scholarly journals Study of Cyclin Proteolysis in Anaphase-Promoting Complex (APC) Mutant Cells Reveals the Requirement for APC Function in the Final Steps of the Fission Yeast Septation Initiation Network

2001 ◽  
Vol 21 (19) ◽  
pp. 6681-6694 ◽  
Author(s):  
Louise Chang ◽  
Jennifer L. Morrell ◽  
Anna Feoktistova ◽  
Kathleen L. Gould
Keyword(s):  
Kinase Activity ◽  
Nuclear Division ◽  
Cyclin B ◽  
Cyclin Dependent Kinase ◽  
Anaphase Promoting Complex ◽  
Wild Type ◽  
Mitotic Cyclin ◽  
Septation Initiation Network ◽  
Mutant Cells ◽  
Null Mutants

ABSTRACT Cytokinesis in eukaryotic cells requires the inactivation of mitotic cyclin-dependent kinase complexes. An apparent exception to this relationship is found in Schizosaccharomyces pombemutants with mutations of the anaphase-promoting complex (APC). These conditional lethal mutants arrest with unsegregated chromosomes because they cannot degrade the securin, Cut2p. Although failing at nuclear division, these mutants septate and divide. Since septation requires Cdc2p inactivation in wild-type S. pombe, it has been suggested that Cdc2p inactivation occurs in these mutants by a mechanism independent of cyclin degradation. In contrast to this prediction, we show that Cdc2p kinase activity fluctuates in APCcut mutants due to Cdc13/cyclin B destruction. In APC-null mutants, however, septation and cutting do not occur and Cdc13p is stable. We conclude that APC cut mutants are hypomorphic with respect to Cdc13p degradation. Indeed, overproduction of nondestructible Cdc13p prevents septation in APC cutmutants and the normal reorganization of septation initiation network components during anaphase.

BIMAAPC3, a component of the Aspergillus anaphase promoting complex/cyclosome, is required for a G2 checkpoint blocking entry into mitosis in the absence of NIMA function

1998 ◽  
Vol 111 (10) ◽  
pp. 1453-1465 ◽  
Author(s):  
C.M. Lies ◽  
J. Cheng ◽  
S.W. James ◽  
N.R. Morris ◽  
M.J. O'Connell ◽  
...  
Keyword(s):  
Kinase Activity ◽  
Cyclin B ◽  
Temperature Sensitive ◽  
Anaphase Promoting Complex ◽  
Unique Point ◽  
Wild Type ◽  
G2 Arrest ◽  
G2 Checkpoint ◽  
Steady State Levels ◽  
Decondensed Chromatin

Temperature sensitive (ts) nimA mutants of Aspergillus nidulans arrest at a unique point in G2 which is post activation of CDC2. Here we show that this G2 arrest is due to loss of nimA function and that it is dependent on BIMAAPC3, a component of the anaphase promoting complex/cyclosome (APC/C). Whereas nimA single mutants arrested in G2 with decondensed chromatin and interphase microtubule arrays, nimA, bimAAPC3 double mutants arrested growth with condensed chromatin and aster-like microtubule arrays. nimA, bimAAPC3 double mutants entered mitosis with kinetics similar to bimAAPC3 single mutants and wild-type cells, indicating a checkpoint-like role for BIMAAPC3 in G2. Even cells which had been depleted for NIMA protein and which contained insignificant levels of NIMA kinase activity entered mitosis on inactivation of bimAAPC3. BIMAAPC3 was present in a >25S complex containing BIMEAPC1, and bimAAPC3 mutants were sensitive to elevated CYCLIN B expression, consistent with BIMAAPC3 being a component of the APC/C. Inactivation of bimAAPC3 had little affect on the steady state levels of the B-type cyclin, NIMECyclin B. Our results indicate that BIMAAPC3, and most likely the APC/C itself, is activated in G2 in nimA mutants. We propose that APC/C activation is part of a novel, late G2 checkpoint, which responds to a defective process or structure in nimA mutants, and which prevents inappropriate entry into mitosis.

scholarly journals A Late Mitotic Regulatory Network Controlling Cyclin Destruction in Saccharomyces cerevisiae

1998 ◽  
Vol 9 (10) ◽  
pp. 2803-2817 ◽  
Author(s):  
Sue L. Jaspersen ◽  
Julia F. Charles ◽  
Rachel L. Tinker-Kulberg ◽  
David O. Morgan
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Regulatory Network ◽  
Growth Defect ◽  
Cyclin Dependent Kinase ◽  
Anaphase Promoting Complex ◽  
Yeast Saccharomyces Cerevisiae ◽  
Mitotic Cyclin ◽  
Low Levels ◽  
Late Mitosis ◽  
Mutant Cells

Exit from mitosis requires the inactivation of mitotic cyclin-dependent kinase–cyclin complexes, primarily by ubiquitin-dependent cyclin proteolysis. Cyclin destruction is regulated by a ubiquitin ligase known as the anaphase-promoting complex (APC). In the budding yeast Saccharomyces cerevisiae, members of a large class of late mitotic mutants, including cdc15,cdc5, cdc14, dbf2, andtem1, arrest in anaphase with a phenotype similar to that of cells expressing nondegradable forms of mitotic cyclins. We addressed the possibility that the products of these genes are components of a regulatory network that governs cyclin proteolysis. We identified a complex array of genetic interactions among these mutants and found that the growth defect in most of the mutants is suppressed by overexpression of SPO12, YAK1, andSIC1 and is exacerbated by overproduction of the mitotic cyclin Clb2. When arrested in late mitosis, the mutants exhibit a defect in cyclin-specific APC activity that is accompanied by high Clb2 levels and low levels of the anaphase inhibitor Pds1. Mutant cells arrested in G1 contain normal APC activity. We conclude that Cdc15, Cdc5, Cdc14, Dbf2, and Tem1 cooperate in the activation of the APC in late mitosis but are not required for maintenance of that activity in G1.

scholarly journals Mechanisms of p34cdc2 regulation.

1993 ◽  
Vol 13 (3) ◽  
pp. 1675-1685 ◽  
Author(s):  
S Atherton-Fessler ◽  
L L Parker ◽  
R L Geahlen ◽  
H Piwnica-Worms
Keyword(s):  
Active Site ◽  
Kinase Activity ◽  
Nucleotide Binding ◽  
Binding Domain ◽  
Cyclin B ◽  
Wild Type ◽  
Nucleotide Binding Domain ◽  
Maximal Inhibition

The kinase activity of human p34cdc2 is negatively regulated by phosphorylation at Thr-14 and Tyr-15. These residues lie within the putative nucleotide binding domain of p34cdc2. It has been proposed that phosphorylation within this motif ablates the binding of ATP to the active site of p34cdc2, thereby inhibiting p34cdc2 kinase activity (K. Gould and P. Nurse, Nature [London] 342:39-44, 1989). To understand the mechanism of this inactivation, various forms of p34cdc2 were tested for the ability to bind nucleotide. The active site of p34cdc2 was specifically modified by the MgATP analog 5'-p-fluorosulfonylbenzoyladenosine (FSBA). The apparent Km for modification of wild-type, monomeric p34cdc2 was 148 microM FSBA and was not significantly affected by association with cyclin B. Tyrosine-phosphorylated p34cdc2 was modified by FSBA with a slightly higher Km (241 microM FSBA). FSBA modification of both tyrosine-phosphorylated and unphosphorylated p34cdc2 was competitively inhibited by ATP, and half-maximal inhibition in each case occurred at approximately 250 microM ATP. In addition to being negatively regulated by phosphorylation, the kinase activity of p34cdc2 was positively regulated by the cyclin-dependent phosphorylation of Thr-161. Mutation of p34cdc2 at Thr-161 resulted in the formation of an enzymatically inactive p34cdc2/cyclin B complex both in vivo and in vitro. However, mutation of Thr-161 did not significantly affect the ability of p34cdc2 to bind nucleotide (FSBA). Taken together, these results indicate that inhibition of p34cdc2 kinase activity by phosphorylation of Tyr-15 (within the putative ATP binding domain) or by mutation of Thr-161 involves a mechanism other than inhibition of nucleotide binding. We propose instead that the defect resides at the level of catalysis.

scholarly journals Initiation of Cytokinesis Is Controlled through Multiple Modes of Regulation of the Sid2p-Mob1p Kinase Complex

2004 ◽  
Vol 24 (8) ◽  
pp. 3262-3276 ◽  
Author(s):  
Ming-Chin Hou ◽  
David A. Guertin ◽  
Dannel McCollum
Keyword(s):  
Kinase Activity ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Related Protein ◽  
Multiple Modes ◽  
Growth Defects ◽  
Similar Region ◽  
Septation Initiation Network ◽  
Self Association ◽  
Mutant Cells

ABSTRACT The Sid2p-Mob1p kinase complex is an important component of the septation initiation network (SIN) in the fission yeast Schizosaccharomyces pombe. However, regulation of this complex is still elusive. Here we show that Mob1p is required not only for the subcellular localization of Sid2p but also for its kinase activity. We identified a region at the amino terminus of Sid2p that is required for Mob1p binding and spindle pole body (SPB) localization. Deletion of this region abolishes Mob1p binding and diminishes SPB localization, whereas this region alone is sufficient to associate with Mob1p and SPBs. We further show that a similar region of the N terminus of the Sid2p-related protein kinase Orb6p binds to the Mob1p-related protein Mob2p, suggesting that this may be a conserved mode of interaction for this family of kinases. Phosphorylation of Ser402 and especially Thr578 is important for Sid2p function. Sid2p with a mutation of Thr578 to Ala (T578A) can no longer rescue sid2-250 mutant cells, and this results in reduction of Mob1p binding. Sid2p mutants mimicking phosphorylation at this site (T578D and T578E) can rescue sid2-250 cells, enhance Sid2p kinase activity, and partially rescue growth defects of upstream sin mutants. Interestingly, Sid2p, but not Mob1p, is self-associated. Our experiments suggest that self-associated Sid2p is inactive. This self-association is mediated by a region that overlaps with Mob1p and SPB binding sites. Overexpression of Mob1p is able to disrupt the self-association of Sid2p. Taken together, our results suggest that Sid2p kinase may utilize multiple modes of regulation including self-association, Mob1p binding, and phosphorylation to achieve its full activity at an appropriate time and place in the cell.

Mechanisms of p34cdc2 regulation

1993 ◽  
Vol 13 (3) ◽  
pp. 1675-1685
Author(s):  
S Atherton-Fessler ◽  
L L Parker ◽  
R L Geahlen ◽  
H Piwnica-Worms
Keyword(s):  
Active Site ◽  
Kinase Activity ◽  
Nucleotide Binding ◽  
Binding Domain ◽  
Cyclin B ◽  
Wild Type ◽  
Nucleotide Binding Domain ◽  
Maximal Inhibition

The kinase activity of human p34cdc2 is negatively regulated by phosphorylation at Thr-14 and Tyr-15. These residues lie within the putative nucleotide binding domain of p34cdc2. It has been proposed that phosphorylation within this motif ablates the binding of ATP to the active site of p34cdc2, thereby inhibiting p34cdc2 kinase activity (K. Gould and P. Nurse, Nature [London] 342:39-44, 1989). To understand the mechanism of this inactivation, various forms of p34cdc2 were tested for the ability to bind nucleotide. The active site of p34cdc2 was specifically modified by the MgATP analog 5'-p-fluorosulfonylbenzoyladenosine (FSBA). The apparent Km for modification of wild-type, monomeric p34cdc2 was 148 microM FSBA and was not significantly affected by association with cyclin B. Tyrosine-phosphorylated p34cdc2 was modified by FSBA with a slightly higher Km (241 microM FSBA). FSBA modification of both tyrosine-phosphorylated and unphosphorylated p34cdc2 was competitively inhibited by ATP, and half-maximal inhibition in each case occurred at approximately 250 microM ATP. In addition to being negatively regulated by phosphorylation, the kinase activity of p34cdc2 was positively regulated by the cyclin-dependent phosphorylation of Thr-161. Mutation of p34cdc2 at Thr-161 resulted in the formation of an enzymatically inactive p34cdc2/cyclin B complex both in vivo and in vitro. However, mutation of Thr-161 did not significantly affect the ability of p34cdc2 to bind nucleotide (FSBA). Taken together, these results indicate that inhibition of p34cdc2 kinase activity by phosphorylation of Tyr-15 (within the putative ATP binding domain) or by mutation of Thr-161 involves a mechanism other than inhibition of nucleotide binding. We propose instead that the defect resides at the level of catalysis.

scholarly journals p18Ink4c, but Not p27Kip1, Collaborates with Men1 To Suppress Neuroendocrine Organ Tumors

2006 ◽  
Vol 27 (4) ◽  
pp. 1495-1504 ◽  
Author(s):  
Feng Bai ◽  
Xin-Hai Pei ◽  
Toru Nishikawa ◽  
Matthew D. Smith ◽  
Yue Xiong
Keyword(s):  
Multiple Endocrine Neoplasia ◽  
Double Mutant ◽  
Kinase Activity ◽  
Suppressor Gene ◽  
Mutant Mice ◽  
Cdk Inhibitors ◽  
Cyclin Dependent Kinase ◽  
Wild Type ◽  
Neuroendocrine Organ ◽  
Stimulation Of

ABSTRACT Mutant mice lacking both cyclin-dependent kinase (CDK) inhibitors p18 Ink4c and p27 Kip1 develop a tumor spectrum reminiscent of human multiple endocrine neoplasia (MEN) syndromes. To determine how p18 and p27 genetically interact with Men1, the tumor suppressor gene mutated in familial MEN1, we characterized p18-Men1 and p27-Men1 double mutant mice. Compared with their corresponding single mutant littermates, the p18 − / −; Men1 +/ − mice develop tumors at an accelerated rate and with an increased incidence in the pituitary, thyroid, parathyroid, and pancreas. In the pituitary and pancreatic islets, phosphorylation of the retinoblastoma (Rb) protein at both CDK2 and CDK4/6 sites was increased in p18 − / − and Men1 +/ − cells and was further increased in p18 − / −; Men1 +/ − cells. The remaining wild-type Men1 allele was lost in most tumors from Men1 +/ − mice but was retained in most tumors from p18 − / −; Men1 +/ − mice. Combined mutations of p27 − / − and Men1 +/ −, in contrast, did not exhibit noticeable synergistic stimulation of Rb kinase activity, cell proliferation, and tumor growth. These results demonstrate that functional collaboration exists between p18 and Men1 and suggest that menin may regulate additional factor(s) that interact with p18 and p27 differently.

scholarly journals Cyclin B Proteolysis and the Cyclin-dependent Kinase Inhibitor rum1p Are Required for Pheromone-induced G1 Arrest in Fission Yeast

1998 ◽  
Vol 9 (6) ◽  
pp. 1309-1321 ◽  
Author(s):  
Bodo Stern ◽  
Paul Nurse
Keyword(s):  
Fission Yeast ◽  
Kinase Activity ◽  
Kinase Inhibitor ◽  
Cyclin B ◽  
G1 Arrest ◽  
Cyclin Dependent Kinase ◽  
Down Regulation ◽  
Cyclin Dependent Kinase Inhibitor ◽  
Pheromone Signaling

The blocking of G1 progression by fission yeast pheromones requires inhibition of the cyclin-dependent kinase cdc2p associated with the B-cyclins cdc13p and cig2p. We show that cyclosome-mediated degradation of cdc13p and cig2p is necessary for down-regulation of B-cyclin–associated cdc2p kinase activity and for phermone-induced G1 arrest. The cyclin-dependent kinase inhibitor rum1p is also required to maintain this G1arrest; it binds both cdc13p and cig2p and is specifically required for cdc13p proteolysis. We propose that rum1p acts as an adaptor targeting cdc13p for degradation by the cyclosome. In contrast, the cig2p–cdc2p kinase can be down-regulated, and the cyclin cig2p can be proteolyzed independently of rum1p. We suggest that pheromone signaling inhibits the cig2p–cdc2p kinase, bringing about a transient G1arrest. As a consequence, rum1p levels increase, thus inhibiting and inducing proteolysis of the cdc13p–cdc2p kinase; this is necessary to maintain G1 arrest. We have also shown that pheromone-induced transcription occurs only in G1 and is independent of rum1p.

scholarly journals The Drosophila Ortholog of MLL3 and MLL4 , trithorax related , Functions as a Negative Regulator of Tissue Growth

2013 ◽  
Vol 33 (9) ◽  
pp. 1702-1710 ◽  
Author(s):  
Hiroshi Kanda ◽  
Alexander Nguyen ◽  
Leslie Chen ◽  
Hideyuki Okano ◽  
Iswar K. Hariharan
Keyword(s):  
Histone H3 ◽  
Tissue Growth ◽  
Negative Regulator ◽  
Cyclin B ◽  
Wild Type ◽  
H3k4 Methylation ◽  
Content Type ◽  
Promote Cell Survival ◽  
Low Levels ◽  
Mutant Cells

The human MLL genes ( MLL1 to MLL4 ) and their Drosophila orthologs, trithorax ( trx ) and trithorax related ( trr ), encode proteins capable of methylating histone H3 on lysine 4. MLL1 and MLL2 are most similar to trx , while MLL3 and MLL4 are more closely related to trr . Several MLL genes are mutated in human cancers, but how these proteins regulate cell proliferation is not known. Here we show that trr mutant cells have a growth advantage over their wild-type neighbors and display changes in the levels of multiple proteins that regulate growth and cell division, including Notch, Capicua, and cyclin B. trr mutant clones display markedly reduced levels of H3K4 monomethylation without obvious changes in the levels of H3K4 di- and trimethylation. The trr mutant phenotype resembles that of Utx , which encodes a H3K27 demethylase, consistent with the observation that Trr and Utx are found in the same protein complex. In contrast to the overgrowth displayed by trr mutant tissue, trx clones are underrepresented, express low levels of the antiapoptotic protein Diap1, and exhibit only modest changes in global levels of H3K4 methylation. Thus, in Drosophila eye imaginal discs, Trr, likely functioning together with Utx, restricts tissue growth. In contrast, Trx appears to promote cell survival.

scholarly journals Cdc2 and Mos Regulate Emi2 Stability to Promote the Meiosis I–Meiosis II Transition

2008 ◽  
Vol 19 (8) ◽  
pp. 3536-3543 ◽  
Author(s):  
Wanli Tang ◽  
Judy Qiju Wu ◽  
Yanxiang Guo ◽  
David V. Hansen ◽  
Jennifer A. Perry ◽  
...  
Keyword(s):  
Kinase Activity ◽  
Cyclin B ◽  
Anaphase Promoting Complex ◽  
Cdc2 Kinase ◽  
Dependent Protein Kinase ◽  
Dependent Protein ◽  
Cytostatic Factor ◽  
Cdc2 Activity ◽  
Phase Entry ◽  
Meiosis I

The transition of oocytes from meiosis I (MI) to meiosis II (MII) requires partial cyclin B degradation to allow MI exit without S phase entry. Rapid reaccumulation of cyclin B allows direct progression into MII, producing a cytostatic factor (CSF)-arrested egg. It has been reported that dampened translation of the anaphase-promoting complex (APC) inhibitor Emi2 at MI allows partial APC activation and MI exit. We have detected active Emi2 translation at MI and show that Emi2 levels in MI are mainly controlled by regulated degradation. Emi2 degradation in MI depends not on Ca2+/calmodulin-dependent protein kinase II (CaMKII), but on Cdc2-mediated phosphorylation of multiple sites within Emi2. As in MII, this phosphorylation is antagonized by Mos-mediated recruitment of PP2A to Emi2. Higher Cdc2 kinase activity in MI than MII allows sufficient Emi2 phosphorylation to destabilize Emi2 in MI. At MI anaphase, APC-mediated degradation of cyclin B decreases Cdc2 activity, enabling Cdc2-mediated Emi2 phosphorylation to be successfully antagonized by Mos-mediated PP2A recruitment. These data suggest a model of APC autoinhibition mediated by stabilization of Emi2; Emi2 proteins accumulate at MI exit and inhibit APC activity sufficiently to prevent complete degradation of cyclin B, allowing MI exit while preventing interphase before MII entry.

scholarly journals The Anaphase-Promoting Complex/Cyclosome Is Required for Anaphase Progression in Multinucleated Ashbya gossypii Cells

2006 ◽  
Vol 6 (2) ◽  
pp. 182-197 ◽  
Author(s):  
Amy S. Gladfelter ◽  
Nicoleta Sustreanu ◽  
A. Katrin Hungerbuehler ◽  
Sylvia Voegeli ◽  
Virginie Galati ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Nuclear Division ◽  
Eukaryotic Cell ◽  
Ashbya Gossypii ◽  
Anaphase Promoting Complex ◽  
Cycle Progression ◽  
Filamentous Ascomycete ◽  
Multinucleated Cells ◽  
Mitotic Cyclin

ABSTRACT Regulated protein degradation is essential for eukaryotic cell cycle progression. The anaphase-promoting complex/cyclosome (APC/C) is responsible for the protein destruction required for the initiation of anaphase and the exit from mitosis, including the degradation of securin and B-type cyclins. We initiated a study of the APC/C in the multinucleated, filamentous ascomycete Ashbya gossypii to understand the mechanisms underlying the asynchronous mitosis observed in these cells. These experiments were motivated by previous work which demonstrated that the mitotic cyclin AgClb1/2p persists through anaphase, suggesting that the APC/C may not be required for the division cycle in A. gossypii. We have now found that the predicted APC/C components AgCdc23p and AgDoc1p and the targeting factors AgCdc20p and AgCdh1p are essential for growth and nuclear division. Mutants lacking any of these factors arrest as germlings with nuclei blocked in mitosis. A likely substrate of the APC/C is the securin homologue AgPds1p, which is present in all nuclei in hyphae except those in anaphase. The destruction box sequence of AgPds1p is required for this timed disappearance. To investigate how the APC/C may function to degrade AgPds1p in only the subset of anaphase nuclei, we localized components and targeting subunits of the APC/C. Remarkably, AgCdc23p, AgDoc1p, and AgCdc16p were found in all nuclei in all cell cycle stages, as were the APC/C targeting factors AgCdc20p and AgCdh1p. These data suggest that the AgAPC/C may be constitutively active across the cell cycle and that proteolysis in these multinucleated cells may be regulated at the level of substrates rather than by the APC/C itself.

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