ets-2 Regulates cdc2 Kinase Activity in Mammalian Cells: Coordinated Expression of cdc2 and Cyclin A

1995 ◽  
Vol 217 (1) ◽  
pp. 8-14 ◽  
Author(s):  
Shau-Ching Wen ◽  
De-Hui Ku ◽  
Antonio De Luca ◽  
Pier Paolo Claudio ◽  
Antonio Giordano ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Kinase Activity ◽  
Cyclin A ◽  
Cdc2 Kinase ◽  
Coordinated Expression

Delayed cyclin A and B1 degradation in non-transformed mammalian cells

1995 ◽  
Vol 108 (7) ◽  
pp. 2599-2608 ◽  
Author(s):  
F. Girard ◽  
A. Fernandez ◽  
N. Lamb
Keyword(s):  
Mammalian Cells ◽  
Marine Invertebrates ◽  
Cyclin A ◽  
Cdc2 Kinase ◽  
Marked Contrast ◽  
Anaphase Onset ◽  
Significant Difference ◽  
Primary Fibroblasts ◽  
Transformed Cell Lines ◽  
Derivatives Of

Cyclins A and B are known to exhibit significant differences in their function, cellular distribution and timing of degradation at mitosis. On the basis of observations in marine invertebrates and Xenopus, it was proposed that cyclin destruction triggers cdc2 kinase inactivation and anaphase onset. However, this model has recently been questioned, both in Xenopus and in budding yeast. In this report, we present evidence for delayed degradation of both cyclins A and B1 in non-transformed mammalian cells. Indeed, by means of indirect immunofluorescence and confocal microscopy, we show that cyclins A and B1 are present up to anaphase in REF52, Hs68, human primary fibroblasts and NRK epithelial cells. In marked contrast, cyclin A is shown to be degraded within metaphase and cyclin B just at the transition to anaphase in HeLa and two transformed cell lines, derivatives of normal NRK and REF52. These results further support the notion that cyclin destruction might be not correlated with anaphase onset in normal cells and highlight a significant difference in the fate of mitotic cyclins between transformed and non-transformed cells.

scholarly journals Activation of the Xenopus cyclin degradation machinery by full-length cyclin A

1996 ◽  
Vol 109 (5) ◽  
pp. 1071-1079 ◽  
Author(s):  
C. Jones ◽  
C. Smythe
Keyword(s):  
Protein Synthesis ◽  
Dna Replication ◽  
Kinase Activity ◽  
Cyclin A ◽  
Cell Types ◽  
Cyclin B1 ◽  
Full Length ◽  
Cyclin B ◽  
Cdc2 Kinase ◽  
Nuclear Envelope Breakdown

The entry into mitosis is dependent on the activation of mitotic forms of cdc2 kinase. In many cell types, cyclin A-associated kinase activity peaks just prior to that of cyclin B, although the precise role of cyclin A-associated kinase in the entry into mitosis is still unclear. Previous work has suggested that while cyclin B is capable of triggering cyclin destruction in Xenopus cell-free systems, cyclin A-associated kinase is not able to support this function. Here we have expressed a full-length human cyclin A in Escherichia coli and purified the protein to homogeneity by virtue of an N-terminal histidine tag. We have found that when added to Xenopus cell-free extracts free of cyclin B and incapable of protein synthesis, the temporal pattern of cyclin A-associated cdc2 kinase activity showed distinct differences that were dependent on the concentration of cyclin A added. When cyclin A was added to a concentration that generated levels of cdc2 kinase activity capable of inducing nuclear envelope breakdown, the histone H1 kinase activity profile was bi-phasic, consisting of an activation phase followed by an inactivation phase. Inactivation was found to be due to cyclin destruction, which was prevented by mos protein. Cyclin destruction was followed by nuclear reassembly and an additional round of DNA replication, indicating that there is no protein synthesis requirement for DNA replication in this embryonic system. It has been suggested that the evolutionary recruitment of cyclin A into an S phase function may have necessitated the loss of an original mitotic ability to activate the cyclin destruction pathway. The results presented here indicate that cyclin A has not lost the ability to activate its own destruction and that cyclin A-mediated activation of the cyclin destruction pathway permitted destruction of cyclin B1 as well as cyclin A, indicating that there are not distinct cyclin A and cyclin B destruction pathways. Thus the ordered progression of the cell cycle requires the careful titration of cyclin. A concentration in order to avoid activation of the cyclin destruction pathway before sufficient active cyclin B/cdc2 kinase has accumulated.

scholarly journals Regulation of Cyclin A-Cdk2 by SCF Component Skp1 and F-Box Protein Skp2

1999 ◽  
Vol 19 (1) ◽  
pp. 635-645 ◽  
Author(s):  
Cain H. Yam ◽  
Raymond W. M. Ng ◽  
Wai Yi Siu ◽  
Anita W. S. Lau ◽  
Randy Y. C. Poon
Keyword(s):  
Cell Cycle ◽  
Mammalian Cells ◽  
Kinase Activity ◽  
Cyclin A ◽  
Cell Cycle Regulators ◽  
Cycle Arrest ◽  
Ubiquitin Ligase E3 ◽  
F Box Protein ◽  
Kinase Phosphorylation

ABSTRACT Cyclin A-Cdk2 complexes bind to Skp1 and Skp2 during S phase, but the function of Skp1 and Skp2 is unclear. Skp1, together with F-box proteins like Skp2, are part of ubiquitin-ligase E3 complexes that target many cell cycle regulators for ubiquitination-mediated proteolysis. In this study, we investigated the potential regulation of cyclin A-Cdk2 activity by Skp1 and Skp2. We found that Skp2 can inhibit the kinase activity of cyclin A-Cdk2 in vitro, both by direct inhibition of cyclin A-Cdk2 and by inhibition of the activation of Cdk2 by cyclin-dependent kinase (CDK)-activating kinase phosphorylation. Only the kinase activity of Cdk2, not of that of Cdc2 or Cdk5, is reduced by Skp2. Skp2 is phosphorylated by cyclin A-Cdk2 on residue Ser76, but nonphosphorylatable mutants of Skp2 can still inhibit the kinase activity of cyclin A-Cdk2 toward histone H1. The F box of Skp2 is required for binding to Skp1, and both the N-terminal and C-terminal regions of Skp2 are involved in binding to cyclin A-Cdk2. Furthermore, Skp2 and the CDK inhibitor p21 Cip1/WAF1 bind to cyclin A-Cdk2 in a mutually exclusive manner. Overexpression of Skp2, but not Skp1, in mammalian cells causes a G1/S cell cycle arrest.

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

1992 ◽  
Vol 118 (5) ◽  
pp. 1109-1120 ◽  
Author(s):  
A Devault ◽  
D Fesquet ◽  
J C Cavadore ◽  
A M Garrigues ◽  
J C Labbé ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Early Development ◽  
Kinase Activity ◽  
Active Form ◽  
Cyclin A ◽  
Cyclin B ◽  
Lag Phase ◽  
Inhibitory Mechanism ◽  
Cdc2 Kinase ◽  
Histone Kinase

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.

scholarly journals In vitro fusion of endocytic vesicles is inhibited by cyclin A-cdc2 kinase.

1993 ◽  
Vol 4 (5) ◽  
pp. 541-553 ◽  
Author(s):  
P G Woodman ◽  
J P Adamczewski ◽  
T Hunt ◽  
G Warren
Keyword(s):  
Indirect Effect ◽  
Mammalian Cells ◽  
Cyclin A ◽  
Vesicle Fusion ◽  
Cyclin B ◽  
Endocytic Vesicle ◽  
Cdc2 Kinase ◽  
Histone Kinase ◽  
Endocytic Vesicles

Receptor-mediated endocytosis and recycling are inhibited in mitotic mammalian cells, and previous studies have shown that inhibition of endocytic vesicle fusion in vitro occurs via cyclin B-cdc2 kinase. To test for the ability of cyclin A-cdc2 kinase to inhibit endocytic vesicle fusion, we employed recombinant cyclin A proteins. Addition of cyclin A to interphase extracts activated a histone kinase and markedly reduced the efficiency of endocytic vesicle fusion. By a number of criteria, inhibition of fusion was shown to be due to the action of cyclin A, via the mitosis-specific cdc2 kinase, and not an indirect effect through cyclin B. Two-stage incubations were used to demonstrate that at least one target of cyclin A-cdc2 kinase is a cytosolic component of the fusion apparatus. Reconstitution experiments showed that this component was also modified in mitotic cytosols and was unaffected by N-ethyl maleimide treatment.

scholarly journals Mutational Analysis of the Cy Motif from p21 Reveals Sequence Degeneracy and Specificity for Different Cyclin-Dependent Kinases

2001 ◽  
Vol 21 (15) ◽  
pp. 4868-4874 ◽  
Author(s):  
James A. Wohlschlegel ◽  
Brian T. Dwyer ◽  
David Y. Takeda ◽  
Anindya Dutta
Keyword(s):  
Inhibitory Activity ◽  
Mammalian Cells ◽  
Mutational Analysis ◽  
Cyclin E ◽  
Cyclin A ◽  
Binding Motif ◽  
Binding Studies ◽  
Alanine Scanning Mutagenesis ◽  
Cyclin Dependent Kinases

ABSTRACT Inhibitors, activators, and substrates of cyclin-dependent kinases (cdks) utilize a cyclin-binding sequence, known as a Cy or RXL motif, to bind directly to the cyclin subunit. Alanine scanning mutagenesis of the Cy motif of the cdk inhibitor p21 revealed that the conserved arginine or leucine (constituting the conserved RXL sequence) was important for p21's ability to inhibit cyclin E-cdk2 activity. Further analysis of mutant Cy motifs showed, however, that RXL was neither necessary nor sufficient for a functional cyclin-binding motif. Replacement of either of these two residues with small hydrophobic residues such as valine preserved p21's inhibitory activity on cyclin E-cdk2, while mutations in either polar or charged residues dramatically impaired p21's inhibitory activity. Expressing p21N with non-RXL Cy sequences inhibited growth of mammalian cells, providing in vivo confirmation that RXL was not necessary for a functional Cy motif. We also show that the variant Cy motifs identified in this study can effectively target substrates to cyclin-cdk complexes for phosphorylation, providing additional evidence that these non-RXL motifs are functional. Finally, binding studies using p21 Cy mutants demonstrated that the Cy motif was essential for the association of p21 with cyclin E-cdk2 but not with cyclin A-cdk2. Taking advantage of this differential specificity toward cyclin E versus cyclin A, we demonstrate that cell growth inhibition was absolutely dependent on the ability of a p21 derivative to inhibit cyclin E-cdk2.

Optogenetic Control of Protein Kinase Activity in Mammalian Cells

2013 ◽  
Vol 3 (5) ◽  
pp. 280-285 ◽  
Author(s):  
Sabrina Wend ◽  
Hanna J. Wagner ◽  
Konrad Müller ◽  
Matias D. Zurbriggen ◽  
Wilfried Weber ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Mammalian Cells ◽  
Kinase Activity ◽  
Protein Kinase Activity ◽  
Optogenetic Control

The Polo-like kinase Plx1 is a component of the MPF amplification loop at the G2/M-phase transition of the cell cycle in Xenopus eggs

1998 ◽  
Vol 111 (12) ◽  
pp. 1751-1757 ◽  
Author(s):  
A. Abrieu ◽  
T. Brassac ◽  
S. Galas ◽  
D. Fisher ◽  
J.C. Labbe ◽  
...  
Keyword(s):  
Phase Transition ◽  
Cell Cycle ◽  
Cyclin A ◽  
Cyclin B ◽  
Cdc2 Kinase ◽  
C Terminus ◽  
M Phase ◽  
Egg Extracts ◽  
Amplification Loop

We have investigated whether Plx1, a kinase recently shown to phosphorylate cdc25c in vitro, is required for activation of cdc25c at the G2/M-phase transition of the cell cycle in Xenopus. Using immunodepletion or the mere addition of an antibody against the C terminus of Plx1, which suppressed its activation (not its activity) at G2/M, we show that Plx1 activity is required for activation of cyclin B-cdc2 kinase in both interphase egg extracts receiving recombinant cyclin B, and cycling extracts that spontaneously oscillate between interphase and mitosis. Furthermore, a positive feedback loop allows cyclin B-cdc2 kinase to activate Plx1 at the G2/M-phase transition. In contrast, activation of cyclin A-cdc2 kinase does not require Plx1 activity, and cyclin A-cdc2 kinase fails to activate Plx1 and its consequence, cdc25c activation in cycling extracts.

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