Structural basis for the ORC1‐Cyclin A association

Progression of cell cycle is regulated by sequential expression of cyclins, which associate with distinct cyclin kinases to drive the transition between different cell cycle phases. The complex of Cyclin A with cyclin‐dependent kinase 2 (CDK2) controls the DNA replication activity through phosphorylation of a set of chromatin factors, which critically influences the S phase transition. It has been shown that the direct interaction between the Cyclin A‐CDK2 complex and origin recognition complex subunit 1 (ORC1) mediates the localization of ORC1 to centrosomes, where ORC1 inhibits cyclin E‐mediated centrosome reduplication. However, the molecular basis underlying the specific recognition between ORC1 and cyclins remains elusive. Here we report the crystal structure of Cyclin A‐CDK2 complex bound to a peptide derived from ORC1 at 2.54 å resolution. The structure revealed that the ORC1 peptide interacts with a hydrophobic groove, termed cyclin binding groove (CBG), of Cyclin A via a KXL motif. Distinct from other identified CBG‐binding sequences, an arginine residue flanking the KXL motif of ORC1 inserts into a neighboring acidic pocket, contributing to the strong ORC1‐Cyclin A association. Furthermore, structural and sequence analysis of cyclins reveals divergence on the ORC1‐binding sites, which may underpin their differential ORC1‐binding activities. This study provides a structural basis of the specific ORC1‐cyclins recognition, with implication in development of novel inhibitors against the cyclin/CDK complexes.     

Cyclin E2, the cycle continues

The eukaryotic cell cycle is regulated by a family of serine/threonine protein kinases known as cyclin-dependent kinases (CDKs). The activation of a CDK is dependent on its association with a cyclin regulatory subunit. The formation of distinct cyclin-CDK complexes controls the progression through the first gap phase (G(1)) and initiation of DNA synthesis (S phase). These complexes are in turn regulated by protein phosphorylation and cyclin-dependent kinase inhibitors (CKIs). Cyclin E2 has emerged as the second member of the E-type cyclin family. Cyclin E2-associated kinase activity is regulated in a cell cycle dependent manner with peak activity at the G(1) to S transition. Ectopic expression of cyclin E2 in human cells accelerates G(1), suggesting that cyclin E2 is rate limiting for G(1) progression. Although the pattern and level of cyclin E2 expression in some primary tumor and normal tissue RNAs are distinct from cyclin E1, both E-type cyclins appear to have inherent functional redundancies. This functional redundancy has facilitated the rapid characterization of cyclin E2 and uncovered unique features associated with each E-type cyclin.     

Integrin Signaling at the M/G1 Transition Induces Expression of Cyclin E

The activities of the mammalian G1 cyclins, cyclin D and cyclin E, during cell cycle progression (G1/S) are believed to be regulated by cell attachment and the presence of growth factors. In order to study the importance of cell attachment and concomitant integrin signaling on the expression of G1 cyclins during the natural adhesion process from mitosis to interphase, protein expression was monitored in cells that were synchronized by mitotic shake off. Here we show that in Chinese hamster ovary (CHO) and neuroblastoma (N2A) cells, expression of cyclin E at the M/G1 transition is regulated by both growth factors and cell attachment, while expression of cyclin D seems to be entirely dependent on the presence of serum. Expression of cyclin E appears to be correlated with the phosphorylation of the retinoblastoma protein, suggesting a link with the activity of the cyclin D/cdk4 complex. Expression of the cdk inhibitors p21^cip1/Waf1 and p27^Kip1 is not changed upon serum depletion or detachment of cells during early G1, suggesting no direct role for these CKIs in the regulation of cyclin activity. Although inhibition of cyclin E/cdk2 kinase activity has been reported previously, this is the first time that cyclin E expression is shown to be dependent on cell attachment.     

Increased G1 cyclin/cdk activity in cells overexpressing the candidate oncogene, MCT-1.

We have recently identified a novel candidate oncogene, MCT-1, in the HUT 78 T-cell line. When overexpressed in NIH3T3 fibroblasts, the MCT-1 gene shortens the G1 phase of the cell cycle and promotes anchorage-independent growth. Progression of cells through a late G1 phase restriction point is regulated by G1 cyclins whose phosphorylation of the retinoblastoma gene product facilitates entry into S phase. Deregulated expression of G1 cyclins and their cognate cdk partners is often found in human tumor cells. In order to address the potential relationship of MCT-1 to cell cycle regulatory molecules, we analyzed the ability of MCT-1 overexpression to modulate cdk4 and cdk6 kinase activity in NIH3T3 fibroblasts constitutively overexpressing MCT-1. We observed an increase in the kinase activity of both cdk4 and cdk6 in asynchronously growing transformed cells compared with the parent cells. This increased kinase activity was accompanied by an elevated level of cyclin D1 protein and increased G1 cyclin/cdk complex formation. We also observed a correlation between increased protein levels of MCT-1 with cyclin D1 expression in a panel of lymphoid cell lines derived from T-cell malignancies. These results demonstrate that constitutive expression of MCT-1 is associated with deregulation of protein kinase-mediated G1 phase checkpoints.     

Inhibition of poly(A) polymerase requires p34cdc2/cyclin B phosphorylation of multiple consensus and non-consensus sites.

We showed previously that p34(cdc2)/cyclin B (MPF) hyperphosphorylates poly(A) polymerase (PAP) during M-phase of the cell cycle, causing repression of its enzymatic activity. Mutation of three cyclin-dependent kinase (cdk) consensus sites in the PAP C-terminal regulatory domain prevented complete phosphorylation and MPF-mediated repression. Here we show that PAP also contains four nearby non-consensus cdk sites that are phosphorylated by MPF. Remarkably, full phosphorylation of all these cdk sites was required for repression of PAP activity, and partial phosphorylation had no detectable effect. The consensus sites were phosphorylated in vitro at a 10-fold lower concentration of MPF than the non-consensus sites. Consistent with this, during meiotic maturation of Xenopus oocytes, consensus sites were phosphorylated prior to the non-consensus sites at metaphase of meiosis I, and remained so throughout maturation, while the non-consensus sites did not become fully phosphorylated until after 12 h of metaphase II arrest. We propose that PAP's multiple cdk sites, and their differential sensitivity to MPF, provide a mechanism to link repression specifically to late M-phase. We discuss the possibility that this reflects a general means to control the timing of cdk-dependent regulatory events during the cell cycle.     

The Drosophila mitotic inhibitor Frühstart specifically binds to the hydrophobic patch of cyclins

The hydrophobic patch of cyclins interacts with cyclin-dependent kinase (Cdk) substrates and p27-type Cdk inhibitors. Although this interaction is assumed to contribute to the specificity of different Cdk-Cyclin complexes, its role in specific steps of the cell cycle has not been demonstrated. Here, we show that in Drosophila the mitotic inhibitor Frühstart (Frs) binds specifically and with high affinity to the hydrophobic patch of cyclins. In contrast to p27-type Cdk inhibitors, Frs does not form a stable interaction with the catalytic centre of Cdk and allows phosphorylation of generic model substrates, such as histone H1. Consistent with a 2.5 times stronger binding to CycA than to CycE in vitro, ectopic expression of frs induces endocycles, in a manner similar to that reported previously for downregulation of CycA or Cdk1. We propose that binding of Frs to cyclins blocks the hydrophobic patch to interfere with Cdk1 substrate recognition.     

APC(ste9/srw1) promotes degradation of mitotic cyclins in G(1) and is inhibited by cdc2 phosphorylation

Fission yeast ste9/srw1 is a WD-repeat protein highly homologous to budding yeast Hct1/Cdh1 and DROSOPHILA: Fizzy-related that are involved in activating APC/C (anaphase-promoting complex/cyclosome). We show that APC(ste9/srw1) specifically promotes the degradation of mitotic cyclins cdc13 and cig1 but not the S-phase cyclin cig2. APC(ste9/srw1) is not necessary for the proteolysis of cdc13 and cig1 that occurs at the metaphase-anaphase transition but it is absolutely required for their degradation in G(1). Therefore, we propose that the main role of APC(ste9/srw1) is to promote degradation of mitotic cyclins when cells need to delay or arrest the cell cycle in G(1). We also show that ste9/srw1 is negatively regulated by cdc2-dependent protein phosphorylation. In G(1), when cdc2-cyclin kinase activity is low, unphosphorylated ste9/srw1 interacts with APC/C. In the rest of the cell cycle, phosphorylation of ste9/srw1 by cdc2-cyclin complexes both triggers proteolysis of ste9/srw1 and causes its dissociation from the APC/C. This mechanism provides a molecular switch to prevent inactivation of cdc2 in G(2) and early mitosis and to allow its inactivation in G(1).     

Cyclin D3 expression in melanoma cells is regulated by adhesion-dependent phosphatidylinositol 3-kinase signaling and contributes to G1-S progression

D-type cyclins regulate G1 cell cycle progression by enhancing the activities of cyclin-dependent kinases (CDKs), and their expression is frequently altered in malignant cells. We and others have previously shown that cyclin D1 is up-regulated in melanoma cells through adhesion-independent MEK-ERK1/2 signaling initiated by mutant B-RAF. Here, we describe the regulation and role of cyclin D3 in human melanoma cells. Cyclin D3 expression was enhanced in a cell panel of human melanoma cell lines compared with melanocytes and was regulated by fibronectin-mediated phosphatidylinositol 3-kinase/Akt signaling but not MEK activity. RNA interference experiments demonstrated that cyclin D3 contributed to G1-S cell cycle progression and proliferation in melanoma cells. Overexpression of cyclin D1 did not recover the effects of cyclin D3 knockdown. Finally, immunoprecipitation studies showed that CDK6 is a major binding partner for cyclin D3, whereas CDK4 preferentially associated with cyclin D1. Together, these findings demonstrate that cyclin D3 is an important regulator of melanoma G1-S cell cycle progression and that D-type cyclins are differentially regulated in melanoma cells.     

Sub-cellular localisation of GFP-tagged tobacco mitotic cyclins during the cell cycle and after spindle checkpoint activation

We have previously shown that the tobacco cyclin B1;1 protein accumulates during the G2 phase of the cell cycle and is subsequently destroyed during mitosis. Here, we investigated the sub-cellular localisation of two different B1-types and one A3-type cyclin during the cell cycle by using confocal imaging and differential interference contrast (DIC) microscopy. The cyclins were visualised as GFP-tagged fusion proteins in living tobacco cells. Both B1-type cyclins were found in the cytoplasm and in the nucleus during G2 but when cells entered into prophase, both cyclins became associated with condensing chromatin and remained on chromosomes until metaphase. As cells exited metaphase, the B1-type cyclins became degraded, as shown by time-lapse images. A stable variant of cyclin B1;1-GFP fusion protein, in which the destruction box had been mutated, maintained its association with the nuclear material at later phases of mitosis such as anaphase and telophase. Furthermore, we demonstrated that cyclin B1;1 protein is stabilised in metaphase-arrested cells after microtubule destabilising drug treatments. In contrast to the B1-type cyclins, the cyclin A3;1 was found exclusively in the nucleus in interphase cells and disappeared earlier than the cyclin B1 proteins during mitosis.     

Epidermal growth factor-induced rapid retinoblastoma phosphorylation at Ser780 and Ser795 is mediated by ERK1/2 in small intestine epithelial cells

The retinoblastoma protein Rb is critical for the regulation of mammalian cell cycle entry. Hypophosphorylated Rb is considered to be the active form and directs G1 arrest, while hyperphosphorylated Rb permits the transition from G1 to S phase for cell proliferation. Upon stimulation by various growth factors, Rb appears to be phosphorylated by a cascade of phosphorylation events mediated mainly by kinases associated with cyclins D and E. Here we report that in prototype small intestine crypt stem cells (RIEC-6), stimulation with either epidermal growth factor or fetal bovine serum results in an unexpected rapid and sustained Rb phosphorylation at sites Ser780, Ser795, and Thr821 which precedes cyclin D1 expression, cyclin D1/cdk4 complex formation, and cdk4 kinase activity. Rb phosphorylation at Ser780 and Ser795 is prevented by MEK, but not phosphatidylinositol 3-kinase, inhibitors. In vitro, Rb is directly phosphorylated by active ERK1/2 as shown by [gamma-32P]ATP labeling. The phosphorylation sites are further directed to Ser780 and Ser795 by kinase assays using recombined active ERK1/2 or immunoprecipitated phospho-ERK1/2 from mitogen stimulated cells. Pull-down assays revealed that Rb interacts with active ERK1/2 but not their inactive unphosphorylated forms. Upon EGF stimulation, phosphorylated ERK1/2 co-immunoprecipitates together with phosphorylated Rb. Collectively, these results demonstrate a novel rapid Rb phosphorylation at specific sites induced by mitogen stimulation in epithelial cells of the small intestine. These data specifically identify ERK1/2 as the kinase responsible for Rb phosphorylation targeted to sites Ser780 and Ser795. It appears that ERK1/2 could be an important link between a mitogenic signal directly to Rb, thereby providing a rapid response mechanism between mitogen stimulation and cell cycle machinery.     

Activity of the APC(Cdh1) form of the anaphase-promoting complex persists until S phase and prevents the premature expression of Cdc20p.

Cell cycle progression is driven by waves of cyclin expression coupled with regulated protein degradation. An essential step for initiating mitosis is the inactivation of proteolysis mediated by the anaphase-promoting complex/cyclosome (APC/C) bound to its regulator Cdh1p/Hct1p. Yeast APC(Cdh1) was proposed previously to be inactivated at Start by G1 cyclin/cyclin-dependent kinase (CDK). Here, we demonstrate that in a normal cell cycle APC(Cdh1) is inactivated in a graded manner and is not extinguished until S phase. Complete inactivation of APC(Cdh1) requires S phase cyclins. Further, persistent APC(Cdh1) activity throughout G1 helps to ensure the proper timing of Cdc20p expression. This suggests that S phase cyclins have an important role in allowing the accumulation of mitotic cyclins and further suggests a regulatory loop among S phase cyclins, APC(Cdh1), and APC(Cdc20).     

Differential regulation of cyclin D1 and D3 expression in the control of astrocyte proliferation induced by endothelin-1

We have previously shown that the mitogenic effect of endothelin-1 (ET-1) in primary astrocytes is dependent on activation of both extracellular signal-regulated kinase (ERK)- and cytoskeleton (CSK)-dependent pathways. In this study, we evaluated the contribution of each of these pathways to the expression and activation of proteins mediating cell cycle progression. Our results suggest that ET-1-induced expression of cyclins D1 and D3 is dependent on the ERK- and CSK-dependent pathways, respectively; moreover, a decrease in the levels of the cyclin-dependent kinase inhibitor (CKI) p27 was observed as a consequence of ERK activation. Expression of both cyclins D1 and D3 together with a decrease in the p27 levels are essential for retinoblastoma protein (pRB) phosphorylation and cyclin A expression. Furthermore, the molecular events responsible for cell-cell contact inhibition of astrocyte proliferation were found to be independent of the mitogenic pathways leading to D-type cyclin expression. Cell growth arrest in confluent astrocytes was found to be correlated with increased expression of CKI p21, resulting in inhibition of D-type cyclin-associated pRB phosphorylation and cyclin A expression. Taken together, these results indicate that cyclins D1 and D3, which constitute the key mediators of the proliferative response of primary astrocytes to ET-1, are regulated by distinct signaling pathways.     

Phosphorylation independent activation of human cyclin-dependent kinase 2 by cyclin A in vitro.

p33cdk2 is a serine-threonine protein kinase that associates with cyclins A, D, and E and has been implicated in the control of the G1/S transition in mammalian cells. Recent evidence indicates that cyclin-dependent kinase 2 (Cdk2), like its homolog Cdc2, requires cyclin binding and phosphorylation (of threonine-160) for activation in vivo. However, the extent to which mechanistic details of the activation process are conserved between Cdc2 and Cdk2 is unknown. We have developed bacterial expression and purification systems for Cdk2 and cyclin A that allow mechanistic studies of the activation process to be performed in the absence of cell extracts. Recombinant Cdk2 is essentially inactive as a histone H1 kinase (< 4 x 10(-5) pmol phosphate transferred.min-1 x microgram-1 Cdk2). However, in the presence of equimolar cyclin A, the specific activity is approximately 16 pmol.mon-1 x microgram-1, 4 x 10(5)-fold higher than Cdk2 alone. Mutation of T160 in Cdk2 to either alanine or glutamic acid had little impact on the specific activity of the Cdk2/cyclin A complex: the activity of Cdk2T160E was indistinguishable from Cdk2, whereas that of Cdk2T160A was reduced by five-fold. To determine if the Cdk2/cyclin A complex could be activated further by phosphorylation of T160, complexes were treated with Cdc2 activating kinase (CAK), purified approximately 12,000-fold from Xenopus eggs. This treatment resulted in an 80-fold increase in specific activity. This specific activity is comparable with that of the Cdc2/cyclin B complex after complete activation by CAK (approximately 1600 pmol.mon-1 x microgram-1). Neither Cdk2T160A/cyclin A nor Cdk2T160E/cyclin A complexes were activated further by treatment with CAK. In striking contrast with cyclin A, cyclin B did not directly activate Cdk2. However, both Cdk2/cyclin A and Cdk2/cyclin B complexes display similar activity after activation by CAK. For the Cdk2/cyclin A complex, both cyclin binding and phosphorylation contribute significantly to activation, although the energetic contribution of cyclin A binding is greater than that of T160 phosphorylation by approximately 5 kcal/mol. The potential significance of direct activation of Cdk2 by cyclins with respect to regulation of cell cycle progression is discussed.     

CycD1, a putative G1 cyclin from Antirrhinum majus, accelerates the cell cycle in cultured tobacco BY-2 cells by enhancing both G1/S entry and progression through S and G2 phases

A putative G1 cyclin gene, Antma;CycD1;1 (CycD1), from Antirrhinum majus is known to be expressed throughout the cell cycle in the meristem and other actively proliferating cells. To test its role in cell cycle progression, we examined the effect of CycD1 expression in the tobacco (Nicotiana tabacum) cell suspension culture BY-2. Green fluorescent protein:CycD1 is located in the nucleus throughout interphase. Using epitope-tagged CycD1, we show that it interacts in vivo with CDKA, a cyclin dependent protein kinase that acts at both the G1/S and the G2/M boundaries. We examined the effect of induced expression at different stages of the cell cycle. Expression in G0 cells accelerated entry into both S-phase and mitosis, whereas expression during S-phase accelerated entry into mitosis. Consistent with acceleration of both transitions, the CycD1-associated cyclin dependent kinase can phosphorylate both histone H1 and Rb proteins. The expression of cyclinD1 led to the early activation of total CDK activity, consistent with accelerated cell cycle progression. Continuous expression of CycD1 led to moderate increases in growth rate. Therefore, in contrast with animal D cyclins, CycD1 can promote both G0/G1/S and S/G2/M progression. This indicates that D cyclin function may have diverged between plants and animals.     

蛋白质的磷酸化作用和泛肽化降解作用与芽殖酵母细胞周期的调控

在芽殖酵母（Ｓａｃｃｈａｒｏｍｙｃｅｓｃｅｒｅｖｉｓｉａｅ）细胞中，Ｇ１期的三种ｃｙｃｌｉｎｓ和Ｓ、Ｍ期的五种ｃｙｃｌｉｎｓ之周期性的合成和分解调节着Ｃｄｃ２８的活性，驱动细胞周期的正常运转。除了ＣＤＫ的磷酸化作用外，蛋白质的泛肽化降解作用间接或直接调控细胞周期：ＣＤＣ３４泛肽化途径通过降解Ｃｄｃ２８的专一抑制子而起始ＤＮＡ复制；ＡＰＣ泛肽化途径通过降解Ｍ期后期的抑制子和Ｍ期ｃｙｃｌｉｎｓ，使姐妹染色体分离和Ｍ期终止。