SERCA1 truncated proteins unable to pump calcium reduce the endoplasmic reticulum calcium concentration and induce apoptosis.

By pumping calcium from the cytosol to the ER, sarco/endoplasmic reticulum calcium ATPases (SERCAs) play a major role in the control of calcium signaling. We describe two SERCA1 splice variants (S1Ts) characterized by exon 4 and/or exon 11 splicing, encoding COOH terminally truncated proteins, having only one of the seven calcium-binding residues, and thus unable to pump calcium. As shown by semiquantitative RT-PCR, S1T transcripts are differentially expressed in several adult and fetal human tissues, but not in skeletal muscle and heart. S1T proteins expression was detected by Western blot in nontransfected cell lines. In transiently transfected cells, S1T homodimers were revealed by Western blot using mildly denaturing conditions. S1T proteins were shown, by confocal scanning microscopy, to colocalize with endogenous SERCA2b into the ER membrane. Using ER-targeted aequorin (erAEQ), we have found that S1T proteins reduce ER calcium and reverse elevation of ER calcium loading induced by SERCA1 and SERCA2b. Our results also show that SERCA1 variants increase ER calcium leakage and are consistent with the hypothesis of a cation channel formed by S1T homodimers. Finally, when overexpressed in liver-derived cells, S1T proteins significantly induce apoptosis. These data reveal a further mechanism modulating Ca(2+) accumulation into the ER of nonmuscle cells and highlight the relevance of S1T proteins to the control of apoptosis.

Cdk2 activity is dispensable for the onset of DNA replication during the first mitotic cycles of the sea urchin early embryo.

Earlier work reported the important role of Cdk2 as a regulator of DNA replication in somatic cells and in Xenopus extracts. In the present report we analyze in vivo the involvement of Cdk2 in DNA replication during early embryogenesis using the first mitotic cycles of sea urchin embryos. Unfertilized Sphaerechinus granularis eggs are arrested after the second meiotic cytokinesis. Fertilization resumes the block and induces DNA replication after a short lag period, making sea urchin early embryo a good model for studying in vivo the onset of DNA replication. We show that Cdk2 as well as its potential partner cyclin A are present in the nucleus in G1 and S phase and therefore available for DNA replication. In accordance with data obtained in Xenopus egg extracts we observed that Cdk2 kinase activity is low and stable during the entire cycle. However, in contrast with this in vitro system in which Cdk2 activity is required for the onset of DNA replication, the specific inhibition of Cdk2 kinase by microinjection of the catalytically inactive Cdk2-K33R or the inhibitor p21(Cip1) does not prevent DNA replication. Because olomoucine, DMAP, and emetine treatments did not preclude DNA synthesis, neither cyclin A/Cdk1 nor cyclin B/Cdk1 kinase activities are necessary to replace the absence of Cdk2 kinase in promoting DNA replication. These data suggest that during early embryogenesis Cdks activities, in particular Cdk2, are dispensable in vivo for the initiation step of DNA replication. However, the specific localization of Cdk2 in the nucleus from the beginning of M phase to the end of S phase suggests its involvement in other mechanisms regulating DNA replication such as inhibition of DNA re-replication and/or that its regulating role is achieved through a pathway independent of the kinase activity. We further demonstrate that even after inhibition of Cdk activities, the permeabilization of the nuclear membrane is required to allow a second round of DNA replication. However, in contrast to Xenopus egg extracts, re-replication can take place in the absence of DMAP-sensitive kinase.

Protein tyrosine phosphorylation in response to fertilization.

The sea urchin egg contains one or more protein tyrosine kinases which are active during the response of the egg to fertilization. In the present study, we have used an antibody specific for phosphotyrosine to determine which egg proteins are phosphorylated on tyrosine in response to fertilization. Analysis of immunoblots prepared from fertilized and unfertilized eggs revealed that fertilization results in a major increase in the phosphotyrosine content of a 350-kDa egg protein. Increased phosphorylation of this protein was detected as early as 1 min after fertilization, at which time it represented the most prominent phosphotyrosine containing protein in the egg. Tyrosine phosphorylation of this protein was transient however, and after 5 min post-insemination, the protein was dephosphorylated or otherwise degraded. Egg membrane proteins of approximately 40, 75, and 145 kDa were also found to act as substrates for protein tyrosine kinases in vitro, but did not exhibit significant changes in phosphotyrosine content during egg activation.

Antipain microinjection prevents progesterone to inhibit adenyl cyclase in Xenopus oocytes.

Microinjection of antipain, an inhibitor of thiol and Ca2+-dependent proteases, in immature Xenopus oocytes inhibited meiotic maturation induced by progesterone, but not by transfer of cytoplasm taken from maturing oocytes. Oocytes could be released from antipain inhibition by increasing progesterone concentration. alpha-32P-ATP was microinjected to study adenylcyclase in ovo. As already reported, neosynthesis of cAMP was decreased following progesterone application. This decrease was not observed, or it was considerably reduced, in oocytes previously injected with antipain. In amphibian, full-grown ovarian oocytes are arrested at first meiotic prophase, and have a large nucleus known as the germinal vesicle. Progesterone induces the production of a cytoplasmic maturation-promoting factor (MPF), which itself triggers germinal vesicle breakdown (GVBD), and subsequent events of meiotic maturation (Masui and Markert, 1971; Gerhart et al., 1984). A considerable body of evidences support the view that release from prophase block is due to inactivation of a cAMP-dependent protein kinase (reviewed by Maller, 1983). On the other hand, progesterone has been shown to induce a transient decrease in cAMP level (Speaker and Butcher, 1977; Schorderet-Slatkine et al., 1982; Cicirelli et al., 1985), and this initial drop of cAMP, along with a number of studies indicating a decrease in adenylate cyclase activity (Mulner et al., 1979; Baltus et al., 1981; Sadler and Maller, 1981; Finidori-Lepicard et al., 1981; Jordana et al., 1981), provided key support to the theory that an early drop in cAMP led to the dephosphorylation of a hypothetical protein which initiates maturation.(ABSTRACT TRUNCATED AT 250 WORDS)

Changes in the activity of the maturation-promoting factor during meiotic maturation and following activation of amphibian and starfish oocytes: their correlations with protein phosphorylation.

Changes in the extent of protein phosphorylation and their possible correlation with changes in the activity of maturation-promoting (MPF) factor were investigated throughout meiotic maturation and following activation of amphibian and starfish oocytes. Despite several exceptions in the pattern of phosphorylation of individual proteins, high and low levels of protein phosphorylation were found to be correlated with high and low levels of MPF activity. Both the extent of protein phosphorylation and MPF activity were found to drop upon parthenogenetic activation and to cycle synchronously thereafter in the amphibian. In contrast no drop in MPF activity or in the extent of protein phosphorylation was observed following activation of starfish oocytes with ionophore A23187. This suggests that changes of protein phosphorylation and of MPF activity are rather related to the progression of the cell cycle than directly to Ca2+-dependent activation reaction. In amphibians global protein kinase activity in homogenates was found to drop with MPF activity following activation. Changes in the ratio of threonine vs serine phosphorylation were also investigated during the course of meiotic maturation and activation in both amphibian and starfish oocytes: changes in the activity of MPF were found to be better correlated with changes in threonine than serine phosphorylation.

Rise and fall of protein phosphorylation during meiotic maturation in oocytes of Sabellaria alveolata (polychaete annelid).

Incorporation of [32P]phosphate into proteins was monitored, in preloaded Sabellaria oocytes, during meiosis. After a fourfold increase during the transition from prophase to metaphase I, the incorporated radioactivity decreased steadily by 25% during completion of meiosis, while it slowly increased in metaphase I-blocked oocytes. Measurements of the amount and specific activity of nucleotide pools showed no variation, while total alkali-labile protein-bound phosphate was found to increase and then decrease during meiosis. Autoradiography of sodium dodecyl sulfate slabgels showed that some proteins have peculiar phosphorylation-dephosphorylation kinetics. The changes in the level of phosphorylation of proteins may be related to similar changes in maturation-promoting factor (MPF) activity.

Activity of the maturation-promoting factor and the extent of protein phosphorylation oscillate simultaneously during meiotic maturation of starfish oocytes.

Maturation-promoting factor (MPF) activity and the protein phosphorylation pattern were monitored throughout the time course of meiotic maturation following hormonal stimulation of prophase-arrested starfish oocytes. MFP activity disappeared or decreased dramatically during the first and second meiotic cleavages. MPF activity came back to a very high level after the first but not the second meiotic cleavage. The state of protein phosphorylation was monitored using both tracer experiments and direct measurements of the absolute amount of phosphate in phosphoproteins. High and low levels of MPF activities were, respectively, associated with high and low levels of protein phosphorylation. It is suggested that the turn over of phosphate already bound to proteins in prophase-blocked oocytes does not change following hormone addition.