR322 DNA and radiolabeled nucleotides. Repair synthesis was monitored by incorporation of label into recovered pBR322 DNA and by a novel method in which the removal of UV photoproducts was determined from the shift of DNA topoisomers that occurs during gel electrophoresis upon repair of these lesions. We investigated the effects of several drugs in the oocyte system and found that although novobiocin, an inhibitor of topoisomerase II, was an effective inhibitor of repair, VM-26, another inhibitor of topoisomerase II, was not. In addition, the topoisomerase I inhibitor camptothecin had no effect on repair in this system. Finally, circular DNA (either supercoiled or nicked circular) was repaired at least 50 times more rapidly than linear DNA.