Changes in the Activity of the Maturation-Promoting Factor Are Correlated with Those of a Major Cyclic AMP and Calcium-Independent Protein Kinase During the First Mitotic Cell Cycles in the Early Starfish Embryo. (cell cycle/maturation-promoting factor/protein kinase/protein synthesis/starfish)

We have examined the time course of protein tyrosine phosphorylation in the meiotic cell cycles of Xenopus laevis oocytes and the mitotic cell cycles of Xenopus eggs. We have identified two proteins that undergo marked changes in tyrosine phosphorylation during these processes: a 42-kDa protein related to mitogen-activated protein kinase or microtubule-associated protein-2 kinase (MAP kinase) and a 34-kDa protein identical or related to p34cdc2. p42 undergoes an abrupt increase in its tyrosine phosphorylation at the onset of meiosis 1 and remains tyrosine phosphorylated until 30 min after fertilization, at which point it is dephosphorylated. p42 also becomes tyrosine phosphorylated after microinjection of oocytes with partially purified M-phase-promoting factor, even in the presence of cycloheximide. These findings suggest that MAP kinase, previously implicated in the early responses of somatic cells to mitogens, is also activated at the onset of meiotic M phase and that MAP kinase can become tyrosine phosphorylated downstream from M-phase-promoting factor activation. We have also found that p34 goes through a cycle of tyrosine phosphorylation and dephosphorylation prior to meiosis 1 and mitosis 1 but is not detectable as a phosphotyrosyl protein during the 2nd through 12th mitotic cell cycles. It may be that the delay between assembly and activation of the cyclin-p34cdc2 complex that p34cdc2 tyrosine phosphorylation provides is not needed in cell cycles that lack G2 phases. Finally, an unidentified protein or group of proteins migrating at 100 to 116 kDa increase in tyrosine phosphorylation throughout maturation, are dephosphorylated or degraded within 10 min of fertilization, and appear to cycle between low-molecular-weight forms and high-molecular-weight forms during early embryogenesis.

Download Full-text

Role of 3':5'-cyclic-AMP-dependent protein kinase in regulation of protein synthesis in reticulocyte lysates.

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.74.4.1463 ◽

1977 ◽

Vol 74 (4) ◽

pp. 1463-1467 ◽

Cited By ~ 42

Author(s):

A. Datta ◽

C. de Haro ◽

J. M. Sierra ◽

S. Ochoa

Keyword(s):

Protein Synthesis ◽

Protein Kinase ◽

Cyclic Amp ◽

Dependent Protein Kinase ◽

Reticulocyte Lysates ◽

Dependent Protein

Download Full-text

Protein kinase C acts downstream of calcium at entry into the first mitotic interphase of Xenopus laevis.

Cell Regulation ◽

10.1091/mbc.1.3.315 ◽

1990 ◽

Vol 1 (3) ◽

pp. 315-326 ◽

Cited By ~ 51

Author(s):

W M Bement ◽

D G Capco

Keyword(s):

Cell Cycle ◽

Protein Kinase C ◽

Protein Kinase ◽

Xenopus Laevis ◽

Mitotic Cell ◽

Cortical Granule ◽

Mitotic Cell Cycle ◽

Cleavage Furrow ◽

Kinase C ◽

Cortical Granule Exocytosis

Transit into interphase of the first mitotic cell cycle in amphibian eggs is a process referred to as activation and is accompanied by an increase in intracellular free calcium [( Ca2+]i), which may be transduced into cytoplasmic events characteristic of interphase by protein kinase C (PKC). To investigate the respective roles of [Ca2+]i and PKC in Xenopus laevis egg activation, the calcium signal was blocked by microinjection of the calcium chelator BAPTA, or the activity of PKC was blocked by PKC inhibitors sphingosine or H7. Eggs were then challenged for activation by treatment with either calcium ionophore A23187 or the PKC activator PMA. BAPTA prevented cortical contraction, cortical granule exocytosis, and cleavage furrow formation in eggs challenged with A23187 but not with PMA. In contrast, sphingosine and H7 inhibited cortical granule exocytosis, cortical contraction, and cleavage furrow formation in eggs challenged with either A23187 or PMA. Measurement of egg [Ca2+]i with calcium-sensitive electrodes demonstrated that PMA treatment does not increase egg [Ca2+]i in BAPTA-injected eggs. Further, PMA does not increase [Ca2+]i in eggs that have not been injected with BAPTA. These results show that PKC acts downstream of the [Ca2+]i increase to induce cytoplasmic events of the first Xenopus mitotic cell cycle.

Download Full-text

Wachstumsparameter in normalen und durch Actinomycin verlängerten Zellzyklen von Tetrahymena / Growth Parameters in Normal and Actinomycin-induced prolonged Cell Cycles of Tetrahymena

Zeitschrift für Naturforschung B ◽

10.1515/znb-1969-1225 ◽

1969 ◽

Vol 24 (12) ◽

pp. 1624-1629 ◽

Cited By ~ 3

Author(s):

Günter Cleffmann

Keyword(s):

Cell Cycle ◽

Protein Synthesis ◽

Cell Division ◽

Dna Replication ◽

Cell Growth ◽

Rna Synthesis ◽

Growth Parameters ◽

Average Duration ◽

Cell Cycles ◽

Growing Cell

Actinomycin in low concentration (0,2 μg/ml — 0,5 μg/ml) prolongs the average duration of the cell cycle of Tetrahymena considerably, but does not inhibit cell division completely. Some parameters of the growing cell have been tested in cell cycles extended in this way and compared to those of normally growing cells. The RNA synthesis of treated cells is reduced to such an extent that the RNA content per cell decreases during the prolonged cell cycle. Nevertheless cell growth, protein synthesis and DNA replication proceed at almost the same rate as in untreated cells. These findings indicate that the presence of actinomycin does not interfere with RNA fractions necessary for growth but reduce the synthesis of RNA fractions which are essential for cell division. Therefore a longer period is needed for their accumulation.

Download Full-text

Cell cycle regulation of the yeast Cdc7 protein kinase by association with the Dbf4 protein.

Molecular and Cellular Biology ◽

10.1128/mcb.13.5.2899 ◽

1993 ◽

Vol 13 (5) ◽

pp. 2899-2908 ◽

Cited By ~ 153

Author(s):

A L Jackson ◽

P M Pahl ◽

K Harrison ◽

J Rosamond ◽

R A Sclafani

Keyword(s):

Cell Cycle ◽

Protein Kinase ◽

Protein Interactions ◽

Kinase Activity ◽

Mitotic Cell ◽

Common Point ◽

Temperature Sensitive ◽

Cdc7 Kinase

Yeast Cdc7 protein kinase and Dbf4 protein are both required for the initiation of DNA replication at the G1/S phase boundary of the mitotic cell cycle. Cdc7 kinase function is stage-specific in the cell cycle, but total Cdc7 protein levels remained unchanged. Therefore, regulation of Cdc7 function appears to be the result of posttranslational modification. In this study, we have attempted to elucidate the mechanism responsible for achieving this specific execution point of Cdc7. Cdc7 kinase activity was shown to be maximal at the G1/S boundary by using either cultures synchronized with alpha factor or Cdc- mutants or with inhibitors of DNA synthesis or mitosis. Therefore, Cdc7 kinase is regulated by a posttranslational mechanism that ensures maximal Cdc7 activity at the G1/S boundary, which is consistent with Cdc7 function in the cell cycle. This cell cycle-dependent regulation could be the result of association with the Dbf4 protein. In this study, the Dbf4 protein was shown to be required for Cdc7 kinase activity in that Cdc7 kinase activity is thermolabile in vitro when extracts prepared from a temperature-sensitive dbf4 mutant grown under permissive conditions are used. In vitro reconstitution assays, in addition to employment of the two-hybrid system for protein-protein interactions, have demonstrated that the Cdc7 and Dbf4 proteins interact both in vitro and in vivo. A suppressor mutation, bob1-1, which can bypass deletion mutations in both cdc7 and dbf4 was isolated. However, the bob1-1 mutation cannot bypass all events in G1 phase because it fails to suppress temperature-sensitive cdc4 or cdc28 mutations. This indicates that the Cdc7 and Dbf4 proteins act at a common point in the cell cycle. Therefore, because of the common point of function for the two proteins and the fact that the Dbf4 protein is essential for Cdc7 function, we propose that Dbf4 may represent a cyclin-like molecule specific for the activation of Cdc7 kinase.

Download Full-text

333 DYNAMICS OF MITOGEN-ACTIVATED PROTEIN KINASE ACTIVITY IN PIG OOCYTES FOLLOWING PARTHENOGENETIC ACTIVATION BY DIFFERENT METHODS

Reproduction Fertility and Development ◽

10.1071/rdv19n1ab333 ◽

2007 ◽

Vol 19 (1) ◽

pp. 282

Author(s):

L. Nanassy ◽

K. Lee ◽

A. Javor ◽

Z. Machaty

Keyword(s):

Cell Cycle ◽

Protein Synthesis ◽

Protein Kinase ◽

Kinase Activity ◽

Mitogen Activated Protein Kinase ◽

Blastocyst Formation ◽

Cell Numbers ◽

Mapk Activity ◽

Mitogen Activated Protein ◽

Pronuclear Formation

Cell cycle progression during mitosis and meiosis is known to be regulated by the M-phase promoting factor (MPF). However, recent findings revealed that mitogen-activated protein kinase (MAPK) also plays an important regulatory role during transition through the cell cycle. At fertilization the activity of MAPK drops shortly after MPF inactivation; the objective of this study was to investigate the dynamics of MAPK activity in pig oocytes after different activation methods. In vitro-matured oocytes were allocated to 3 groups. In group 1 (EP), the oocytes were activated by 2 DC pulses of 1.2 kV cm-1, 60 �s each. In the second group (EP + BU), the oocytes were electroporated and incubated for 4 h in 100 �M butyrolactone I (BU, an inhibitor of cdc2 kinase). In group 3 (EP + CHX), the oocytes were electroporated and treated for 5 h with 10 �g mL-1 cycloheximide (CHX, a protein synthesis inhibitor). After electroporation all oocytes were incubated in 7.5 �g mL-1 cytochalasin B for 4 h. Some oocytes were used to determine MAPK activity at 0, 1, 2, 3, 4, 5, and 6 h after electroporation using a MAPK assay kit. The assay measures MAPK activity by determining the phosphorylation of myelin basic protein by MAPK using the transfer of the γ-phosphate of [γ-32P] ATP. Pronuclear formation was evaluated at 6 h after electroporation; blastocyst formation and total cell numbers per embryo were determined after a 7-day culture in PZM-3 medium. Pronuclear formation was compared by the chi-square test, blastocyst formation was assessed using ANOVA, and the kinase activity was evaluated using the Student t-test. Pronuclear formation was highest in the combined methods [69.39% (EP) vs. 86.32% (EP + BU) and 87.56 % (EP + CHX); P < 0.05]. Similarly, the combined methods supported better development to the blastocyst stage [25.06 � 7.96% (EP), 58.32 � 7.62% (EP + BU), and 63.91 � 6.35% (EP + CHX); P < 0.05], whereas the average cell numbers of the blastocysts did not differ (47.11 � 3.12, 46.56 � 2.33, and 44.04 � 1.86, respectively). The initial MAPK activity was 0.123 � 0.017 pmol/min/oocyte which, after 1 h, dropped in all cases to values of 0.069 � 0.009 (EP), 0.072 � 0.007 (EP + BU), and 0.077 � 0.012 (EP + CHX) pmol/min/oocyte (P < 0.05). The MAPK activity in the EP group reached its lowest level at 3 h (0.057 � 0.007 pmol/min/oocyte); however, at 4 h it started to recover and by 6 h the activity (0.079 � 0.022 pmol/min/oocyte) did not differ from that of the non-activated oocytes. In the other groups, MAPK activity stayed low, and by the end of the experimental period it was significantly lower than that in the nontreated metaphase II oocytes (P < 0.05). The results indicate that electroporation followed by protein kinase inhibition or protein synthesis inhibition leads to the efficient inactivation of MAPK activity, and confirm our earlier findings that these combined treatments support superior embryo development after oocyte activation.

Download Full-text