Cyclin A


Cyclin A in invertebrates

In starfish, fertilization occurs naturally at late meiosis I. In the absence of fertilization, however, oocytes complete meiosis I and II, resulting in mature eggs, which are still fertilizable, arrested at the pronucleus stage. In this study, cDNAs of starfish cyclin A and Cdc2 were isolated and the cell cycle dynamics of cyclin A and cyclin B levels and their associated Cdc2 kinase activity were monitored. Tyr phosphorylation of Cdc2, and Cdc25 phosphorylation states were examined throughout meiotic and early embryonic cleavage cycles in vivo. In meiosis I, cyclin A is undetectable and cyclin B/Cdc2 alone exhibits histone H1 kinase activity; thereafter, both cyclin A/Cdc2 and cyclin B/Cdc2 kinase activity oscillates along with the cell cycle. Cyclin B-associated Cdc2 (but not cyclin A-associated Cdc2) is subjected to regulation via Tyr phosphorylation. With some exceptions, phosphorylation states of Cdc25 correlate with cyclin B/Cdc2 kinase activity. Between meiosis I and II and at the pronucleus stage, cyclin A and B levels remain low, Cdc2 Tyr phosphorylation is undetectable, and Cdc25 remains phosphorylated depending on MAP kinase activity, showing a good correlation between these two stages. Upon fertilization of mature eggs, Cdc2 Tyr phosphorylation reappears and Cdc25 is dephosphorylated. In the first cleavage cycle, under conditions which prevent Cdc25 activity, cyclin A/Cdc2 is activated with a normal time course and then cyclin B/Cdc2 is activated with a significant delay, resulting in the delayed completion of M-phase. Thus, in contrast to meiosis I, both cyclin A and cyclin B appear to be involved in the embryonic cleavage cycles. It is proposed that regulation of cyclin A/Cdc2 and cyclin B/Cdc2 is characteristic of meiotic and early cleavage cycles (Okano-Uchida, 1998).

The coordination of cell proliferation and cell fate determination is critical during development but the mechanisms through which this is accomplished are unclear. This study presents evidence that the Snail-related transcription factor CES-1 of Caenorhabditis elegans coordinates these processes in a specific cell lineage. CES-1 can cause loss of cell polarity in the NSM neuroblast. By repressing the transcription of the BH3-only gene egl-1, CES-1 can also suppress apoptosis in the daughters of the NSM neuroblasts. CES-1 also affects cell cycle progression in this lineage. Specifically, it was found that CES-1 can repress the transcription of the cdc-25.2 gene, which encodes a Cdc25-like phosphatase, thereby enhancing the block in NSM neuroblast division caused by the partial loss of cya-1, which encodes Cyclin A. The results indicate that CDC-25.2 and CYA-1 control specific cell divisions and that the over-expression of the ces-1 gene leads to incorrect regulation of this functional 'module'. Finally, evidence is provided that dnj-11 MIDA1 not only regulate CES-1 activity in the context of cell polarity and apoptosis but also in the context of cell cycle progression. In mammals, the over-expression of Snail-related genes has been implicated in tumorigenesis. These findings support the notion that the oncogenic potential of Snail-related transcription factors lies in their capability to, simultaneously, affect cell cycle progression, cell polarity and apoptosis and, hence, the coordination of cell proliferation and cell fate determination (Yan, 2013).

Cyclin A and the mid-blastula transition in Xenopus

Previous work identified a developmental timer that controls the stability of cyclin A protein in interphase-arrested Xenopus embryos. Cyclins A1 and A2 abruptly become unstable in hydroxyurea-treated embryos at the time that untreated embryos are beginning gastrulation, termed early gastrulation transition (EGT). Cyclins A1 and A2 are degraded at the equivalent of the EGT by the ICE-like caspases (See Drosophila Death caspase) that are responsible for programmed cell death. The cleavage site is identified as DEPD, located between residues 87 to 90. Analysis of embryos treated with hydroxyurea or cycloheximide shows widespread cellular apoptosis coincident with cyclin A cleavage. These data further indicate that the apoptotic pathway is present in Xenopus embryos prior to the EGT; however, it is maintained in an inactive state in early cleaving embryos by maternally encoded inhibitors. In normal embryos, suppression of apoptosis is timed to begin with the initiation of zygotic transcription, at the mid-blastula transition (MBT). The decreased biosynthetic capacity of embryos treated with hydroxyurea or cycloheximide most likely interferes with the ability to maintain sufficient levels of apoptotic inhibitors and results in widespread apoptosis. These results suggest a scenario whereby the apoptotic pathway is suppressed in the early cleaving embryo by maternally contributed inhibitors. Degradation at the EGT of maternal RNAs encoding these inhibitors is compensated for by new zygotic transcription beginning at the MBT. This indicates that the interval between the MBT and the EGT represents a critical developmental period during which the regulation of embryonic cellular processes is transferred from maternal to zygotic control (Stack, 1997).

At the midblastula transition, the Xenopus laevis embryonic cell cycle is remodeled from rapid alternations between S and M phases to become the complex adult cell cycle. Cell cycle remodeling occurs after zygotic transcription initiates and is accompanied by terminal downregulation of maternal cyclins A1 and B2. The disappearance of both cyclin A1 and B2 proteins is preceded by the rapid deadenylation of their mRNAs. A specific mechanism triggers this deadenylation. This mechanism depends upon discrete regions of the 3' untranslated regions and requires zygotic transcription. Together, these results strongly suggest that zygote-dependent deadenylation of cyclin A1 and cyclin B2 mRNAs is responsible for the downregulation of these proteins. These studies also raise the possibility that zygotic control of maternal cyclins plays a role in establishing the adult cell cycle (Audic, 2001).

Activation of cyclin-dependent kinases

Activation of the cyclin-dependent kinase to promote cell cycle progression requires their association with cyclins as well as phosphorylation of a threonine residue. This phosphorylation is carried out by the Cdk-activating kinase (CAK). Purification of CAK from mammals, starfish, and Xenopus has identified it as a heterotrimeric complex composed of a catalytic subunit, p40MO15/cdk7, a regulatory subunit, cyclin H, and an assembly factor, MAT1. CAK phosphorylates not only c34cdc2 (see Drosophila cdc2) but also other Cdks, including p33cdk2 and cdk4, which function earlier in the cell cycle. Additionally, the CAK subunits are components of TFIIH, a basal transcription factor involved in the initiation of transcription, phosphorylation of the C-terminal domain of the large subunit of RNA polymerase II and DNA repair. The cloning of the CAK from S. cerevisiae raises the possibility that the the predominant CAK in vertebrate cell extracts, may not function as a physiological CAK. S. cerevisiae CAK is active as a monomer and is not a component of the basal transcription factor (Kaldis, 1996 and references).

Interaction with Retinoblastoma-related pocket proteins, cdk inhibitors, and E2F transcription factor

The Retinoblastoma-related protein p107, like the p21 family of cdk inhibitors, can inhibit the phosphorylation of target substrates by Cyclin A/cdk2 and Cyclin E/cdk2 complexes (See Drosophila Retinoblastoma-family protein). The associations of p107 and p21 with cyclin/cdk2 rely on a structurally and functionally related interaction domain. Interactions between p107 and p21 are mutually exclusive: p21 causes a dissociation of p107/cyclin/cdk2 complexes to yield p21/cyclin/cdk2 complexes. The activation of the p107-bound cyclin/cdk kinases leads to dissociation of p107 from the transcription factor E2F. It has been suggested that p107 functions similarly to Rb, causing growth arrest of sensitive cells in the G1 phase of the cell cycle. The p107 molecule can be dissected into two domains, each one capable of independently blocking the cell cycle progression. One domain corresponds to the sequences needed for interaction with transcription E2F, and the other corresponds to the interaction domain for Cyclin A or Cyclin E complexes (Zhu, 1995 a and b).

The retinoblastoma (pRB) family of proteins includes three proteins known to suppress growth of mammalian cells. Growth suppression by two of these proteins, p107 and p130, could result from the inhibition of associated cyclin-dependent kinases (cdks). One important unresolved issue, however, is the mechanism by which inhibition occurs. In vivo and in vitro evidence suggests that p107 is a bona fide inhibitor of both cyclin A-cdk2 and cyclin E-cdk2. p107 exhibits an inhibitory constant (Ki) comparable to that of the cdk inhibitor p21/WAF1. In contrast, pRB is unable to inhibit cdks. Further reminiscent of p21, a second cyclin-binding site was mapped to the amino-terminal portions of p107 and p130. This amino-terminal domain is capable of inhibiting cyclin-cdk2 complexes, although it is not a potent substrate for these kinases. In contrast, a carboxy-terminal fragment of p107 that contains the previously identified cyclin-binding domain serves as an excellent kinase substrate although it is unable to inhibit either kinase. Clustered point mutations suggest that the amino-terminal domain is functionally important for cyclin binding and growth suppression. Moreover, peptides spanning the cyclin-binding region are capable of interfering with p107 binding to cyclin-cdk2 complexes and kinase inhibition. The ability to distinguish between p107 and p130 as inhibitors rather than simple substrates suggests that these proteins may represent true inhibitors of cdks (Castano, 1998).

p107 and p130 immune complexes exhibit kinase activity. Such immune complexes were tested with four substrates commonly utilized to assay Cdk activity, including all three known members of the retinoblastoma family. Immunodepletion reveals this kinase activity can be abolished by removal of either cyclin A or Cdk2 but is unaffected by removal of Cdk4 or any D-type cyclin. The appearance of p107 associated activity follows the accumulation of p107 protein. In contrast, the kinase activity associated with p130 immune complexes becomes apparent after mid-G1, coincident with p130 hyperphosphorylation. GST-Rb, GST-p107, and GST-p130 (where GST indicates glutathione S-transferase) are equally suitable substrates in p107 and p130 immune complex kinase assays, yielding activity equal to 25% of the cyclin A activity present. The p107 and p130 associated activity is unable to phosphorylate histone H1, suggesting the p107 and p130 associated cyclin A/Cdk2 may represent a distinct pool with a distinct substrate specificity. The p107 and p130 associated activity is released from the immune complexes upon incubation with ATP and Mg2+ and exhibits the same substrate preference observed with the untreated immune complex. These data suggest that p107 and p130 recognize, or form by association, a distinct pool of cyclin A/Cdk2 that preferentially phosphorylates retinoblastoma family members (Hauser, 1997).

Cyclin A/CDK2 binds directly to E2F-1 and inhibits the DNA binding activity of E2F-1/DP-1 by phosphorylation. The DNA-binding activity of the E2F-1/DP-1 complex is inhibited following phosphorylation of Cyclin A/CDK2 (Xu, 1994).

Elevated levels of the p21(WAF1) (p21) cyclin-dependent kinase inhibitor induce growth arrest. A panel of monoclonal antibodies against human p21 has been characterized in an effort to understand the dynamic regulatory interactions between this and other cellular proteins during the cell cycle. The kinase activity of cyclin A/Cdk2 associated with p21 is significantly lower than that of cyclin A/Cdk2 free of p21, suggesting that p21 abolishes its activity in vivo, and the use of multiple antibodies has enabled a study of the molecular architecture of p21 complexes in vivo. In addition, human fibroblasts released from a quiescent state display abundant amounts of p21 devoid of associated proteins ("free" p21), the levels of which decrease as cells approach S phase. Cyclin A levels increase as the amount of monomeric p21 decreases, resulting in an excess of cyclin A/Cdk2 complexes that are not bound to, or inactivated by, p21. These data strengthen the notion that the G1-to-S phase transition in human fibroblasts occurs when the concentration of cyclin A/Cdk2 surpasses that of p21 (Cai, 1998).

The cyclin-dependent kinase (CDK) inhibitor p27 binds and inhibits the kinase activity of several CDKs. The level of p27 is elevated in cells arrested in G0 by growth factor deprivation or contact inhibition. In G0, p27 is predominantly monomeric, although some portion is associated with residual Cyclin A/Cdk2 heterodimer. During G1, all of p27 is associated with Cyclin D1/Cdk4 and is then redistributed to Cyclin A/Cdk2 as cells entered S phase. p27 binds better to Cyclin/CDK complexes than to monomeric CDKs. In growing cells, the majority of p27 is associated with Cyclin D1; levels of p27 are significantly lower than levels of Cyclin D1. In cells arrested in G1 with lovastatin, Cyclin D1 is degraded and p27 redistributes to Cyclin A/Cdk2. The level of p27 is reduced after UV irradiation, but because Cyclin D1 is degraded more rapidly than p27, there is a transient increase in binding of p27 to Cyclin A/Cdk2 (Poon, 1995).

Although p27(Kip1) has been considered a general inhibitor of G1 and S phase cyclin-dependent kinases, the interaction of p27 with two such kinases, cyclin A-Cdk2 and cyclin D-Cdk4, is different. In Mv1Lu cells containing a p27 inducible system, a 6-fold increase over the basal p27 level completely inhibits Cdk2 and cell cycle progression. In contrast, the same or a larger increase in p27 levels does not inhibit Cdk4 or its homolog Cdk6, despite extensive binding to these kinases. A p27-cyclin A-Cdk2 complex formed in vitro is essentially inactive, whereas a p27-cyclin D2-Cdk4 complex is active as a retinoblastoma kinase and serves as a substrate for the Cdk-activating kinase Cak. High concentrations of p27 inhibit cyclin D2-Cdk4, apparently through the conversion of active complexes into inactive ones by the binding of additional p27 molecules. In contrast to their differential interaction, cyclin A-Cdk2 and cyclin D2-Cdk4 are similarly inhibited by bound p21(Cip1/Waf1). The role of cyclin A-Cdk2 as a p27 target, and the role of cyclin D2-Cdk4 as a p27 reservoir may result from the differential ability of bound p27 to inhibit the kinase subunit in these complexes (Blain, 1997).

Adenovirus E1A can stimulate transcription of Cyclin A in the absence of exogenous growth factors. Required for this activity is conserved region 2 (CR2), while both the N-terminal parts of E1A and CR1 are dispensable. This indicates that activation of Cyclin A gene expression requires the binding of E1A to p107, while binding to either pRB or p300 is not involved in transcriptional activation. p107 represses the Cyclin A promoter through its cell cycle-regulatory E2F binding site. 12S E1A can activate the Cyclin A promoter, essentially by counteracting its repression by p107. Since Cr2 is required for cell transformation, transcriptional activation of the Cyclin A gene by E1A appears to be important for its capacity to override control of cellular growth (Zerfass, 1996).

Transcription of the human proto-oncogene MYC (Drosophila homolog: dmyc) is repressed in quiescent or non-dividing cells. Upon mitogenic stimulation, expression of MYC is rapidly and transiently induced, maintained throughout G1, and declines to a basal level throughout further cell cycle transitions. Regulation of MYC promoter activity critically depends on the presence of a binding site for transcription factor E2F. Transcription from the MYC P2 promoter is induced efficiently by E2F-1, but repressed by Rb. Furthermore, overexpression of Cyclin A strongly activates the MYC promoter. This effect is further enhanced by coexpression of E2F-1 and Cyclin A. Expression of G1-phase Cyclin D1 leads to an E2F binding site-dependent trans-activation of the MYC promoter. Such activation is abrogated by overexpression of Rb. The interaction of D-type G1 Cyclins with Rb resembles that of the adenovirus E1A protein with Rb in that it can disrupt inhibitory E2F-Rb complexes. These results support a model in which intervention of distinct cyclins and their respective associated kinases promotes transcriptional activation of MYC throughout the cell cycle either by conversion of E2F within multimeric complexes into an active transcription factor or by liberation of free functional E2F (Oswald, 1994).

Human cyclin A1, a newly discovered cyclin, is expressed in testis and is thought to function in the meiotic cell cycle. The expression of human cyclin A1 and cyclin A1-associated kinase activities is regulated during the mitotic cell cycle. In the osteosarcoma cell line MG63, cyclin A1 mRNA and protein are present at very low levels in cells at the G0 phase. They increase during the progression of the cell cycle and reach the highest levels in the S and G2/M phases. Furthermore, the cyclin A1-associated histone H1 kinase activity peaks at the G2/M phase. Cyclin A1 can bind to important cell cycle regulators: the Rb family of proteins, the transcription factor E2F-1, and the p21 family of proteins. The in vitro interaction of cyclin A1 with E2F-1 is greatly enhanced when cyclin A1 is complexed with CDK2. Associations of cyclin A1 with Rb and E2F-1 are observed in vivo in several cell lines. When cyclin A1 is coexpressed with CDK2 in sf9 insect cells, the CDK2-cyclin A1 complex has kinase activities for histone H1, E2F-1, and the Rb family of proteins. These results suggest that the Rb family of proteins and E2F-1 may be important targets for phosphorylation by the cyclin A1-associated kinase. Cyclin A1 may function in the mitotic cell cycle in certain cells (Yang, 1999).

Interaction with cyclin-dependent kinases

The formation of cdk-cyclin complexes has been investigated at the molecular level and quantified using spectroscopic approaches. In the absence of phosphorylation, cdk2, cdc2, and cdk7 (see Drosophila Cyclin-dependent kinase 7) form highly stable complexes with their "natural" cyclin partners, with dissociation constants in the nanomolar range. In contrast, nonphosphorylated cdc2-cyclin H, cdk2-cyclin H, and cdk7-cyclin A complexes present a 25-fold lower stability. On the basis of both the structure of the cdk2-cyclin A complex and on kinetic results, it is suggested that interaction of any cyclin with any cdk involves the same hydrophobic contacts and induces a marked conformational change in the catalytic cleft of the cdks. Although cdks bind ATP strongly, they remain in a catalytically inactive conformation. In contrast, binding of the cyclin induces structural rearrangements that result in the selective reorientation of ATP, a concomitant 3-fold increase in its affinity, and a 5-fold decrease of its release from the active site of cdks (Heitz, 1997).

CDK2 (another cyclin dependent kinase) activity, like that of CDC2, oscillates during the cell cycle in cultured mammalian fibroblasts. Unlike CDC2 activity that peaks during mitosis, CDK2 activity rises in late G1 or early S phase, and declines during mitosis. Active S-phase CDK2 migrates in multiple large complexes on gel filtration, and the CDK2 in one of these complexes is associated with Cyclin A. These findings suggest that CDK2 and CDC2, in association with distinct cyclins, regulate separate functions in the mammalian cell cycle (Rosenblatt, 1992).

Cyclin A is required at two phases during the human cell cycle. In association with E2F it helps to terminate activation of transcription of genes involved in DNA replication. Later, depletion of Cyclin A arrests cells in G2, and it is required for progression from G2 to M. Cyclin A is associated with both cdk2 and cdc2. The cdc2 band associated with Cyclin A in early S is hyperphosphorylated. This suggests that Cyclin A binding is accompanied by tyrosine phosphorylation of cdc2, which keeps this kinase inactive until G2. The binding of cdk2 to Cyclin A increases steadily during the cell cycle and occurs in both S and G2-enriched cell fractions (Pagano, 1992).

Cyclins are regulatory subunits of p34cdc2 protein kinase. p34 controls critical cell cycle transitions through coordinate action with cyclins. Another human p34 homolog, cyclin-dependent kinase 2 (CDK2) is 66% identical to CDC2Hs and 89% identical to the Xenopus Eg1 gene, forming a distinct subfamily of CDC2-related protein kinases. CDK2, like CDC2s, encodes a 33-kDa Cyclin A-associated protein kinase that contains phosphotyrosine. However, the subunit composition of these two protein kinase complexes can vary in different cell types; they have different in vitro substrate preferences, and CDK2mRNA is observed much earlier than CDC2mRNA when lymphocytes are stimulated to enter the cell cycle (Elledge, 1992).

Tyrosine phosphorylation of cdc2 occurs on Tyr15, a residue located within the ATP binding site of the protein: this is required for maintaining the cdc2-Cyclin B complex inactive until DNA replication is completed. At the end of G2, the activation of the cdc25 phosphatase (known as String in Drosophila) causes cdc2 dephosphorylation and the activation of the histone H1 kinase activity (Krek, 1991).

The activation of cyclin-dependent kinases (CDKs) requires the phosphorylation of a conserved threonine (Thr160 in Cdk2) by CDK-activating kinase (CAK). Human KAP (also called Cdi1), a CDK-associated phosphatase, dephosphorylates Thr160 in human Cdk2. KAP does not dephosphorylate Tyr15 and only dephosphorylates Thr160 in native monomeric Cdk2. The binding of Cyclin A to Cdk2 inhibited the dephosphorylation of Thr160 by KAP but does not preclude the binding of KAP to the Cyclin A-Cdk2 complex. Moreover, the dephosphorylation of Thr160 by KAP prevents Cdk2 kinase activity upon subsequent association with Cyclin A. These results suggest that KAP binds to Cdk2 and dephosphorylates Thr160 when the associated cyclin subunit is degraded or dissociates (Poon, 1995).

It has been proposed that the functions of the cyclin-dependent kinase inhibitors p21(Cip1/Waf1) and p27Kip1 are limited to cell cycle control at the G1/S-phase transition and in the maintenance of cellular quiescence. To test the validity of this hypothesis, p21 was expressed in a diverse panel of cell lines, thus isolating the effects of p21 activity from the pleiotropic effects of upstream signaling pathways that normally induce p21 expression. The data show that at physiological levels of accumulation, p21, in addition to its role in negatively regulating the G1/S transition, contributes to regulation of the G2/M transition. Both G1- and G2-arrested cells are observed in all cell types, with different preponderances. Preponderant G1 arrest in response to p21 expression correlates with the presence of functional pRb. G2 arrest is more prominent in pRb-negative cells. The arrest distribution does not correlate with the p53 status, and the proliferating-cell nuclear antigen (PCNA) binding activity of p21 does not appear to be involved, since p27, which lacks a PCNA binding domain, produces a similar arrest profile. In addition, DNA endoreduplication occurs in pRb-negative but not in pRb-positive cells, suggesting that functional pRb is necessary to prevent DNA replication in cells arrested in G2 by p21. These results suggest that the primary target of the Cip/Kip family of inhibitors leading to efficient G1 arrest as well as to blockade of DNA replication from either G1 or G2 phase is the pRb regulatory system. The tendency of Rb-negative cells to undergo endoreduplication cycles when p21 is expressed may have negative implications in the therapy of Rb-negative cancers with genotoxic agents that activate the p53/p21 pathway (Niculescu, 1998).

The predominant G2 arrest observed in pRb-negative cells and limited G2 arrest observed in pRb-positive cells is most likely due to p21-mediated inhibition of cyclin A-Cdk2. The dynamics of arrest in pRb-negative cells probably reflects the inefficiency of inhibition of cyclin E-Cdk2. It is unlikely that inhibition of cyclin B1-associated kinase by p21 contributes to G2 arrest, since there is no evidence for significant amounts of cyclin B1 associated with p21. Nevertheless, it has been observed that in p21-induced G2-arrested cells, cyclin B1-Cdk2 is inhibited by phosphorylation of Cdc2. Although the mechanism for this indirect inhibition by p21 is not yet known, a similar phenomenon has been observed in Xenopus egg extracts, where inhibition of Cdk2 leads to inhibitory phosphorylation of Cdc2 and concomitant G2 arrest. pRb is necessary to block endoreduplication. After arrest in G2, a significant subpopulation of pRb-negative cells responding to p21 or p27 expression undergo cycles of endoreduplicative DNA replication. Although a significant fraction of pRb-positive cells arrests in G2, endoreduplication is never observed. Two conclusions can be drawn from these observations: (1) p21 can arrest cells in G2 in a physiological environment that is permissive for entering the S phase without an intervening mitosis. This would seem to be a violation of the normal safeguards that prevent cell cycle events from occurring out of order. (2) Initiation of the S phase, however, whether from G1 or G2, requires neutralization of the inhibitory functions of pRb. This cannot happen in the presence of both pRb and p21. The incomplete inhibition of cyclin E- and/or cyclin A-associated kinase activities may allow sufficient activity for initiation of replication but not sufficient to proceed to mitosis. The appropriate balance may not be met in every cell, since many apparently do not undergo endoreduplication. Licensing of origins may not be efficient, since endoreduplicative replication appears to proceed slowly. But the fact that pRb appears to be capable of blocking endoreduplication in G2-arrested cells suggests a role in enforcing the order of cell cycle events. Whatever critical functions downstream of pRb are required for replication after passage through G1 appear to be required again if replication is to occur from G2. The regulation of pRb phosphorylation, in fact, may be an important G2 function of p21 in response to DNA damage (Niculescu, 1998).

Cell-cycle phase transitions are controlled by cyclin-dependent kinases (Cdks). Key to the regulation of these kinase activities are Cdk inhibitors -- proteins that are induced in response to various antiproliferative signals but that can also oscillate during cell-cycle progression, leading to Cdk inactivation. A current dogma is that kinase complexes containing the prototype Cdk inhibitor p21 transit between active and inactive states: Cdk complexes associated with one p21 molecule remain active until they associate with additional p21 molecules. However, using a number of different techniques including analytical ultracentrifugation of purified p21/cyclin A/Cdk2 complexes, it has been demonstrated unambiguously that a single p21 molecule is sufficient for kinase inhibition and that p21-saturated complexes contain only one stably bound inhibitor molecule. Even phosphorylated forms of p21 remain efficient inhibitors of Cdk activities. Therefore the level of Cdk inactivation by p21 is determined by the fraction of kinase complexed with the inhibitor and not by the stoichiometry of inhibitor bound to the kinase or the phosphorylation state of the Cdk inhibitor (Hengst, 1998).

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, the potential regulation of cyclin A-Cdk2 activity by Skp1 and Skp2 was investigated. 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 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 (Yam, 1999).

Interaction of Cyclin A with CCAAT displacement protein/cut homeodomain protein

Developmental control of bone tissue-specific genes requires positive and negative regulatory factors to accommodate physiological requirements for the expression or suppression of the encoded proteins. Osteocalcin (OC) gene transcription is restricted to the late stages of osteoblast differentiation. OC gene expression is suppressed in nonosseous cells and osteoprogenitor cells and during the early proliferative stages of bone cell differentiation. The rat OC promoter contains a homeodomain recognition motif within a highly conserved multipartite promoter element (OC box I) that contributes to tissue-specific transcription. The CCAAT displacement protein (CDP), a transcription factor related to the Cut homeodomain protein in Drosophila, may regulate bone-specific gene transcription in immature proliferating osteoblasts. Using gel shift competition assays and DNase I footprinting, CDP/cut is shown to recognize two promoter elements (TATA and OC box I) of the bone-related rat OC gene. Overexpression of CDP/cut in ROS 17/2.8 osteosarcoma cells results in repression of OC promoter activity; this repression is abrogated by mutating OC box I. Gel shift immunoassays show that CDP/cut forms a proliferation-specific protein/DNA complex in conjunction with cyclin A and p107, a member of the retinoblastoma protein family of tumor suppressors. These findings suggest that CDP/cut may represent an important component of a cell signaling mechanism that provides cross-talk between developmental and cell cycle-related transcriptional regulators to suppress bone tissue-specific genes during proliferative stages of osteoblast differentiation (van Gurp, 1999).

Previous experiments with peptide fusion proteins suggested that cyclin A/Cdk1 and Cdk2 might exhibit similar yet distinct phosphorylation specificities. Using a physiological substrate, CDP/Cux, this notion has been confirmed. Proteolytic processing of CDP/Cux by cathepsin L generates the CDP/Cux p110 isoform at the beginning of S phase. CDP/Cux p110 makes stable interactions with DNA during S phase but is inhibited in G2 following the phosphorylation of serine 1237 by cyclin A/Cdk1. It is proposed that differential phosphorylation by cyclin A/Cdk1 and cyclin A/Cdk2 enables CDP/Cux p110 to exert its function as a transcriptional regulator specifically during S phase. Like cyclin A/Cdk1, cyclin A/Cdk2 interacts efficiently with recombinant CDP/Cux proteins that contain the Cut homeodomain and an adjacent cyclin-binding motif (Cy). In contrast to cyclin A/Cdk1, however, cyclin A/Cdk2 does not efficiently phosphorylate CDP/Cux p110 on serine 1237 and does not inhibit its DNA binding activity in vitro. Accordingly, co-expression with cyclin A/Cdk2 in cells does not inhibit the DNA binding and transcriptional activities of CDP/Cux p110. To confirm that the sequence surrounding serine 1237 is responsible for the differential regulation by Cdk1 and Cdk2, four amino acids flanking the phosphorylation site were replaced to mimic a known Cdk2 phosphorylation site present in the Cdc6 protein. Both cyclin A/Cdk2 and Cdk1 efficiently phosphorylates the CDP/Cux(Cdc6) mutant and inhibits its DNA binding activity. Altogether these results help explain why the DNA binding activity of CDP/Cux p110 is maximal during S phase and decreases in G2 phase (Santaguida, 2005).

Regulation of Cyclin A transcription

Continued: Evolutionary homologs see part 2/3 | part 3/3

Cyclin A: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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