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T Lorca

Publications and source records attributed to T Lorca.

At least 19 recordsLinked to original sources
PubMed2005

The zebrafish bcl-2 homologue Nrz controls development during somitogenesis and gastrulation via apoptosis-dependent and -independent mechanisms.

Although the role of the b-cell lymphoma (Bcl)-2 family of apoptosis inhibitors is well documented in tumor cells and tissue morphogenesis, their role during the early development of vertebrates is unknown. Here, we characterize Nrz, a new Bcl-2-related inhibitor of apoptosis in zebrafish. Nrz is a mitochondrial protein, antagonizing the death-accelerator Bax. The nrz gene is mainly expressed during gastrulation and somitogenesis. The knockdown of nrz with antisense morpholinos leads to alterations of the somites, correlated with an increase in apoptosis. In addition, earlier during development, in the zebrafish gastrula, nrz knockdown results in an increase of snail-1 expression at the margin and frequent gastrulation arrest at the shield stage, independently of apoptosis. Together these data suggest that Nrz, in addition to its effect on apoptosis, contributes to cell movements during gastrulation by negatively regulating the expression of Snail-1, a transcription factor that controls cell adhesion.

Amino Acid Sequence↗
PubMed2001

Mps1 is a kinetochore-associated kinase essential for the vertebrate mitotic checkpoint.

The mitotic checkpoint acts to inhibit entry into anaphase until all chromosomes have successfully attached to spindle microtubules. Unattached kinetochores are believed to release an activated form of Mad2 that inhibits APC/C-dependent ubiquitination and subsequent proteolysis of components needed for anaphase onset. Using Xenopus egg extracts, a vertebrate homolog of yeast Mps1p is shown here to be a kinetochore-associated kinase, whose activity is necessary to establish and maintain the checkpoint. Since high levels of Mad2 overcome checkpoint loss in Mps1-depleted extracts, Mps1 acts upstream of Mad2-mediated inhibition of APC/C. Mps1 is essential for the checkpoint because it is required for recruitment and retention of active CENP-E at kinetochores, which in turn is necessary for kinetochore association of Mad1 and Mad2.

Amino Acid Sequence↗
PubMed2001

c-Mos and cyclin B/cdc2 connections during Xenopus oocyte maturation.

Fully-grown G2 arrested Xenopus oocytes can be induced to enter and progress into meiotic cell cycle by progesterone stimulation. This process is termed oocyte maturation. An early response to progesterone is the synthesis of the onco-protein c-Mos, defined as the candidate initiator of Xenopus oocyte maturation, which triggers the MAPK cascade, MPF activation and promotes CSF activity. Here we review our current knowledge on the synthesis, activation and functions of c-Mos in connection with MPF activation during maturation. We also discuss our recent results concerning the dispensability of cyclin B degradation in meiosis I-meiosis II transition and the stabilization of c-Mos through its direct phosphorylation by cyclin B/cdc2.

Anaphase-Promoting Complex-Cyclosome↗
PubMed2001

[When chromosomal dynamics control cell division].

In most tumor cells a chromosomal instability leads to an abnormal chromosome number (aneuploidy). The mitotic checkpoint is essential for ensuring accurate chromosome segregation by allowing mitotic delay in response to a spindle defect. This checkpoint delays the onset of anaphase until all the chromosomes are correctly aligned on the mitotic spindle. When unattached kinetochores are present, the metaphase/anaphase transition is not allowed and the time available for chromosome-microtubule capture increases. Genes required for this delay were first identified in Saccharomyces cerevisiae (the MAD, BUB and MPS1 genes) and subsequently, homologs have been identified in higher eucaryotes showing that the spindle checkpoint pathway is highly conserved. The checkpoint functions by preventing an ubiquitin ligase called the anaphase-promoting complex/cyclosome (APC) from ubiquitinylating proteins whose destruction is required for anaphase onset.

Aneuploidy↗
PubMed2001

The APC is dispensable for first meiotic anaphase in Xenopus oocytes.

Here we show that segregation of homologous chromosomes and that of sister chromatids are differentially regulated in Xenopus and possibly in other higher eukaryotes. Upon hormonal stimulation, Xenopus oocytes microinjected with antibodies against the anaphase-promoting complex (APC) activator Fizzy or the APC core subunit Cdc27, or with the checkpoint protein Mad2, a destruction-box peptide or methylated ubiquitin, readily progress through the first meiotic cell cycle and arrest at second meiotic metaphase. However, they fail to segregate sister chromatids and remain arrested at second meiotic metaphase when electrically stimulated or when treated with ionophore A34187, two treatments that mimic fertilization and readily induce chromatid segregation in control oocytes. Thus, APC is required for second meiotic anaphase but not for first meiotic anaphase.

Anaphase↗
PubMed2001

Cyclin B/cdc2 induces c-Mos stability by direct phosphorylation in Xenopus oocytes.

The c-Mos proto-oncogene product plays an essential role during meiotic divisions in vertebrate eggs. In Xenopus, it is required for progression of oocyte maturation and meiotic arrest of unfertilized eggs. Its degradation after fertilization is essential to early embryogenesis. In this study we investigated the mechanisms involved in c-Mos degradation. We present in vivo evidence for ubiquitin-dependent degradation of c-Mos in activated eggs. We found that c-Mos degradation is not directly dependent on the anaphase-promoting factor activator Fizzy/cdc20 but requires cyclin degradation. We demonstrate that cyclin B/cdc2 controls in vivo c-Mos phosphorylation and stabilization. Moreover, we show that cyclin B/cdc2 is capable of directly phosphorylating c-Mos in vitro, inducing a similar mobility shift to the one observed in vivo. Tryptic phosphopeptide analysis revealed a practically identical in vivo and in vitro phosphopeptide map and allowed identification of serine-3 as the largely preferential phosphorylation site as previously described (Freeman et al., 1992). Altogether, these results demonstrate that, in vivo, stability of c-Mos is directly regulated by cyclin B/cdc2 kinase activity.

Animals↗
PubMed2001

Interaction between cyclin T1 and SCF(SKP2) targets CDK9 for ubiquitination and degradation by the proteasome.

CDK9 paired with cyclin T1 forms the human P-TEFb complex and stimulates productive transcription through phosphorylation of the RNA polymerase II C-terminal domain. Here we report that CDK9 is ubiquitinated and degraded by the proteasome whereas cyclin T1 is stable. SCF(SKP2) was recruited to CDK9/cyclin T1 via cyclin T1 in an interaction requiring its PEST domain. CDK9 ubiquitination was modulated by cyclin T1 and p45(SKP2). CDK9 accumulated in p45(SKP2-/-) cells, and its expression during the cell cycle was periodic. The transcriptional activity of CDK9/cyclin T1 on the class II major histocompatibility complex promoter could be regulated by CDK9 degradation in vivo. We propose a novel mechanism whereby recruitment of SCF(SKP2) is mediated by cyclin T1 while ubiquitination occurs exclusively on CDK9.

Anaphase-Promoting Complex-Cyclosome↗
PubMed2000

Part of Xenopus translin is localized in the centrosomes during mitosis.

During oogenesis, maternal mRNAs are synthesised and stored in a translationally dormant form due to the presence of regulatory elements at the 3' untranslated regions (3'UTR). In Xenopus oocytes, several studies have described the presence of RNA-binding proteins capable to repress maternal-mRNA translation. The testis-brain RNA-binding protein (TB-RBP/Translin) is a single-stranded DNA- and RNA-binding protein which can bind the 3' UTR regions (Y and H elements) of stored mRNAs and can suppress in vitro translation of the mRNAs that contain these sequences. Here we report the cloning of the Xenopus homologue of the TB-RBP/Translin protein (X-translin) as well as its expression, its localisation, and its biochemical association with the protein named Translin associated factor X (Trax) in Xenopus oocytes. The fact that this protein is highly present in the cytoplasm from stage VI oocytes until 48 h embryos and that it has been described as capable to inhibit paternal mRNA translation, indicates that it could play an important role in maternal mRNA translation control during Xenopus oogenesis and embryogenesis. Moreover, we investigated X-translin localisation during cell cycle in XTC cells. In interphase, although a weak and diffuse nuclear staining was observed, X-translin was mostly present in the cytoplasm where it exhibited a prominent granular staining. Interestingly, part of X-translin underwent a remarkable redistribution throughout mitosis and associated with centrosomes, which may suggest a new unknown role for this protein in cell cycle.

Amino Acid Sequence↗
PubMed2000

The polo-like kinase Plx1 prevents premature inactivation of the APC(Fizzy)-dependent pathway in the early Xenopus cell cycle.

Members of the polo-like family of protein kinases have been involved in the control of APC (anaphase-promoting complex) during the cell cycle, yet how they activate APC is not understood in any detail. In Xenopus oocytes, Ca2+-dependent degradation of cyclin B associated with release from arrest at second meiotic metaphase was demonstrated to require the polo-like kinase Plx1. The aim of the present study was to examine, beyond Ca2+-dependent resumption of meiosis, the possible role of Plx1 in the control of cyclin degradation during the early mitotic cell cycle. Plx1 was found to be dispensable for MPF to turn on the cyclin degradation machinery. However, it is required to prevent premature inactivation of the APC-dependent proteolytic pathway. Microcystin suppresses the requirement for Plx1 in both Ca2+-dependent exit from meiosis, associated with degradation of both cyclin B and A downstream of CaMK2 activation, and prevention of premature APC(Fizzy) inactivation in the early mitotic cell cycle. These results are consistent with the view that Plx1 antagonizes an unidentified microcystin-sensitive phosphatase that inactivates APC(Fizzy).

Anaphase-Promoting Complex-Cyclosome↗
PubMed2000

The Xenopus XMAP215 and its human homologue TOG proteins interact with cyclin B1 to target p34cdc2 to microtubules during mitosis.

Cytoskeleton reorganization, leading to mitotic spindle formation, is an M-phase-specific event and is controlled by maturation promoting factor (MPF: p34cdc2-cyclinB1 complex). It has previously been demonstrated that the p34cdc2-cyclin B complex associates with mitotic spindle microtubules and that microtubule-associated proteins (MAPs), in particular MAP4, might be responsible for this interaction. In this study, we report that another ubiquitous MAP, TOG in human and its homologue in Xenopus XMAP215, associates also with p34cdc2 kinase and directs it to the microtubule cytoskeleton. Costaining of Xenopus cells with anti-TOGp and anti-cyclin B1 antibodies demonstrated colocalization in interphase cells and also with microtubules throughout the cell cycle. Cyclin B1, TOG/XMAP215, and p34cdc2 proteins were recovered in microtubule pellets isolated from Xenopus egg extracts and were eluted with the same ionic strength. Cosedimentation of cyclin B1 with in vitro polymerized microtubules was detected only in the presence of purified TOG protein. Using a recombinant C-terminal TOG fragment containing a Pro-rich region, we showed that this domain is sufficient to mediate cosedimentation of cyclin B1 with microtubules. Finally, we demonstrated interaction between TOG/XMAP215 and cyclin B1 by co-immunoprecipitation assays. As XMAP215 was shown to be the only identified assembly promoting MAP which increases the rapid turnover of microtubules, the TOG/XMAP215-cyclin B1 interaction may be important for regulation of microtubule dynamics at mitosis.

Animals↗
PubMed1999

Biogenic amine survey and organoleptic changes in fresh, stored, and temperature-abused bluefish (Pomatomus saltatrix).

Changes in histamine, putrescine, and cadaverine concentrations in bluefish filets (Pomatomus saltatrix) stored at 5, 10, and 15 degrees C were determined using high-performance liquid chromatography. An organoleptic assessment was conducted simultaneously with the biogenic amine analyses. The histamine levels found in fresh bluefish obtained from wholesale seafood distributors ranged between <1 ppm and 99 with an average of 39 ppm. Putrescine and cadaverine were not found in fresh bluefish. Fish fillets stored at each of the three temperatures developed histamine. The greatest accumulation of histamine was observed in fish stored at 15 degrees C, which developed histamine levels as high as 2,200 ppm. Putrescine levels increased at each temperature during storage. Cadaverine was present only in uninoculated bluefish stored at 15 degrees C. Histamine achieved higher levels in bluefish pieces inoculated with Morganella morganii, which demonstrates that bluefish support bacterial histamine formation. Histamine levels at each temperature exceeded the 50-ppm advisory level established by the Food and Drug Administration before 100% sensory rejection. Standard plate counts increased during storage of fish at all temperatures, but the correlation between histamine levels and standard plate count was not significant.

Animals↗
PubMed1998

Fizzy is required for activation of the APC/cyclosome in Xenopus egg extracts.

The Xenopus homologue of Drosophila Fizzy and budding yeast CDC20 has been characterized. The encoded protein (X-FZY) is a component of a high molecular weight complex distinct from the APC/cyclosome. Antibodies directed against FZY were produced and shown to prevent calmodulin-dependent protein kinase II (CaMKII) from inducing the metaphase to anaphase transition of spindles assembled in vitro in Xenopus egg extracts, and this was associated with suppression of the degradation of mitotic cyclins. The same antibodies suppressed M phase-promoting factor (MPF)-dependent activation of the APC/cyclosome in interphase egg extracts, although they did not appear to alter the pattern or extent of MPF-dependent phosphorylation of APC/cyclosome subunits. As these phosphorylations are thought to be essential for APC/cyclosome activation in eggs and early embryos, we conclude that at least two events are required for MPF to activate the APC/cyclosome, allowing both chromatid segregation and full degradation of mitotic cyclins. The first one, which does not require FZY function, is the phosphorylation of APC/cyclosome subunits. The second one, that requires FZY function (even in the absence of MAD2 protein and when the spindle assembly checkpoint is not activated) is not yet understood at its molecular level.

Amino Acid Sequence↗
PubMed1997

Involvement of the Ca2+/calmodulin-dependent protein kinase II pathway in the Ca2+-mediated regulation of the capacitative Ca2+ entry in Xenopus oocytes.

Activation of the phosphoinositide transduction pathway induces capacitative Ca2+ entry in Xenopus oocytes. This can also be evoked by intracellular injection of Ins(1,4.5)P3, external application of thapsigargin and/or incubation in a Ca2+-free medium. Readmission of Ca2+ to voltage-clamped, thapsigargin-treated Xenopus oocytes triggers Ca2+-dependent Cl- current variations that reflect capacitative Ca2+ entry. Inhibition of Ca2+/calmodulin-dependent protein kinase II (CaMKII) by specific peptides markedly increased the amplitude of the transients, suggesting an involvement of the CaMKII pathway in the regulation of capacitative Ca2+ entry. Biochemical studies provide evidence for the activation of CaMKII in response to the development of capacitative Ca2+ entry. In effect, a CaMKII assay in vivo allows us to postulate that readmission of Ca2+ to thapsigargin-treated oocytes can induce a burst of CaMKII activity. Finally, analysis of the Cl- transient kinetics at high resolution of time suggests that CaMKII inhibition blocks the onset of the inactivation process without affecting the activation rate. We therefore postulate that CaMKII might participate in a negative feedback regulation of store-depletion-evoked Ca2+ entry in Xenopus oocytes.

Amino Acid Sequence↗
PubMed1996

MAP kinase does not inactivate, but rather prevents the cyclin degradation pathway from being turned on in Xenopus egg extracts.

Unfertilized frog eggs arrest at the second meiotic metaphase, due to cytostatic activity of the c-mos proto-oncogene (CSF). MAP kinase has been proposed to mediate CSF activity in suppressing cyclin degradation. Using an in vitro assay to generate CSF activity, and recombinant CL 100 phosphatase to inactivate MAP kinase, we confirm that the c-mos proto-oncogene blocks cyclin degradation through MAP kinase activation. We further show that for MAP kinase to suppress cyclin degradation, it must be activated before cyclin B-cdc2 kinase has effectively promoted cyclin degradation. Thus MAP kinase does not inactivate, but rather prevents the cyclin degradation pathway from being turned on. Using a constitutively active mutant of Ca2+/calmodulin dependent protein kinase II, which mediates the effects of Ca2+ at fertilization, we further show that the kinase can activate cyclin degradation in the presence of both MPF and the c-mos proto-oncogene without inactivating MAP kinase.

Animals↗
PubMed1994

The proteolysis-dependent metaphase to anaphase transition: calcium/calmodulin-dependent protein kinase II mediates onset of anaphase in extracts prepared from unfertilized Xenopus eggs.

It has been shown, using spindles assembled in vitro in extracts containing CSF (the cytostatic factor responsible for arresting unfertilized vertebrate eggs at metaphase), that onset of anaphase requires Ca(2+)-dependent activation of the ubiquitin-dependent proteolytic pathway that destroys both mitotic cyclins and an unknown protein responsible for metaphase arrest (Holloway et al., 1993, Cell, 73, 1382-1402). We showed recently that Ca2+/calmodulin-dependent protein kinase II (CaM KII) activates the ubiquitin-dependent cyclin degradation pathway in CSF extracts (Lorca et al., 1993, Nature, 366, 270-273), but did not investigate its possible effect on sister chromatid segregation. In this work we identify CaM KII as the only target of Ca2+ in inducing anaphase in CSF extracts, and further show that transition to anaphase does not require the direct phosphorylation of metaphase spindle components by CaM KII. A possible interpretation of the above results could have been that the ubiquitin-dependent degradation pathway is required for onset of anaphase only when spindles are clamped at metaphase due to CSF activity, and not in the regular cell cycle that occurs in the absence of CSF activity. We ruled out this possibility by showing that competitive inhibition of the ubiquitin-dependent degradation pathway still prevents the onset of anaphase in cycling extracts that lack CSF and do not require Ca2+ for sister chromatid separation.

Anaphase↗
PubMed1994

Differential sensitivity of FOS and JUN family members to calpains.

Degradation of c-fos protein (c-FOS) in the cytoplasm is very rapid in vivo and constitutes a crucial regulation of the nuclear steady-state level through the control of the amount of full-length molecules available for nuclear transport. Using cytoplasmic extracts from various origins, we report herein that c-FOS degradation can be initiated in a calcium-dependent manner which involves cysteine proteases called milli- and micro-calpain. Interestingly, FOS-B, a member of the fos multigene family, as well as all members of the jun family (JUN-B, c-JUN and JUN-D) are also sensitive to calpains albeit to different extents. FRA-2, which is a c-FOS-related protein, is resistant to micro- but not to milli-calpain whereas FRA-1, another member of the fos family, is resistant to both proteases. Given the fact that a work by others (Hiraï et al., 1991b) suggests that calpains can be involved in c-FOS and c-JUN degradation in vivo, our observations raises the possibility of a novel contribution to the regulation of AP-1 transcription complex activity through a differential control of the steady-state level of some of its components that involves calpains.

Animals↗
PubMed1993

Calmodulin-dependent protein kinase II mediates inactivation of MPF and CSF upon fertilization of Xenopus eggs.

In vertebrates, unfertilized eggs are arrested at second meiotic metaphase by a cytostatic factor (CSF), an essential component of which is the product of the c-mos proto-oncogene. CSF prevents ubiquitin-dependent degradation of mitotic cyclins and thus inactivation or the M phase-promoting factor (MPF). Fertilization or parthenogenetic activation triggers a transient increase in the cytoplasmic free Ca2+ (reviewed in refs 5 and 6), inactivates both CSF and MPF, and releases eggs from meiotic metaphase arrest. A calmodulin-dependent process is required for cyclin degradation to occur in cell-free extracts prepared from metaphase II-arrested eggs (CSF extracts) when the free Ca2+ concentration is transiently raised in the physiological micromolar range. Here we show that when a constitutively active mutant of calmodulin-dependent protein kinase II (CaM KII) is added to a CSF extract, cyclin degradation and Cdc2 kinase inactivation occur even in the absence of Ca2+, and the extract loses its ability to cause metaphase arrest when transferred into embryos. Furthermore, specific inhibitors of CaM KII prevent cyclin degradation after calcium addition. Finally, the direct microinjection of constitutively active CaM KII into unfertilized eggs inactivates Cdc2 kinase and CSF, even in the absence of a Ca2+ transient. The target for Ca(2+)-calmodulin is thus CaM KII.

Amino Acid Sequence↗