Intracellular localization of MAP2-related protein (O-map) in prophase I and metaphase II oocytes of Xenopus

An antibody directed against rat brain microtubule-associated protein 2 (MAP2) immunoprecipitated a protein of 240 kDa from a Xenopus oocyte extract. In contrast, in Xenopus brain extract, this antibody recognizes a protein of 280 kDa. The oocyte MAP2-related protein (called O-MAP) is present in both prophase I and metaphase II-blocked oocytes as demonstrated by immunoblotting experiments; it is in vivo phosphorylated. Immunocytochemical studies using the anti-rat brain MAP2 antibody demonstrated that the O-MAP colocalizes within the cortical microtubular array present in both prophase I and metaphase II oocytes. However, O-MAP is not associated with the microtubular structures which are organized during the oocyte prophase-metaphase transition, i.e., a giant cytoplasmic network and both the meiotic spindles. O-MAP therefore appears as a microtubule-associated protein oocyte specific and may play a role in the regulation of microtubule stability and the organization of the oocyte cytoskeleton.     

Isolation and Characterization of Goldfish cdc2, a Catalytic Component of Maturation-Promoting Factor

We have isolated a cdc2 cDNA from a library constructed from immature goldfish oocytes. The isolated clone has a PSTAVR sequence, instead of the PSTAIR sequence common to cdc2 in other species. Its product was characterized by monoclonal antibodies against its C-terminal amino acid sequence. The antibodies recognized an anti-PSTAIR-reactive 35 kDa protein in immature oocyte extracts, which was not recognized by anti-goldfish cdk2 antibody. In addition to the 35 kDa cdc2, mature oocytes contained a 34 kDa cdc2, which was a component of MPF purified from carp eggs. Upon gel filtration column, the 35 kDa cdc2 migrated at monomeric position, while the 34 kDa cdc2 migrated at around 100 kDa, where cyclin B also comigrated. These results strongly suggest that the 35 kDa protein is monomeric inactive cdc2, while the 34 kDa protein is cyclin B-bound active cdc2. The finding that the 35 kDa inactive cdc2 does not form a complex with any other proteins in immature oocytes is in contrast to the situation in Xenopus and starfish, in which cdc2-cyclin B complex exists already as pre-MPF in immature oocytes.     

Identification and characterization of B"-subunits of protein phosphatase 2 A in Xenopus laevis oocytes and adult tissues.

Protein phosphatase 2A is a phosphoserine/threonine phosphatase implicated in many cellular processes. The core enzyme comprises a catalytic and a PR65/A-subunit. The substrate specificity and subcellular localization are determined by a third regulatory B-subunit (PR55/B, PR61/B' and PR72/130/B"). To identify the proteins of the B" family in Xenopus laevis oocytes, a prophase Xenopus oocyte cDNA library was screened using human PR130 cDNA as a probe. Three different classes of cDNAs were isolated. One class is very similar to human PR130 and is probably the Xenopus orthologue of PR130 (XPR130). A second class of clones (XN73) is identical to the N-terminal part of XPR130 but ends a few amino acids downstream of the putative splicing site of PR130. To investigate how this occurs, the genomic structure of the human PR130 gene was determined. This novel protein does not act as a PP2A subunit but might compete with the function of PR130. The third set of clones (XPR70) is very similar to human PR48 but has an N-terminal extension. Further analysis of the human EST-database and the human PR48 gene structure, revealed that the human PR48 clone published is incomplete. The Xenopus orthologue of PR48 encodes a protein of 70 kDa which like the XPR130, interacts with the A-subunit in GST pull-down assays. XPR70 is ubiquitously expressed in adult tissues and oocytes whereas expression of XPR130 is very low in brain and oocytes. Expression of XN73 mainly parallels XPR130 with the exception of the brain.     

Xenopus Rbm9 is a novel interactor of XGld2 in the cytoplasmic polyadenylation complex

During early development, control of the poly(A) tail length by cytoplasmic polyadenylation is critical for the regulation of specific mRNA expression. Gld2, an atypical poly(A) polymerase, is involved in cytoplasmic polyadenylation in Xenopus oocytes. In this study, a new XGld2-interacting protein was identified: Xenopus RNA-binding motif protein 9 (XRbm9). This RNA-binding protein is exclusively expressed in the cytoplasm of Xenopus oocytes and interacts directly with XGld2. It is shown that XRbm9 belongs to the cytoplasmic polyadenylation complex, together with cytoplasmic polyadenylation element-binding protein (CPEB), cleavage and polyadenylation specificity factor (CPSF) and XGld2. In addition, tethered XRbm9 stimulates the translation of a reporter mRNA. The function of XGld2 in stage VI oocytes was also analysed. The injection of XGld2 antibody into oocytes inhibited polyadenylation, showing that endogenous XGld2 is required for cytoplasmic polyadenylation. Unexpectedly, XGld2 and CPEB antibody injections also led to an acceleration of meiotic maturation, suggesting that XGld2 is part of a masking complex with CPEB and is associated with repressed mRNAs in oocytes.     

Purification, immunological and biochemical characterization of Ap4A binding protein from Xenopus laevis oocytes.

Diadenosine 5',5'-P1,P4-tetraphosphate (Ap4A) binding protein specifically binds Ap4A. The protein has been purified from Xenopus laevis oocytes and presents an estimated molecular weight of 100,000 by gel filtration. In the first stages of the purification, the Ap4A binding activity is found associated to DNA polymerase alpha-DNA primase, forming heterogeneous high molecular weight complexes. A monoclonal antibody has been prepared against the purified Ap4A binding protein. The antibody partially neutralizes the Ap4A binding activity. Using the immunoblot technique, it has been shown that the antibody is able to recognize either native or SDS-denatured Ap4A binding protein. The monoclonal antibody immunoreacted with a polypeptide of 90,000 which coincides with the molecular weight obtained by gel chromatography and indicates that the native Ap4A binding protein from Xenopus oocytes is probably a monomeric protein.     

Purification and identification of a novel subunit of protein serine/threonine phosphatase 4

The catalytic subunit of protein serine/threonine phosphatase 4 (PP4C) has greater than 65% amino acid identity to the catalytic subunit of protein phosphatase 2A (PP2AC). Despite this high homology, PP4 does not appear to associate with known PP2A regulatory subunits. As a first step toward characterization of PP4 holoenzymes and identification of putative PP4 regulatory subunits, PP4 was purified from bovine testis soluble extracts. PP4 existed in two complexes of approximately 270-300 and 400-450 kDa as determined by gel filtration chromatography. The smaller PP4 complex was purified by sequential phenyl-Sepharose, Source 15Q, DEAE2, and Superdex 200 gel filtration chromatographies. The final product contained two major proteins: the PP4 catalytic subunit plus a protein that migrated as a doublet of 120-125 kDa on SDS-polyacrylamide gel electrophoresis. The associated protein, termed PP4R1, and PP4C also bound to microcystin-Sepharose. Mass spectrometry analysis of the purified complex revealed two major peaks, at 35 (PP4C) and 105 kDa (PP4R1). Amino acid sequence information of several peptides derived from the 105 kDa protein was utilized to isolate a human cDNA clone. Analysis of the predicted amino acid sequence revealed 13 nonidentical repeats similar to repeats found in the A subunit of PP2A (PP2AA). The PP4R1 cDNA clone engineered with an N-terminal Myc tag was expressed in COS M6 cells and PP4C co-immunoprecipitated with Myc-tagged PP4R1. These data indicate that one form of PP4 is similar to the core complex of PP2A in that it consists of a catalytic subunit and a "PP2AA-like" structural subunit.     

Regulation of Greatwall kinase during Xenopus oocyte maturation

Greatwall kinase has been identified as a key element in M phase initiation and maintenance in Drosophila, Xenopus oocytes/eggs, and mammalian cells. In M phase, Greatwall phosphorylates endosulfine and related proteins that bind to and inhibit protein phosphatase 2A/B55, the principal phosphatase for Cdk-phosphorylated substrates. We show that Greatwall binds active PP2A/B55 in G2 phase oocytes but dissociates from it when progesterone-treated oocytes reach M phase. This dissociation does not require Greatwall kinase activity or phosphorylation at T748 in the presumptive T loop of the kinase. A mutant K71M Greatwall, also known as Scant in Drosophila, induces M phase in the absence of progesterone when expressed in oocytes, despite its reduced stability and elevated degradation by the proteasome. M phase induction by Scant Greatwall requires protein synthesis but is not associated with altered binding or release of PP2A/B55 as compared to wild-type Greatwall. However, in vitro studies with Greatwall proteins purified from interphase cells indicate that Scant, but not wild-type Greatwall, has low but detectable activity against endosulfine. These results demonstrate progesterone-dependent regulation of the PP2A/B55-Greatwall interaction during oocyte maturation and suggest that the cognate Scant Greatwall mutation has sufficient constitutive kinase activity to promote M phase in Xenopus oocytes.     

Purification of GVBD-Inducing Protein from the Ciliate Tetrahymenathermophila

Germinal-vesicle-breakdown (GVBD) was induced if a 132,000-g supernatant of Tetrahymena thermophila homogenates was injected into Xenopus oocytes. Using this induction of GVBD as a bioassay system, a GVBD-inducing substance was purified from the Tetrahymena by ultra-filtration, liquid chromatography, and electroelution from a band on native-PAGE gel. Proteins eluted from the single band on the native-PAGE gel induced GVBD in the absence of oocyte protein synthesis. This band resolved into two bands on SDS-PAGE: 60 and 112 kDa. The 60 kDa protein was the active fraction inducing GVBD. Immunoprecipitation of the 60 kDa protein prevented the GVBD-inducing activity, supporting the conclusion that the 60 kDa protein is the GVBD-inducing substance. An immunoblot with anti-60 kDa monoclonal antibody and PSTAIR antibody showed that p13suc1-beads could remove cdc2 homologues from T. thermophila supernatant but could not remove the GVBD-inducing activity. The 60-kDa protein appeared at the same time as micronuclear division and disappeared at the beginning of the macronuclear division during synchronous cell division. The cyclic appearance of the 60-kDa protein in the T. thermophila cell cycle suggests that this protein has a cell cycle function.     

The insulin-like growth factor 1 (IGF-1) receptor is responsible for mediating the effects of insulin, IGF-1, and IGF-2 in Xenopus laevis oocytes.

Competitive hormone binding studies with membrane and partially purified receptors from Xenopus laevis oocytes revealed that the oocyte possesses high affinity (KD = 1-3 nM) binding sites for both insulin growth factors 1 and 2 (IGF-1 and IGF-2), but not for insulin. Consistent with these findings, IGF-1 activates hexose uptake by Xenopus oocytes with a KA (3 nM) identical with its KD, while IGF-2 and insulin activate hexose uptake with KA values of 50 nM and 200-250 nM, respectively, suggesting activation mediated through an IGF-1 receptor. Both IGF-1 and insulin activate receptor beta-subunit autophosphorylation and, thereby, protein substrate (reduced and carboxyamidomethylated lysozyme, i.e. RCAM-lysozyme) phosphorylation with KA values comparable to their respective KD values for ligand binding and KA values for activation of hexose uptake. The autophosphorylated beta-subunit(s) of the receptor were resolved into two discrete components, beta 1 and beta 2 (108 kDa and 94 kDa, respectively), which were phosphorylated exclusively on tyrosine and which exhibited similar extents of IGF-1-activated autophosphorylation. When added prior to autophosphorylation, RCAM-lysozyme blocks IGF-1-activated autophosphorylation and, thereby, IGF-1-activated protein substrate (RCAM-lysozyme) phosphorylation. Based on these findings, we conclude that IGF-1-stimulated autophosphorylation of its receptor is a prerequisite for catalysis of protein substrate phosphorylation by the receptor's tyrosine-specific protein kinase. The IGF-1 receptor kinase is implicated in signal transmission from the receptor, since anti-tyrosine kinase domain antibody blocks IGF-1-stimulated kinase activity in vitro and, when microinjected into intact oocytes, prevents IGF-1-stimulated hexose uptake.     

Primary structure and developmental expression of Dp ZP2, a vitelline envelope glycoprotein homolog of mouse ZP2, in Discoglossus pictus, one of the oldest living Anuran species

A glycoprotein of the Xenopus vitelline envelope, gp 69/64, which mediates sperm binding, is closely related to the components of ZPA family, such as the mouse zona pellucida ZP2. To test the generality of these findings, we studied Discoglossus pictus, a species evolutionary distant from Xenopus and identified as a protein of 63 kDa in the vitelline envelope. Preliminary studies suggest that this protein may bind sperm at fertilization. We found that the 63-kDa protein is glycosylated and contains both N- and O-linked chains. We have cloned the cDNA encoding the Discoglossus protein of 63 kDa (Dp ZP2) by screening a Discoglossus cDNA library using Xenopus gp 69/64 cDNA as a probe. Analysis of the deduced sequence of Discoglossus protein revealed 48% identity with Xenopus gp 69/64 and 37-40% identity with mouse ZP2. The sequence conservation included a ZP domain, a potential furin cleavage site and a putative transmembrane domain. The N-terminus region of Dp ZP2 was 40% identical to the corresponding region of Xenopus gp 69/64 which has been shown to be essential for sperm binding to the VE. Although, as of yet, there is no evidence for sperm binding at the Dp ZP2 N-terminus, it is interesting that in this region three potential O-glycosylation sites are conserved in both species, in contrast to N-glycosylation sites. It was found that the Dp ZP2 mRNA is expressed in stage 1 oocytes and in the follicle cells surrounding the oocyte. Similarly, in Xenopus oocytes, the gp 69/64m RNA, was found in the oocytes, as well as in the somatic cells. Mol. Reprod. Dev. 59:133-143, 2001.     

Purification and characterization of protein phosphatase 2A from petals of the tulip Tulipa gesnerina

The holoenzyme of protein phosphatase (PP) from tulip petals was purified by using hydrophobic interaction, anion exchange and microcystin affinity chromatography to analyze activity towards p-nitrophenyl phosphate (p-NPP). The catalytic subunit of PP was released from its endogenous regulatory subunits by ethanol precipitation and further purified. Both preparations were characterized by immunological and biochemical approaches to be PP2A. On SDS-PAGE, the final purified holoenzyme preparation showed three protein bands estimated at 38, 65, and 75 kDa while the free catalytic subunit preparation showed only the 38 kDa protein. In both preparations, the 38 kDa protein was identified immunologically as the catalytic subunit of PP2A by using a monoclonal antibody against the PP2A catalytic subunit. The final 623- and 748- fold purified holoenzyme and the free catalytic preparations, respectively, exhibited high sensitivity to inhibition by 1 nM okadaic acid when activity was measured with p-NPP. The holoenzyme displayed higher stimulation in the presence of ammonium sulfate than the free catalytic subunit did by protamine, thereby suggesting different enzymatic behaviors.     

Maturation-promoting factor and p34cdc2 kinase during oocyte maturation of the Japanese quail

Maturation-promoting factor and a homolog of fission yeast cdc2+ gene product (p34cdc2) were investigated during the final 24 hr of maturation of quail oocytes. Kinase activity of p34cdc2 in the oocyte germinal disk (GD) increased 15 times at maturation. Two bands, at 32 and 34 kDa, were detected in immature oocytes by immunoblotting of SDS-PAGE with anti-p34cdc2 monoclonal antibody. A new band, which is close to the 32-kDa band but with a slightly faster mobility, appeared during maturation. No p34cdc2 could be detected outside the GD. Microinjection of GD extract from mature oocytes caused maturation of Xenopus oocytes.     

The mechanism and regulation of deadenylation: identification and characterization of Xenopus PARN.

In Xenopus oocytes, the deadenylation of a specific class of maternal mRNAs results in their translational repression. Here we report the purification, characterization, and molecular cloning of the Xenopus poly(A) ribonuclease (xPARN). xPARN copurifies with two polypeptides of 62 kDa and 74 kDa, and we provide evidence that the 62-kDa protein is a proteolytic product of the 74-kDa protein. We have isolated the full-length xPARN cDNA, which contains the tripartite exonuclease domain conserved among RNase D family members, a putative RNA recognition motif, and a domain found in minichromosome maintenance proteins. Characterization of the xPARN enzyme shows that it is a poly(A)-specific 3' exonuclease but does not require an A residue at the 3' end. However, the addition of 25 nonadenylate residues at the 3' terminus, or a 3' terminal phosphate is inhibitory. Western analysis shows that xPARN is expressed throughout early development, suggesting that it may participate in the translational silencing and destabilization of maternal mRNAs during both oocyte maturation and embryogenesis. In addition, microinjection experiments demonstrate that xPARN can be activated in the oocyte nucleus in the absence of cytoplasmic components and that nuclear export of deadenylated RNA is impeded. Based on the poly(A) binding activity of xPARN in the absence of catalysis, a model for substrate specificity is proposed.     

Purification and characterization of maturation-promoting factor in fish.

Maturation-promoting factor (MPF) activity has been demonstrated for the first time in fish oocytes. We purified MPF from a 100,000g supernatant of crushed, naturally spawned carp oocytes using four chromatography columns: Q-Sepharose Fast-Flow, p13suc1-affinity Sepharose, Mono S, and Superose 12. The final preparation was purified over 1000-fold with a recovery of about 1%. On Superose 12, MPF eluted as a single peak with an apparent molecular weight of 100 kDa. SDS-PAGE analysis of the active fractions after Superose 12 revealed the presence of four proteins of 33, 34, 46, and 48 kDa. A monoclonal antibody against the PSTAIR sequence of cdc2 kinase recognized the 33- and 34-kDa proteins for which the 46- and 48-kDa proteins are endogenous substrates. The 46- and 48-kDa proteins were recognized by a monoclonal antibody against Escherichia coli-produced goldfish cyclin B, but not by an anti-cyclin A antibody. When oocytes were matured in the presence of 32P, the labeling was seen with the 34-kDa protein, but not with the 33-kDa protein. The 34-kDa protein corresponded to the MPF activity, but the 33-kDa protein did not. These findings indicate that carp MPF is a complex of cdc2 kinase and cyclin B, and further that active MPF contains the phosphorylated form of cdc2 kinase.     

Binding of porcine sperm plasma membrane proteins to sheep, hamster and mouse oocyte plasma membrane

Four porcine sperm plasma membrane proteins were previously identified as putative ligands for the oocyte plasma membrane. The present study examined the binding of these proteins and two additional porcine sperm membrane proteins to oocytes from sheep, mice and hamsters as a first step in assessing potential conservation of these putative sperm ligands across species and across mammalian orders. Plasma membrane vesicles were isolated from porcine sperm, solubilised, and the proteins separated by one-dimensional gel electrophoresis. The 7, 27, 39 and 62 kDa porcine sperm protein bands demonstrating predominant binding of the porcine oocyte plasma membrane on ligand blots, a 90 kDa protein band demonstrating minor binding, and a 97 kDa protein band that did not bind the oocyte plasma membrane probe were electroeluted. Proteins were biotinylated, and incubated with zona-free oocytes. Bound biotinylated protein was labelled with fluorescent avidin and the oocytes examined with a confocal microscope. The 7 kDa, 27 kDa and the 39 kDa proteins bound to the sheep oocytes but not to a majority of the hamster or mouse oocytes. The 62 kDa protein bound to sheep oocytes and mouse oocytes but not to a majority of the hamster oocytes. The 90 kDa protein bound to oocytes from all three species. The 97 kDa protein, which did not recognise the porcine oocyte probe on a Western ligand blot, did not bind to oocytes from any species and served as a negative control. These observations are consistent with significant conservation of molecule and function among species within the same mammalian order. Hence, one species may be a good model for other species from the same order. Only limited conservation of binding activity of porcine sperm plasma membrane proteins to rodent oocytes was observed, suggesting a greater divergence either in molecular structure or in function among species from different orders.     

Characterization of protein kinase C in Xenopus oocytes.

Protein kinase C (PKC) was partially purified from Xenopus laevis oocytes by ammonium sulfate fractionation followed by DEAE-cellulose and hydroxyapatite column chromatography. In the latter chromatography, two distinct PKC activities were identified. Both PKC fractions contained an 80 kDa protein which was recognized by three antisera raised against the conserved regions of mammalian PKC. However, specific antisera against alpha, beta I, beta II, and gamma-subspecies of rat PKC did not recognize the protein. Kinetic properties of the Xenopus PKCs were very similar to those of the rat alpha PKC, and only a subtle difference was found in the mode of activation by arachidonic acid. When oocytes were treated with the tumor promoter, phorbol 12-myristate 13-acetate, one of the Xenopus PKCs was found to disappear very rapidly, while the other remained unchanged up to 2 hr.     

Cyclin B in Xenopus oocytes: implications for the mechanism of pre-MPF activation.

Using a polyclonal antibody raised against B2 cyclin from Xenopus laevis, we show that prophase-arrested Xenopus oocytes contain a stockpile of cyclin B2 protein. During progesterone-induced maturation, an increase in the synthesis of cyclin B2 is observed, although Western blotting experiments show that this new synthesis does not significantly increase the mass of cyclin over the maternal stockpile. In the oocyte cyclin B2 is already present in two forms which differ in the extent of phosphorylation, but the phosphorylated form becomes predominant as oocytes progress towards germinal vesicle breakdown (GVBD), coincident with cdc2 protein kinase activation. These two events do not depend upon formation of a new complex between cyclin and cdc2 protein kinase, since these two proteins are already found associated in resting oocytes, prior to activation of the kinase.     

Okadaic acid overcomes the blocked cell cycle caused by depleting Cdc2-related kinases in Trypanosoma brucei

Mitosis and cytokinesis are highly coordinated in eukaryotic cells. But procyclic-form Trypanosoma brucei under G1 or mitotic arrest is still capable of dividing, resulting in anucleate daughter cells (zoids). Okadaic acid (OKA), an inhibitor of protein phosphatases PP1 and PP2A, is known to inhibit kinetoplast replication and cell division yielding multinucleate cells with single kinetoplasts. However, when OKA was applied to cells arrested in G1 or G2/M phase via RNAi knockdown of specific cdc2-related kinases (CRKs), DNA synthesis and nuclear division were resumed without kinetoplast replication or cell division, resulting in multinucleate cells as in the wild type. Cells arrested in G2/M via depleting the mitotic cyclin CycB2 or an aurora B kinase homologue TbAUK1 were, however, not released by OKA treatment. The phenomenon is thus similar to the OKA activation of Cdc2 in Xenopus oocyte by inhibiting PP2A [Maton, et al., Differential regulation of Cdc2 and Aurora-A in Xenopus oocytes: a crucial role of phosphatase 2A. J. Cell Sci. 118 (2005) 2485-2494]. A simultaneous knockdown of the seven PP1s or the PP2A catalytic subunit in T. brucei by RNA interference did not, however, result in multinucleate cells. This could be explained by assuming a negative regulation, either directly or indirectly, of CRK by an OKA-sensitive phosphatase, which could be a PP2A as in the Xenopus oocyte and a positive regulation of kinetoplast replication by an OKA-susceptible protein(s). Test of a PP2A-specific inhibitor, fostriecin, on cells arrested in G2/M via CRK depletion or a knockdown of the PP2A catalytic subunit from the CRK-depleted cells both showed a partial lift of the G2/M block without forming multinucleate cells. These observations support the abovementioned assumption and suggest the presence of a novel OKA-sensitive protein(s) regulating kinetoplast replication that still remains to be identified.     

Xenopus MAP kinase activator: identification and function as a key intermediate in the phosphorylation cascade.

MAP kinase is thought to play a pivotal role not only in the growth factor-stimulated signalling pathway but also in the M phase phosphorylation cascade downstream of MPF. MAP kinase is fully active only when both tyrosine and threonine/serine residues are phosphorylated. We have now identified and purified a Xenopus MAP kinase activator from mature oocytes that is able to induce activation and phosphorylation on tyrosine and threonine/serine residues of an inactive form of Xenopus MAP kinase. The Xenopus MAP kinase activator itself is a 45 kDa phosphoprotein and is inactivated by protein phosphatase 2A treatment in vitro. Microinjection of the purified activator into immature oocytes results in immediate activation of MAP kinase. Further experiments using microinjection as well as cell free extracts have shown that Xenopus MAP kinase activator is an intermediate between MPF and MAP kinase. Thus, MAP kinase activator plays a key role in the phosphorylation cascade.