Calcium-mediated transduction of the hormonal message in meiosis reinitiation of starfish oocytes : Modulation following injection of cholera to toxin and cAMP-dependent protein kinase

Injections of the regulatory subunit of type I cAMP-dependent protein kinase, of the heat-stable inhibitor protein of cAMP-dependent protein kinase and of calmodulin have no effect on meiosis reinitiation. Drugs, including theophylline, caffeine and procaine, which have been shown previously to inhibit 1-methyladenine (1-MeAde)-induced Ca²⁺ release, both in living starfish oocytes and from plasma membrane-rich fractions obtained from isolated cortices, inhibit meiosis reinitiation when added before—but not after—the end of the hormone-dependent period (period when presence of the hormone in the medium is required for meiosis to occur). In the same conditions, theophylline suppresses 1-MeAde-induced stimulation of protein phosphorylation. Injection of cholera toxin subunit A increases oocyte sensitivity to 1-MeAde. Catalytic subunit of cAMP-dependent protein kinase (C) inhibits meiosis reinitiation when injected before the end of the hormone-dependent period. Oocytes can be released from inhibition due to C injection by raising 1-Me-Ade concentration. These findings support the view that Ca²⁺ release occurs until transduction of the hormonal message (i.e., its intramembrane transfer) has been completed and show that protein phosphorylation remains under plasma membrane control until that time. They also suggest that transduction of the hormonal message might be modulated by intracellular cAMP and membrane phosphorylation, although cAMP content does not change following 1-MeAde addition.     

Starfish oocyte maturation: evidence for a cyclic AMP-dependent inhibitory pathway

Oocyte maturation (meiosis reinitiation) in starfish is induced by the natural hormone 1-methyladenine (1-MeAde). Cyclic AMP seems to play a negative role in maturation since 1-MeAde triggers a decrease of the oocyte cAMP concentration and since intracellular microinjections of cAMP delay or inhibit maturation. Cyclic GMP is also inhibitory but other nucleotides such as cCMP, cIMP, and cUMP are inactive. The involvement of cAMP and cGMP in the control of oocyte maturation has been further investigated by the use of the stereoisomers of the phosphodiesterase-stable adenosine and guanosine 3',5'-phosphorothioates (cAMPS and cGMPS). The Sp isomers of cAMPS and cGMPS respectively activate cAMP-dependent protein kinase and cGMP-dependent kinase, while the Rp isomers inhibit the kinases. Extracellular addition of these cAMPS and cGMPS isomers has no effect on the oocytes. Intracellular microinjection of the kinase-activating (Sp)-cAMPS and (Sp)-cGMPS delays or inhibits 1-MeAde-induced maturation in a concentration-dependent manner (I50, 30 and 300 microM, respectively). Microinjections of (Rp)-cAMPS and (Rp)-cGMPS have no inhibitory effects and neither trigger nor facilitate maturation. Using various analogs, we found that the delaying or inhibiting effect is restricted to the compounds activating cAMP-dependent kinase, while the compounds inactive on or inhibiting the kinase have no effects on maturation. The inhibitory effect of (Sp)-cAMPS can be reversed by comicroinjection of the heat-stable inhibitor of cAMP-dependent protein kinase, by comicroinjection of the antagonist (Rp)-cAMPS, or by an increase in the 1-MeAde concentration. The negative effects of (Sp)-cAMPS or (Sp)-cGMPS are observed only when these isomers are microinjected during the hormone-dependent period. These results suggest that a cAMP-dependent inhibitory pathway participates in the maintenance of the prophase arrest of oocytes and that 1-MeAde acts both by inhibiting this negative pathway (dis-inhibitory pathway) and by stimulating a parallel activatory pathway leading to oocyte maturation. The generality of this mechanism is discussed.     

Ammonia and other weak bases applied at any time of the hormone-dependent period inhibit 1-methyladenine-induced meiosis reinitiation of starfish oocytes

The effect of weak bases on starfish oocyte maturation induced by 1-methyladenine (1-MeAde) was investigated. Ammonium chloride, nicotine, and procaine inhibited meiosis reinitiation induced by either 1-MeAde or dithiothreitol. The inhibitory effect of the weak bases was highly dependent on the pH of sea water and increased with the increase in pH. These weak bases neither inhibited nor delayed meiosis reinitiation when given after the end of the hormone-dependent period, the period during which 1-MeAde is required for meiosis to occur, whereas they blocked hormone action when added just before the end of the hormone-dependent period.     

Starfish oocyte maturation: 1-methyladenine triggers a drop of cAMP concentration related to the hormone-dependent period

Oocyte maturation (meiosis reinitiation) in starfish is induced by the natural hormone 1-methyladenine (1-MeAde). Oocytes of Evasterias troschelii contain 0.43 pmole cyclic AMP/mg protein and 0.47 pmole cyclic GMP/mg protein. Upon stimulation by 1-MeAde the oocytes undergo a moderate (10-30%) decrease in their cAMP concentration. The concentration of cGMP remains unaltered. Oocytes treated with forskolin, an activator of adenylate cyclase, increase their cAMP concentration over 35-fold, up to 16 pmole cAMP/mg protein. When stimulated by 1-MeAde these forskolin-pretreated oocytes undergo a major (50-70%) decrease in their cAMP concentration. A similar decrease is triggered by mimetics of 1-MeAde, such as dithiothreitol, arachidonic acid (AA), and 8-hydroxyeicosatetraenoic acid (8-HETE), but not by adenine which is inactive. 1-MeAde-stimulated oocytes of Pisaster ochraceus also undergo a decrease in cAMP content, the size of which is increased by forskolin. Although a decrease in cAMP begins at sub-threshold 1-MeAde concentrations, the maximal decrease occurs at the same concentration of 1-MeAde needed for maturation induction and a further 1000-fold increase of the 1-MeAde concentration has no further effect. Upon removal of 1-MeAde, the cAMP concentration immediately increases to its original level. Sequential addition and removal of 1-MeAde triggers a sequential decrease and increase of the cAMP concentration, illustrating the continuous requirement for 1-MeAde for eliciting the decrease. Successive additions of 1-MeAde, however, do not trigger further decreases of the cAMP concentration. The temperature dependences of the cAMP concentration decrease and of the hormone-dependent period (HDP; the time of contact with 1-MeAde required for induction of maturation) are closely related. Forskolin, which increases the cAMP concentration, also increases the duration of the HDP (2.5-fold), delays the time course of protein phosphorylation burst and germinal vesicle breakdown, and inhibits AA- and 8-HETE-induced maturation. We conclude that 1-MeAde triggers a drop in cAMP concentration, which is tightly associated with the hormone-dependent period of oocyte maturation.     

Intracellular microinjection of alkaline buffers reversibly inhibits the initial phase of hormone action in meiosis reinitiation of starfish oocytes

Intracellular microinjection of alkaline Hepes-KOH buffers, which increases intracellular pH (pHI) from 6.92 to 7.70 in fully grown prophase-blocked oocytes of the starfish Marthasterias glacialis, like external application of ammonia and other weak bases (M. Doree, K. Sano, and H. Kanatani, 1982, Dev. Biol.90, 13–17), inhibited meiosis reinitiation induced by 1-methyladenine (1-MeAde) or dithiothreitol (DTT), a mimetic of the hormone. Oocytes could be released from inhibition by raising the concentration of hormone or of its mimetic. Increasing pHI to 7.70 neither inhibited nor delayed meiosis reinitiation when pH was clamped after the end of the hormone-dependent period, the period during which 1-MeAde is required in the external medium for meiosis to occur, whereas it blocked the action of the hormone at low concentration when performed before the end of the hormone-dependent period. When hormone concentration was higher, germinal vesicle breakdown (GVBD) occurred, but duration of the hormone-dependent period was increased. Delay introduced by alkalinization for oocytes to reach GVBD after 1-MeAde addition was smaller at high than at medium concentrations of the hormone. Increasing pHI did not inhibit action of MPF, the cytoplasmic maturation factor which induces GVBD and the subsequent process of meiotic maturation following hormonal treatment of prophase-blocked oocytes.     

cAMP,not Ca2+/calmodulin,regulates the phosphorylation of acetylcholine receptor in Torpedo californica electroplax

The regulation of the phosphorylation of the acetylcholine receptor in electroplax membranes from Torpedo californica and of purified acetylcholine receptor was investigated. The phosphorylation of the membrane-bound acetylcholine receptor was not stimulated by Ca²⁺/calmodulin, nor was it inhibited by EGTA, but it was stimulated by the catalytic subunit of cAMP-dependent protein kinase, and was blocked by the protein inhibitor of cAMP-dependent protein kinase. Purified acetylcholine receptor was not phosphorylated by Ca²⁺/calmodulin-dependent protein kinase activity in electroplax membranes, nor by partially purified Ca²⁺/calmodulin-dependent protein kinases from soluble or particulate fractions from the electroplax. Of the four acetylcholine receptor subunits, termed α, β, γ and δ, only the γ- and δ-subunits were phosphorylated by the cAMP-dependent protein kinase (+cAMP), or by its purified catalytic subunits.     

cAMP, not Ca/calmodulin, regulates the phosphorylation of acetylcholine receptor in Torpedo californica electroplax

The regulation of the phosphorylation of the acetylcholine receptor in electroplax membranes from Torpedo californica and of purified acetylcholine receptor was investigated. The phosphorylation of the membrane-bound acetylcholine receptor was not stimulated by Ca²⁺/calmodulin, nor was it inhibited by EGTA, but it was stimulated by the catalytic subunit of cAMP-dependent protein kinase, and was blocked by the protein inhibitor of cAMP-dependent protein kinase. Purified acetylcholine receptor was not phosphorylated by Ca²⁺/calmodulin-dependent protein kinase activity in electroplax membranes, nor by partially purified Ca²⁺/calmodulin-dependent protein kinases from soluble or particulate fractions from the electroplax. Of the four acetylcholine receptor subunits, termed α, β, γ and δ, only the γ- and δ-subunits were phosphorylated by the cAMP-dependent protein kinase (+cAMP), or by its purified catalytic subunits.     

Differential regulation of histone H1 and ribosomal S6 kinases during sea star oocyte maturation

In the preceding paper [Pelech, S.L., Meijer, L., & Krebs, E.G. (1987) Biochemistry (preceding paper in this issue)], at least three activated kinases were detected in soluble extracts from sea star oocytes induced to undergo maturation by 1-methyladenine (1-MeAde). Coincident with nuclear envelope breakdown (20 min after exposure to 1-MeAde), there was a rapid activation of a histone H1 kinase that eluted from DEAE-Sephacel with a conductivity of approximately 6 mmho. By contrast, 60-min treatment of the oocytes with 1-MeAde was required for maximal activation of two kinases, each of which phosphorylated a synthetic peptide, Arg-Arg-Leu-Ser-Ser-Leu-Arg-Ala (RRLSSLRA), patterned after a phosphorylation site sequence from ribosomal protein S6. These RRLSSLRA kinases were released from DEAE-Sephacel with elution conductivities of approximately 6 and approximately 10.5 mmho. The 1-MeAde dose-response curves for maturation induction and activation of the histone H1 and RRLSSLRA kinases were superimposable. Both oocyte maturation and the activation of the kinases required the presence of 1-MeAde during the hormone-dependent period. When 1-MeAde was removed after this period, full histone H1 kinase activation still occurred and maturation was induced. Forskolin pretreatment of the oocytes, by elevating the basal cAMP level more than 35-fold, doubled the hormone-dependent period and similarly delayed the onset of histone H1 kinase activation by 1-MeAde. However, postmaturation activation of the RRLSSLRA kinases was completely blocked by forskolin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hormone-induced phosphorylation of a 16000 Dalton polypeptide following meiosis reinitiation in starfish oocytes

In vitro phosphorylation of endogenous proteins is increased in homogenates prepared from 1-methyladenine-treated starfish oocytes when compared with control oocytes, although addition of the hormone to homogenates from control oocytes has no such effect. Following hormonal stimulation the best endogenous substrate is by far a 16 000 dalton (D) protein, the content of which also seems to increase, perhaps through proteolysis of a 21 000 D precursor. cAMP-dependent protein kinases are not involved in either basal or hormone-stimulated phosphorylations, as demonstrated by the lack of effect of either cAMP or of the heat-stable inhibitor of cAMP-dependent protein kinase on the extent of phosphorylation of individual endogenous substrates. Addition of 0.1 mM Ca²⁺ decreases to some extent the protein kinase activity in starfish homogenates and specifically suppresses the phosphorylation of a 40 000 D membrane protein. Starfish oocytes appear to contain myosin light chain kinase activity, as shown by the ability of homogenates to catalyse phosphorylation of exogenous 20 000 D myosin light chains.     

Arachidonic acid, 12- and 15-hydroxyeicosatetraenoic acids, eicosapentaenoic acid, and phospholipase A2 induce starfish oocyte maturation

In starfish oocyte maturation (meiosis reinitiation) is induced by the natural hormone 1-methyladenine (1-Me-Ade). This paper shows that arachidonic acid (AA) induces oocyte maturation at concentrations above 0.5 microM. This maturation shares many characteristics with 1-MeAde-induced maturation: same kinetics, same required contact time, same stimulations of protein phosphorylation and sodium influx. Although calcium facilitates the AA-induced but not the 1-MeAde-induced maturation, AA, like 1-MeAde, does not stimulate the uptake of calcium. Calcium does not facilitate the uptake of AA by oocytes. Out of 36 different fatty acids (saturated and unsaturated), only eicosatetraenoic (AA) and eicosapentaenoic acids were found to mimic 1-MeAde. Calcium-dependent phospholipases A2 from bee venom and Naja venom also induce maturation (0.1-1 unit/ml) when added externally to the oocytes. Phospholipase A2 inhibitors (quinacrine, bromophenacylbromide) block maturation; inhibition is reversed by increasing the 1-MeAde concentration and only occurs during the hormone-dependent period. AA is usually metabolized through oxidation by cyclooxygenase or lipoxygenase. Cyclooxygenase inhibitors (acetylsalicylic acid, indomethacin, tolazoline) do not block maturation; prostaglandins E2, D2, F2 alpha, I2, and thromboxane B2 do not induce meiosis reinitiation. On the other hand, lipoxygenase inhibitors (quercetin, butylated hydroxytoluene, and eicosatetraynoic acid) block 1-MeAde-induced maturation; although leukotrienes (A4, B4, C4, D4, E4) have no effects on oocytes, two other lipoxygenase products, 12- and 15-hydroxyeicosatetraenoic acids (and their corresponding hydroperoxy-) induce oocyte maturation (around 1 microM). The possible mode of action of the fatty acids inducing oocyte maturation is discussed.     

Inhibition of Starfish Oocyte Maturation by Tumor-Promoting Phorbol Esters*

Effect of tumor promoters including phorbol esters and teleocidin on 1-methyladenine (1-MeAde)-induced oocyte maturation was studied in the starfish. When isolated immature oocytes were treated with 1-MeAde and 12-O-tetradecanoylphorbol-13-acetate (TPA), 1-MeAde-induced maturation was completely inhibited at more than 2.5 μg/ml. However, if TPA was added after the hormone-dependent period (the minimum period wherein 1-MeAde is required), such maturation-inhibiting effect was no longer observed. Pretreatment with TPA for 5 min showed that its inhibitory action is irreversible. However, when TPA-injected oocytes were treated with 1-MeAde, all oocytes underwent germinal vesicle breakdown (GVBD). GVBD was induced in TPA-treated oocytes upon injection of the cytoplasm of maturing oocytes containing maturation-promoting factor (MPF). These facts show that TPA acts on the oocyte surface to inhibit the production of MPF. Retinoids including retinal, retinol and retinoic acid reversed the inhibitory effect of TPA on 1-MeAde-induced maturation. Experiments with various phorbol esters showed a good correlation between their maturation-inhibiting activity and their known tumor-promoting activity. Further, telecoidin, which is structurally unrelated to phorbol esters, inhibited 1-MeAde action. Since both tumor-promoting phorbol esters and teleocidin are known to activate Ca²⁺ -activated, phospholipid-dependent protein kinase (protein kinase C) and their activation effect is inhibited by retinoids, it appears that the activation of protein kinase C by tumor promoters is involved in blocking of 1-MeAde action.     

A Membrane Receptor Mechanism for Steroid Hormones Reinitiating Meiosis in Xenopus Laevis Oocytes*

There is a membrane progesterone receptor in Xenopus laevis oocytes that undergo meiosis under steroid exposure. Early responses include a decrease of leucine uptake, a decrease of adenylate cyclase and alkaline phosphatase activities, and a decrease of the phosphorylation of a specific p48 protein in the membrane. These results are compatible with a decrease of membrane fluidity brought about by the hormonal message. However the above-cited effects as well as the Ca₂₊-changes are not yet enough well understood to be able to precisely delineate their role in meiosis reinitiation.     

Regulation of rat cardiac nuclei-associated Mg2+-NTPase by phosphorylation

A nucleoside triphosphatase (NTPase) activity appeared to be associated with a highly purified nuclear preparation from rat cardiac ventricles. Different nucleoside triphosphates (UTP > GTP > ITP > CTP) supported this enzymic activity, which was stimulated by Mg` but not by Call. The nuclear NTPase activity could be down regulated by endogenous phosphorylation of a 55,000 M_r protein. Maximal phosphorylation of the 55,000 M_r protein occurred in the presence of Mg²⁺-ATP. Addition of cAMP, cGMP, Ca²⁺, Ca²⁺/phospholipid, Ca²⁺/calmodulin, and catalytic subunit of cAMP-dependent protein kinase was not associated with any further phosphorylation of the 55,000 M_r protein. However, in the presence of Ca²⁺/calmodulin or the catalytic subunit of the cAMP-dependent protein kinase additional proteins became phosphorylated, but these had no effect on the Mg²⁺-NTPase activity. These results indicate that a protein with M_r 55,000 may be involved in the regulation the Mg²⁺-NTPase activity associated with rat cardiac nuclei.Abbreviations Hg Hemoglobin - GAR Goat Anti-Rabbit antibody - SR Sarcoplasmic Reticulum - NTP Nucleoside Triphosphate - TCA Trichloroacetic acid - PAGE Polyacrylamide gel electrophoresis     

Hormone-induced parthenogenetic activation of mature starfish oocytes

1-Methyladenine, which has been previously shown to be the hormone responsible for meiosis reinitiation in starfish oocytes, triggers parthenogenetic activation when applied to matured starfish oocytes after emission of the second polar body and formation of the pronucleus. In Marthasterias glacialis and Asterias rubens oocytes parthenogenetic activation includes elevation of a fertilization membrane, cleavage and the formation of normal bipinnaria larvae. Activation is likely to result from 1-methyladenine interaction with the category of stereospecific membrane receptors involved in meiosis reinitiation, since structural requirements of this compound are identical for both biological responses. Appearance of oocyte responsiveness to 1-MeAde after, but not before emission of the second polar body cannot be accounted for by their increased sensitivity to intracellular Ca²⁺ at that time, although it is shown that Ca²⁺ mediates hormone effect in inducing parthenogenetic activation. Pretreatment of immature oocytes with the free hormone in excess strongly inhibits the 1-methyladenine-induced parthenogenetic activation of the oocytes when they have completed maturation.It is suggested that reappearance of 1-MeAde sensitivity when oocytes form a pronucleus depends either upon recruitment or new receptor units or on the reactivation of pre-existing inactivated receptors at this stage of oocyte maturation.     

Regulation of Ca2+/calmodulin-dependent protein kinase kinase β by cAMP signaling

BackgroundCa²⁺/calmodulin-dependent protein kinase kinase (CaMKK) is a pivotal activator of CaMKI, CaMKIV and 5’-AMP-activated protein kinase (AMPK), controlling Ca²⁺-dependent intracellular signaling including various neuronal, metabolic and pathophysiological responses. Recently, we demonstrated that CaMKKβ is feedback phosphorylated at Thr144 by the downstream AMPK, resulting in the conversion of CaMKKβ into Ca²⁺/CaM-dependent enzyme. However, the regulatory phosphorylation of CaMKKβ at Thr144 in intact cells and in vivo remains unclear.MethodsAnti-phosphoThr144 antibody was used to characterize the site-specific phosphorylation of CaMKKβ in immunoprecipitated samples from mouse cerebellum and in transfected mammalian cells that were treated with various agonists and protein kinase inhibitors. CaMKK activity assay and LC-MS/MS analysis were used for biochemical characterization of phosphorylated CaMKKβ.ResultsOur data suggest that the phosphorylation of Thr144 in CaMKKβ is rapidly induced by cAMP/cAMP-dependent protein kinase (PKA) signaling in CaMKKβ-transfected HeLa cells, that is physiologically relevant in mouse cerebellum. We confirmed that the catalytic subunit of PKA was capable of directly phosphorylating CaMKKβ at Thr144 in vitro and in transfected cells. In addition, the basal phosphorylation of CaMKKβ at Thr144 in transfected HeLa cells was suppressed by AMPK inhibitor (compound C). PKA-catalyzed phosphorylation reduced the autonomous activity of CaMKKβ in vitro without significant effect on the Ca²⁺/CaM-dependent activity, resulting in the conversion of CaMKKβ into Ca²⁺/CaM-dependent enzyme.ConclusioncAMP/PKA signaling may confer Ca²⁺-dependency to the CaMKKβ-mediated signaling pathway through direct phosphorylation of Thr144 in intact cells.General significanceOur results suggest a novel cross-talk between cAMP/PKA and Ca²⁺/CaM/CaMKKβ signaling through regulatory phosphorylation.     

The gene coding the human S11 surface antigens maps between the loci for HPRT and G6PD on the X-chromosome

1-Methyladenine, which has been previously shown to be the hormone responsible for meiosis reinitiation in starfish oocytes, triggers parthenogenetic activation when applied to matured starfish oocytes after emission of the second polar body and formation of the pronucleus. In Marthasterias glacialis and Asterias rubens oocytes parthenogenetic activation includes elevation of a fertilization membrane, cleavage and the formation of normal bipinnaria larvae. Activation is likely to result from 1-methyladenine interaction with the category of stereospecific membrane receptors involved in meiosis reinitiation, since structural requirements of this compound are identical for both biological responses. Appearance of oocyte responsiveness to 1-MeAde after, but not before emission of the second polar body cannot be accounted for by their increased sensitivity to intracellular Ca²⁺ at that time, although it is shown that Ca²⁺ mediates hormone effect in inducing parthenogenetic activation. Pretreatment of immature oocytes with the free hormone in excess strongly inhibits the 1-methyladenine-induced parthenogenetic activation of the oocytes when they have completed maturation.It is suggested that reappearance of 1-MeAde sensitivity when oocytes form a pronucleus depends either upon recruitment or new receptor units or on the reactivation of pre-existing inactivated receptors at this stage of oocyte maturation.     

Initiation of meiosis in yeast mutants defective in adenylate cyclase and cyclic AMP-dependent protein kinase

Control of the initiation of meiosis was examined in diploids of yeast homozygous for two temperature-sensitive mutations, cyr1 and CYR3, which are defective in adenylate cyclase and cAMP-dependent protein kinase, respectively. The cyr1 and CYR3 mutations permitted the initiation of meiosis, but resulted in the frequent production of two-spored asci at the restrictive temperature. Unlike the wild-type diploid cells, the cyr1 and CYR3 homozygous diploid cells were capable of initiating meiosis even in nutrient growth media. This unique feature of the cyr1 and CYR3 mutants suggests that these mutations relate to the choice between mitotic and meiotic processes. In diploids homozygous for the bcy1 mutation that results in deficiency of the regulatory subunit of cAMP-dependent protein kinase and production of a high level of the catalytic subunit of this enzyme, no premeiotic DNA replication and commitment to intragenic recombination occurred, and no spores were formed. We conclude that the initiation of meiosis may be dependent upon the repression of cAMP production and the inactivation of cAMP-dependent protein kinase.     

cAMP-dependent phosphorylation and hexamethylene-bis-acetamide induced dephosphorylation of p19 in murine erythroleukemia cells

The objective of this study was to investigate cyclic-adenosinemonophosphate (cAMP)-dependent phosphorylation in murine erythroleukemia (MEL) cells and to identify either direct substrates of cAMP-dependent kinase or downstream effectors of cAMP dependent phosphorylation with a potential function in growth and differentiation. MEL-cells rendered deficient in cAMP-dependent protein kinase (A-kinase) activity by stable transfection with DNA encoding for either a mutant regulatory subunit or a specific peptide inhibitor of A-Kinase (PKI) are unable to differentiate normally in response to chemical inducers. We have identified by 2-D Western blotting 2 phosphorylated forms of p19, a highly conserved 18-19 kDa cytosolic protein that is frequently upregulated in transformed cells and undergoes phosphorylation in mammalian cells upon activation of several signal transduction pathways. The phosphorylation of the more acidic phosphorylated form is increased in a cAMP-dependent fashion and impaired in cells deficient in cAMP-dependent kinase (A-kinase). Treatment of MEL-cells with the chemical inducer of differentiation hexamethylene-bisacetamide (HMBA) led to dephosphoryation of this phosphoform. Our data are compatible with previous observations which imply that phosphorylation of Ser 38 in p19 by p34cdc2-kinase leads to a more basic phosphoform and simultaneous phosphorylation by mitogen-activated kinase of Ser 25 in response to protein kinase C and the cAMP- dependent kinase creates the more acidic species.     

Protein phosphorylation and oocyte maturation : II. Inhibition of starfish oocyte maturation by intracellular microinjection of protein phosphatases 1 and 2A and alkaline phosphatase

Oocyte maturation (meiosis re-initiation) in starfish is induced by the natural hormone 1-methyladenine (1-MeAde). Following hormonal stimulation of the oocyte, an intracellular Maturation Promoting Factor (MPF) appears in the cytoplasm which triggers nuclear envelope breakdown and maturation divisions. Microinjection of pure preparations of the catalytic subunits of protein phosphatases 1 and 2A inhibits 1-MeAde-induced maturation in a dose-dependent manner. Calmodulin-dependent protein phosphatase 2B is inefficient. Maturation induced by mimetics of 1-MeAde, such as dithiothreitol (DTT), methylglyoxal-bis(guanylhydrazone) (MGBG), 8-hydroxyeicosatetraenoic acid (8 HETE) and arachidonic acid (AA) is also inhibited by these protein phosphatases. In all cases inhibition can be reversed by increasing the concentration of 1-Me-Ade or of mimetic. Alkaline phosphatase also inhibits maturation in a dose-dependent way and in a reversible manner. Microinjection of protein phosphatase is still effective when preformed long after the end of the hormone-dependent period, and can even be effective a few minutes before the breakdown of the nuclear envelope. No detectable MPF activity is found in 1-MeAde-treated phosphatase-injected oocytes. However, microinjection of phosphatase 2A simultaneously with MPF (obtained from 1-MeAde-treated donors) does not result in inhibition. These results constitute direct evidence for the necessity of an elevated level of phosphorylated proteins for MPF activity and maturation. The mode of action of 1-MeAde in inducing starfish oocyte maturation is discussed in relation to protein phosphorylation.