Biomedical subjects

X Johné Liu

Publications and source records attributed to X Johné Liu.

12 recordsLinked to original sources

PubMed2006

Cdc42 activation couples spindle positioning to first polar body formation in oocyte maturation.

During vertebrate egg maturation, cytokinesis initiates after one pole of the bipolar metaphase I spindle attaches to the oocyte cortex, resulting in the formation of a polar body and the mature egg. It is not known what signal couples the spindle pole positioning to polar body formation. We approached this question by drawing an analogy to mitotic exit in budding yeast, as asymmetric spindle attachment to the appropriate cortical region is the common regulatory cue. In budding yeast, the small G protein Cdc42 plays an important role in mitotic exit following the spindle pole attachment . We show here that inhibition of Cdc42 activation blocks polar body formation. The oocytes initiate anaphase but fail to properly form and direct a contractile ring. Endogenous Cdc42 is activated at the spindle pole-cortical contact site immediately prior to polar body formation. The cortical Cdc42 activity zone, which directly overlays the spindle pole, is circumscribed by a cortical RhoA activity zone; the latter defines the cytokinetic contractile furrow . As the RhoA ring contracts during cytokinesis, the Cdc42 zone expands, maintaining its complementary relationship with the RhoA ring. Cdc42 signaling may thus be an evolutionarily conserved mechanism that couples spindle positioning to asymmetric cytokinesis.

Animals↗

PubMed2006

Protein kinase A(PKA)-restrictive and PKA-permissive phases of oocyte maturation.

Substantial evidence has indicated that cAMP-dependent protein kinase (protein kinase A or PKA) plays a critical role in maintaining meiotic prophase arrest in vertebrate oocytes. However, PKA activity dynamic and its physiological substrate profile during oocyte maturation remain poorly defined. We have recently developed a novel PKA substrate construct which we employ to monitor PKA activity in live oocytes. We demonstrated here that, during progesterone-induced oocyte maturation, PKA was inactivated within 30 min of the addition of progesterone and thereafter remained inactivated throughout the entire maturation process. However, artificial reactivation of endogenous PKA had differential consequences, depending on the timing of PKA reactivation. Reactivation of endogenous PKA at any time prior to germinal vesicle breakdown (GVBD) inhibited progesterone-induced GVBD. PKA reactivation at GVBD, or thereafter, did not interfere with meiosis I to meiosis II transition, nor did it interfere with metaphase II arrest. These results demonstrate for the first time a PKA-restrictive and a PKA-permissive phase in oocyte maturation.

Animals↗

PubMed2006

Oocyte isolation and enucleation.

Xenopus laevis oocytes are popular cells in experimental biology. Fully grown oocytes are large (approximately 1.3-mm diameter) with an enormous nucleus (approximately 300-microm diameter). Oocytes are generally isolated by either manual dissection (manual defolliculation) or enzymatic (mainly with collagenase preparations) digestion of the extracellular connective tissues. In this chapter, we describe both procedures, which are routinely used in our laboratory. However, manual defolliculation does not actually remove the innermost layer of follicle cells, which are anchored to the vitelline membrane. To remove these follicle cells, further mechanical or enzymatic treatment is required. On the other hand, many have experienced nonspecific effects with collagenase-treated oocytes, including spontaneous oocyte maturation and reduced oocyte health. We discuss possible explanations and solutions to these problems. Finally, we also describe procedures we employ routinely to isolate oocyte nuclei and enucleated oocytes.

Animals↗

PubMed2006

Monitoring protein kinase A activities using expressed substrate in live cells.

Protein kinase A (PKA) activity is regulated by intracellular cyclic adenosine monophosphate. Conventional protein kinase assays after cell lysis are hence not suitable for analyzing PKA activities. In this chapter, we describe a new method for monitoring PKA activity in live cells. A triparti substrate for PKA (Myr-HA-beta2AR-C) is constructed that contains an N-terminal myristylation sequence followed by an antigenic hemagglutinin epitope tag and a substrate motif (the C-terminal tail of human beta2 adrenergic receptor). The PKA phosphorylation status of the substrate in frog oocytes is determined either by two-dimensional electrophoresis followed by HA epitope immunoblotting or by direct SDS-PAGE followed by immunoblotting using anti-P-beta2 adrenergic receptor antibodies specifically recognizing the PKA-phosphorylated C-terminus. We also describe the application of this strategy in mammalian somatic cells through DNA transfection. Myr-HA-beta2AR-C should be widely adaptable as an in vivo PKA activity indicator.

Animals↗

PubMed2005

Transcription-dependent and transcription-independent functions of the classical progesterone receptor in Xenopus ovaries.

Two forms of the classical progesterone receptors (PR), XPR-1 and XPR-2, have been cloned in Xenopus laevis. Their relative roles in mediating progesterone action in the ovaries are not clear. Using antibodies generated against the cloned XPR-2, we demonstrated here that the somatic follicle cells expressed an 80-kDa PR protein, termed XPR-1. Treatment of follicle cells with progesterone resulted in disappearance of this protein, consistent with proteosome-mediated XPR-1 protein degradation. A smaller (approximately 70 kDa) PR protein, termed XPR-2, was expressed in the oocytes, but not in follicle cells. XPR-2 underwent progesterone-induced phosphorylation but not protein degradation. Treating isolated ovarian fragments with progesterone caused oocyte maturation and the release of the mature oocytes from the ovarian tissues ("ovulation"). Inhibition of transcription, with actinomycin D, did not interfere with progesterone-induced oocyte maturation but blocked "ovulation" so that mature oocytes were trapped in the follicles. These results suggest that the dual functions of progesterone, transcription-dependent follicle rupture and transcription-independent oocyte maturation, are mediated by the two forms of PR proteins differentially expressed in the follicle cells and the oocytes, respectively.

Amino Acid Sequence↗

PubMed2005

Co-operation of Gsalpha and Gbetagamma in maintaining G2 arrest in Xenopus oocytes.

Progesterone-induced oocyte maturation is thought to involve the inhibition of an oocyte adenylyl cyclase and reduction of intracellular cAMP. Our previous studies demonstrated that injection of inhibitors of G protein betagamma complex induces hormone-independent oocyte maturation. In contrast, over-expression of Xenopus Gbeta1 (xGbeta1), alone or together with bovine Ggamma2, elevates oocyte cAMP and inhibits progesterone-induced oocyte maturation. To further investigate the mechanism of Gbetagamma-induced oocyte maturation, we generated a mutant xGbeta1, substituting Asp-228 for Gly (D228G). An equivalent mutation in the mammalian Gbeta1 results in the loss of its ability to activate adenylyl cyclases. Indeed, co-injection of xGbeta1D228G with Ggamma2 failed to increase oocyte cAMP or inhibit progesterone-induced oocyte maturation. To directly demonstrate that oocytes contained a Gbetagamma-regulated adenylyl cyclase, we analyzed cAMP formation in vitro by using oocyte membrane preparations. Purified brain Gbetagamma complexes significantly activated membrane-bound adenylyl cyclase activities. Multiple adenylyl cyclase isoforms were identified in frog oocytes by PCR using degenerate primers corresponding to highly conserved catalytic amino acid sequences. Among these we identified a partial Xenopus adenylyl cyclase 7 (xAC7) that was 65% identical in amino acid sequence to human AC7. A dominant-negative mutant of xAC7 induced hormone-independent oocyte maturation and accelerated progesterone-induced oocyte maturation. Theses findings suggest that xAC7 is a major component of the G2 arrest mechanism in Xenopus oocytes.

Adenylyl Cyclases↗

PubMed2005

A serotonin receptor antagonist induces oocyte maturation in both frogs and mice: evidence that the same G protein-coupled receptor is responsible for maintaining meiosis arrest in both species.

Accumulating evidence has indicated that vertebrate oocytes are arrested at late prophase (G2 arrest) by a G protein coupled receptor (GpCR) that activates adenylyl cyclases. However, the identity of this GpCR or its regulation in G2 oocytes is unknown. We demonstrated that ritanserin (RIT), a potent antagonist of serotonin receptors 5-HT2R and 5-HT7R, released G2 arrest in denuded frog oocytes, as well as in follicle-enclosed mouse oocytes. In contrast to RIT, several other serotonin receptor antagonists (mesulergine, methiothepine, and risperidone) had no effect on oocyte maturation. The unique ability of RIT, among serotonergic antagonists, to induce GVBD did not match the antagonist profile of any known serotonin receptors including Xenopus 5-HT7R, the only known G(s)-coupled serotonin receptor cloned so far in this species. Unexpectedly, injection of x5-HT7R mRNA in frog oocytes resulted in hormone-independent frog oocyte maturation. The addition of exogenous serotonin abolished x5-HT7R-induced oocyte maturation. Furthermore, the combination of x5-HT7R and exogenous serotonin potently inhibited progesterone-induced oocyte maturation. These results provide the first evidence that a G-protein coupled receptor related to 5-HT7R may play a pivotal role in maintaining G2 arrest in vertebrate oocytes.

Animals↗

PubMed2004

Progesterone inhibits protein kinase A (PKA) in Xenopus oocytes: demonstration of endogenous PKA activities using an expressed substrate.

3'-5' cyclic adenosine monophosphate (cAMP)-dependent protein kinase, PKA, is thought to be a key enzyme that controls prophase arrest in vertebrate oocytes. It has long been established that overexpression of the catalytic subunit of PKA inhibits hormone-induced frog oocyte maturation whereas overexpression of the regulatory subunits induces hormone-independent oocyte maturation. However, the activities of endogenous oocyte PKA, or its regulation by the maturation-inducing hormone progesterone, have never been directly demonstrated in frog oocytes. We have developed a novel expressed substrate for PKA in live oocytes by constructing a fusion protein containing an N-terminal myristylation sequence (derived from the Src tyrosine kinase) followed by an antigenic epitope tag and a substrate motif (the C-terminal cytoplasmic domain of beta2 adrenergic receptor). Following mRNA injection, the phosphorylation status of the substrate was determined by two-dimensional electrophoresis followed by epitope immunoblotting, or alternatively by SDS-PAGE followed by immunoblotting using antibodies specifically recognizing the PKA-phosphorylated form of the substrate. In prophase oocytes, the expressed protein, myr-HA-beta2AR-C, was fully phosphorylated on a single PKA site (Ser346 of human beta2 adrenergic receptor). Within one hour of the addition of progesterone, the PKA site became mostly dephosphorylated. No re-phosphorylation of the PKA site, and therefore no reactivation of PKA, was observed throughout the entire maturation process. To demonstrate the generality of this PKA substrate, we analyzed its phosphorylation status in COS-7 cells following transfection. We show that dibutyryl cAMP rapidly stimulates phosphorylation of the PKA site. These results represent the first biochemical demonstration of regulation of endogenous Xenopus oocyte PKA by progesterone. Furthermore, myr-HA-beta2AR-C should be widely adaptable as an in vivo PKA activity indicator.

Animals↗

PubMed2003

A G protein-coupled receptor kinase induces Xenopus oocyte maturation.

Several recent studies have suggested that resumption of oocyte meiosis, indicated by germinal vesicle breakdown or GVBD, involves inhibition of endogenous heterotrimeric G proteins in both frogs and mice. These studies imply that a heterotrimeric G protein(s), and hence its upstream activator (a G protein-coupled receptor or GpCR), is activated in prophase oocytes and is responsible for maintaining meiosis arrest. To test the existence and function of this putative GpCR, we utilized a mammalian G-protein-coupled receptor kinase (GRK3) and beta-arrestin-2, which together are known to cause GpCR desensitization. Injection of mRNA for rat GRK3 caused hormone-independent GVBD. The kinase activity of GRK3 was essential for GVBD induction as its kinase-dead mutant (GRK3-K220R) was completely ineffective. Another GRK3 mutant (GRK3-DeltaC), which lacked the C-terminal G(betagamma)-binding domain and which was not associated with oocyte membranes, also failed to induce GVBD. Furthermore, injection of rat beta-arrestin-2 mRNA also induced hormone-independent GVBD. Several inhibitors of clathrin-mediated receptor endocytosis (the clathrin-binding domain of beta-arrestin-2, concanavalin A, and monodansyl cadaverine) significantly reduced the abilities of GRK3/beta-arrestin-2 to induce GVBD. These results support the central role of a yet-unidentified GpCR in maintaining prophase arrest in frog oocytes and provide a potential means for its molecular identification.

Animals↗

PubMed2003

Biphasic activation of Aurora-A kinase during the meiosis I- meiosis II transition in Xenopus oocytes.

Xenopus Aurora-A (also known as Eg2) is a member of the Aurora family of mitotic serine/threonine kinases. In Xenopus oocytes, Aurora-A phosphorylates and activates a cytoplasmic mRNA polyadenylation factor (CPEB) and therefore plays a pivotal role in MOS translation. However, hyperphosphorylation and activation of Aurora-A appear to be dependent on maturation-promoting factor (MPF) activation. To resolve this apparent paradox, we generated a constitutively activated Aurora-A by engineering a myristylation signal at its N terminus. Injection of Myr-Aurora-A mRNA induced germinal vesicle breakdown (GVBD) with the concomitant activation of MOS, mitogen-activated protein kinase, and MPF. Myr-Aurora-A-injected oocytes, however, appeared to arrest in meiosis I with high MPF activity and highly condensed, metaphase-like chromosomes but no organized microtubule spindles. No degradation of CPEB or cyclin B2 was observed following GVBD in Myr-Aurora-A-injected oocytes. In the presence of progesterone, the endogenous Aurora-A became hyperphosphorylated and activated at the time of MPF activation. Following GVBD, Aurora-A was gradually dephosphorylated and inactivated before it was hyperphosphorylated and activated again. This biphasic pattern of Aurora-A activation mirrored that of MPF activation and hence may explain meiosis I arrest by the constitutively activated Myr-Aurora-A.

Animals↗

PubMed2002

Inhibition of MEK or cdc2 kinase parthenogenetically activates mouse eggs and yields the same phenotypes as Mos(-/-) parthenogenotes.

Mammalian eggs are arrested in metaphase II of meiosis until fertilization. Arrest is maintained by cytostatic factor (CSF) activity, which is dependent on the MOS-MEK-MAPK pathway. Inhibition of MEK1/2 with a specific inhibitor, U0126, parthenogenetically activated mouse eggs, producing phenotypes similar to Mos(-/-) parthenogenotes (premature, unequal cleavages and large polar bodies). U0126 inactivated MAPK in eggs within 1 h, in contrast to the 5 h required after fertilization, while the time course of MPF inactivation was similar in U0126-activated and fertilized eggs. We also found that inactivation of MPF by the cdc2 kinase inhibitor roscovitine induced parthenogenetic activation. Inactivation of MPF by roscovitine resulted in the subsequent inactivation of MAPK with a time course similar to that following fertilization. Notably, roscovitine also produced some Mos(-/-)-like phenotypes, indistinguishable from U0126 parthenogenotes. Simultaneous inhibition of both MPF and MAPK in eggs treated with roscovitine and U0126 produced a very high proportion of eggs with the more severe phenotype. These findings confirm that MEK is a required component of CSF in mammalian eggs and imply that the sequential inactivation of MPF followed by MAPK inactivation is required for normal spindle function and polar body emission.

Animals↗

PubMed2002

GIPC participates in G protein signaling downstream of insulin-like growth factor 1 receptor.

Several recent studies have demonstrated that insulin-like growth factor (IGF)-1-induced mitogen-activated protein kinase (MAP kinase) activation is abolished by pertussis toxin, suggesting that trimeric G proteins of the G(i) class are novel cellular targets of the IGF-1 signaling pathway. We report here that the intracellular domain of the Xenopus IGF-1 receptor is capable of binding to the Xenopus homolog of mammalian GIPC, a PDZ domain-containing protein previously identified as a binding partner of G(i)-specific GAP (RGS-GAIP). Binding of xGIPC to xIGF-1 receptor is independent of the kinase activity of the receptor and appears to require the PDZ domain of xGIPC. Injection of two C-terminal truncation mutants that retained the PDZ domain blocked IGF-1-induced Xenopus MAP kinase activation and oocyte maturation. While full-length xGIPC injection did not significantly alter insulin response, it greatly enhanced human RGS-GAIP in stimulating the insulin response in frog oocytes. This represents the first demonstration that GIPC x RGS-GAIP complex acts positively in IGF-1 receptor signal transduction.

Adaptor Proteins, Signal Transducing↗