Okadaic acid mimics a nuclear component required for cyclin B-cdc2 kinase microinjection to drive starfish oocytes into M phase.

G2-arrested oocytes contain cdc2 kinase as an inactive cyclin B-cdc2 complex. When a small amount of highly purified and active cdc2 kinase, prepared from starfish oocytes at first meiotic metaphase, is microinjected into Xenopus oocytes, it induces activation of the inactive endogenous complex and, as a consequence, drives the recipient oocytes into M phase. In contrast, the microinjected kinase undergoes rapid inactivation in starfish oocytes, which remain arrested at G2. Endogenous cdc2 kinase becomes activated in both nucleated and enucleated starfish oocytes injected with cytoplasm taken from maturing oocytes at the time of nuclear envelope breakdown, but only cytoplasm taken from nucleated oocytes becomes able thereafter to release second recipient oocytes from G2 arrest, and thus contains M phase-promoting factor (MPF) activity. Both nucleated and enucleated starfish oocytes produce MPF activity when type 2A phosphatase is blocked by okadaic acid. If type 2A phosphatase is only partially inhibited, neither nucleated nor enucleated oocytes produce MPF activity, although both do so if purified cdc2 kinase is subsequently injected as a primer to activate the endogenous kinase. The nucleus of starfish oocytes contains an inhibitor of type 2A phosphatase, but neither active nor inactive cdc2 kinase. Microinjection of the content of a nucleus into the cytoplasm of G2-arrested starfish oocytes activates endogenous cdc2 kinase, produces MPF activity, and drives the recipient oocytes into M phase. Together, these results show that the MPF amplification loop is controlled, both positively and negatively, by cdc2 kinase and type 2A phosphatase, respectively. Activation of the MPF amplification loop in starfish requires a nuclear component to inhibit type 2A phosphatase in cytoplasm.

cdc25 is a nuclear protein expressed constitutively throughout the cell cycle in nontransformed mammalian cells.

A family of proteins homologous to the cdc25 gene product of the fission yeast bear specific protein tyrosine phosphatase activity involved in the activation of the p34cdc2-cyclin B kinase. Using affinity-purified antibodies raised against a synthetic peptide corresponding to the catalytic site of the cdc25 phosphatase, we show that cdc25 protein is constitutively expressed throughout the cell cycle of nontransformed mammalian fibroblasts and does not undergo major changes in protein level. By indirect immunofluorescence, cdc25 protein is found essentially localized in the nucleus throughout interphase and during early prophase. Just before the complete nuclear envelope breakdown at the prophase-prometaphase boundary, cdc25 proteins are redistributed throughout the cytoplasm. During metaphase and anaphase, cdc25 staining remains distributed throughout the cell and excludes the condensed chromosomes. The nuclear locale reappears during telophase. In light of the recent data describing the cytoplasmic localization of cyclin B protein (Pines, J., and T. Hunter. 1991. J. Cell Biol. 115:1-17), the data presented here suggest that separation in two distinct cellular compartments of the cdc25 phosphatase and its substrate p34cdc2-cyclin B may be of importance in the regulation of the cdc2 kinase activity.

Cyclin A potentiates maturation-promoting factor activation in the early Xenopus embryo via inhibition of the tyrosine kinase that phosphorylates cdc2.

We have produced human cyclin A in Escherichia coli and investigated how it generates H1 kistone kinase activity when added to cyclin-free extracts prepared from parthenogenetically activated Xenopus eggs. Cyclin A was found to form a major complex with cdc2, and to bind cdk2/Eg1 only poorly. No lag phase was detected between the time when cyclin A was added and the time when H1 histone kinase activity was produced in frog extracts, even in the presence of 2 mM vanadate, which blocks cdc25 activity. Essentially identical results were obtained using extracts prepared from starfish oocytes. We conclude that formation of an active cyclin A-cdc2 kinase during early development escapes an inhibitory mechanism that delays formation of an active cyclin B-cdc2 kinase. This inhibitory mechanism involves phosphorylation of cdc2 on tyrosine 15. Okadaic acid (OA) activated cyclin B-cdc2 kinase and strongly reduced tyrosine phosphorylation of cyclin B-associated cdc2, even in the presence of vanadate. 6-dimethylamino-purine, a reported inhibitor of serine-threonine kinases, suppressed OA-dependent activation of cyclin B-cdc2 complexes. This indicates that the kinase(s) which phosphorylate(s) cdc2 on inhibitory sites can be inactivated by a phosphorylation event, itself antagonized by an OA-sensitive, most likely type 2A phosphatase. We also found that cyclin B- or cyclin A-cdc2 kinases can induce or accelerate conversion of the cyclin B-cdc2 complex from an inactive into an active kinase. Cyclin B-associated cdc2 does not undergo detectable phosphorylation on tyrosine in egg extracts containing active cyclin A-cdc2 kinase, even in the presence of vanadate. We propose that the active cyclin A-cdc2 kinase generated without a lag phase from neo-synthesized cyclin A and cdc2 may cause a rapid switch in the equilibrium of cyclin B-cdc2 complexes to the tyrosine-dephosphorylated and active form of cdc2 during early development, owing to strong inhibition of the cdc2-specific tyrosine kinase(s). This may explain why early cell cycles are so rapid in many species.

Microinjection of a conserved peptide sequence of p34cdc2 induces a Ca2+ transient in oocytes.

The product of the yeast cell cycle control gene cdc2, and its homologs in higher eukaryotes (p34cdc2), all contain a perfectly conserved sequence of 16 amino acids that has not been found in any other protein sequence. Microinjection of this peptide triggers a specific increase in the concentration of intracellular free Ca2+ that originates from intracellular stores in both starfish and Xenopus oocytes. Thus, p34cdc2 might interact through its conserved peptide domain with some component of the Ca2(+)-regulatory system.

Cloning, expression and subcellular localization of the human homolog of p40MO15 catalytic subunit of cdk-activating kinase.

Transitions of the cell cycle are controlled by cyclin-dependent protein kinases (cdks) whose phosphorylation on the Thr residue included in the conserved sequence YTHEVV dramatically increases the activity. A kinase responsible for this specific phosphorylation, called CAK for cdk-activating kinase, has been recently purified from starfish and Xenopus oocytes and shown to contain the MO15 gene product as a catalytic subunit. In the present paper, we have cloned the human homolog of Xenopus p40MO15 by probing a HeLa cell cDNA library with degenerate oligonucleotides deduced from Xenopus and starfish MO15 sequences. Human and Xenopus MO15 displayed a strong homology showing 86% identity with regard to amino acid sequences. Northern blot analysis of RNA extracts from a series of human tissues as well as from cultured rodent fibroblasts revealed a unique 1.4 kb MO15 mRNA. No variation in the amount of MO15 transcript or protein was found along the entire course of the fibroblast cell cycle. Fluorescence in situ hybridization on human lymphocyte metaphases showed two distinct chromosomal locations of human MO15 gene at 5q12-q13 and 2q22-q24. By using gene tagging and mammalian cell transfection, we demonstrate that the KRKR motif located at the carboxy terminal end of MO15 is required for nuclear targeting of the protein. Mutation of KRKR to NGER retains MO15 in the cytoplasmic compartment, whilst the wild-type protein is detected exclusively in the nucleus. Interestingly, we demonstrate that the nuclear targeting of MO15 is necessary to confer the protein its CAK activity. In contrast to the wild-type, the NLS-mutated MO15 expressed in Xenopus oocytes is unable to generate CAK as long as the nuclear envelope is not broken. The nuclear localization of both the MO15 gene product and CAK activity may imply that cdks activation primarily occurs in the cell nucleus.

Specific sulfonation of tyrosine, tryptophan and hydroxy-amino acids in peptides.

1. ClSO3H in trifluoroacetic acid rapidly converts serine and threonine into O-sulfate ester derivatives while tyrosine and tryptophan are converted into arylsulfonic acids. 2. H2SO4 in trifluoroacetic acid reacts more slowly with serine, threonine and tyrosine while is not able to modify tryptophan. 3. All other amino acids are perfectly stable under the above reaction conditions. 4. Peptides containing susceptible amino acid residues are specifically converted into the corresponding sulfonated derivatives in high or quantitative yield.

Evidence for two distinct adenylate cyclase catalysts in rat brain.

The Lubrol-soluble adenylate cyclase activity of brain synaptosomal membranes appeared, upon gel filtration or sucrose gradient centrifugation, as two overlapping peaks. Fractions corresponding to the peak of the largest Stokes radius (Biogel pool 1) or highest s value (gradient pool 1) contained an adenylate cyclase activity which could be detected whatever the enzyme assay conditions. In contrast, in fractions from the second peak (Biogel pool 2 or gradient pool 2), forskolin was needed to reveal adenylate cyclase activity. The enzyme activity of each Biogel pool was retained by forskolin-agarose and eluted by forskolin with a 34-83% yield. A polypeptide of 155 kDa made up 80% of the forskolin-agarose eluate 1, whereas it was almost absent from eluate 2. Since data from various groups point to the 155 kDa polypeptide as a brain adenylate cyclase catalyst, still another distinct catalyst of lower molecular mass is likely to be present in brain.

[Synthesis and activation of N-aminopolyacrylamide derivatives as carriers in solid phase degradation of proteins]. / Synthèse et activation de dérivés N-aminopolyacrylamide comme support dans la dégradation en phase solide des protéines.

N-amino polyacrylamide derivatives were synthesized and their activation by different techniques were examined. The use of these derivatives as solid support in sequential degradation of proteins is proposed. An example is reported.

On the activation of the canine pepsinogens.

Acidification induces a conversion of canine pepsinogens by a sequential mechanism to the active pepsins. Activation in the presence of pepstatin, which strongly inhibits the pepsins but does not prevent the first step of activation, allows the isolation of the peptide released in this first step. This peptide inhibits the milk clotting activity of canine and also porcine pepsin. Canine pepsins obtained in the absence of pepstatin were characterized by amino acid composition, molecular weight, and activity against hemoglobin and milk and compared with those of other mammalian pepsins.

Calcium-calmodulin-dependent phosphorylations in the control of muscular contraction?

Muscular contraction is triggered by the increase in free calcium concentration and modulated by cyclic nucleotide-dependent phosphorylation. Beside a direct trigger of sarcomeric muscle contraction through binding of troponin C, calcium ions trigger or modulate contractility through calcium-calmodulin-dependent myosin light chain kinases, and increase the rate of relaxation through the calmodulin-dependent phosphorylation of phospholamban, the activator of the cardiac sarcoplasmic reticulum calcium pump. In both cases, a concerted regulation by calcium and cyclic nucleotides is observed. Hyperactivation of the calcium pump is brought about by additional phosphorylation of phospholamban by cAMP-dependent protein kinase. Similarly myofibrillar myosin light chain kinases from smooth and skeletal muscles are substrates of the cAMP-dependent protein kinase. The calmodulin-dependent protein kinases are probably organized into supramolecular regulatory complexes.

Modulation of the accumulation of inositol phosphates and the mobilization of calcium in aortic myocytes.

In vascular smooth muscle cells the phorbol ester, 12-O-tetradecanoyl phorbol 13-acetate (TPA), a potent activator of C-kinase, inhibited the accumulation of inositol phosphates and the mobilization of calcium produced by several agonists. In the same way, TPA inhibited the fluoride-induced activation of phosphoinositide metabolism. These results suggest a C-kinase action at a post-receptor level. Moreover, the fluoride-induced accumulation of inositol phosphates shows the presence of one or more guanine nucleotide-binding proteins (G-proteins) in the regulation of receptor-phospholipase C coupling. This was confirmed by the use of N-ethylmaleimide and pertussis toxin. These results support the view that, in addition to the induction of sustained contractions, C-kinase can activate negative feedback mechanisms in aortic myocytes.

cdk5 expression and association with p35nck5a in early stages of rat cerebellum neurogenesis; tyrosine dephosphorylation and activation in post-mitotic neurons.

We have examined the expression of cyclin dependent kinase (cdk) 5 protein kinase and p35nck5a, its activator subunit, during postnatal neurogenesis in rat cerebellum, using mono-specific antibodies. Both cdk5 and p35nck5a are present and associated in proliferative stages, although cdk5-p35 kinase activity is barely detectable. Cdk5-p35 activity, but not the expression of either subunit, increases up to 6-fold during neuronal differentiation. Since we observe that cdk5 is phosphorylated on tyrosine in proliferative, but not in post-mitotic stages, we suggest that post-translational regulatory mechanisms control cdk5-p35 protein kinase activity during neurogenesis.

The effects of maitotoxin on phosphoinositides and calcium metabolism in a primary culture of aortic smooth muscle cells.

Maitotoxin, a potent marine toxin isolated from toxic tropical dinoflagellates and poisonous fishes induces contraction of different smooth muscle preparations. Actions of maitotoxin on phosphoinositides and calcium metabolism were studied using a primary culture of aortic smooth muscle cells. Maitotoxin induced a very large increase of cytosolic calcium concentration as evaluated by fura-2 acetoxymethyl ester fluorescence. This increase was concomitant with stimulation of inositol-phosphate accumulation and loss of viability of aortic smooth muscle cells. These responses to maitotoxin were abolished in Ca2+-free medium, and were mimicked by saponin. Calcium ionophores or K+ depolarisation did not induce inositol-phosphate formation. These results suggest that maitotoxin acts by altering smooth muscle cells permeability allowing a sustained calcium influx which is able to activate inositol-phosphate formation and which is lethal for the cells.

Immunocytochemical localization of myosin, tropomyosin and actin in vestibular hair cells of human fetuses and cats.

Semithin sections (1 micron) of human fetuses and young cat vestibular epithelium embedded in Epon were reacted with primary antibodies raised against myosin, tropomyosin, and actin. The results were very similar in the two species. Myosin and tropomyosin were colocalized and strictly limited to an apical superficial corona arranged in a punctiform pattern. The stereocilia and cuticular plate were unreactive to myosin and tropomyosin antibodies. Actin antibodies stained the stereocilia, cuticular plate, and a marginal ring surrounding and underlying the cuticular plate. At this level, myosin and tropomyosin were not detected. This result suggests that the circumferential actin ring has a structural role. Colocalization of myosin, tropomyosin, and actin in a superficial area around the stereocilia bundle and along the apical surface of the hair cell imply that active processes could exist at the apex of certain sensory vestibular hair cells. The specificity of myosin antibodies and their cross-reactivity with different types of myosin are discussed.