DP1 phosphorylation in multimeric complexes: weaker interaction with cyclin A through the E2F1 cyclin A binding domain leads to more efficient phosphorylation than stronger interaction through the p107 cyclin A binding domain.

Stable enzyme-substrate interaction has been recognized as a major mechanism underlying the substrate preferences of cyclin-dependent kinases (Cdks). To learn the relationship between stability of physical association and efficiency of phosphorylation, we studied DP1 phosphorylation by cyclin A-Cdk2 in multiprotein complexes. When DP1 was connected to cyclin A-Cdk2 through E2F4 and p107, its phosphorylation was very inefficient, although its association with cyclin A-Cdk2 was stable. In contrast, DP1 was efficiently phosphorylated when weakly connected to cyclin A-Cdk2 via E2F1 or E2F4 with a fused cyclin A binding domain of E2F1. The transactivation activity of E2F4-DP1 heterodimers was reduced when DP1 was phosphorylated, while a phosphorylation deficient mutant of DP1 resisted this down-regulation. Phosphorylation and functional regulation of DP1 were not due to nuclear localization. Thus, stronger physical association between the kinase and the substrate does not necessarily lead to more efficient phosphorylation than weaker interaction does.     

Interaction of Xenopus Cdc2 x cyclin A1 with the origin recognition complex

The initiation of DNA replication in eukaryotes is regulated in a minimum of at least two ways. First, several proteins, including origin recognition complex (ORC), Cdc6 protein, and the minichromosome maintenance (MCM) protein complex, need to be assembled on chromatin before initiation. Second, cyclin-dependent kinases regulate DNA replication in both a positive and a negative way by inducing the initiation of DNA replication at G(1)/S transition and preventing further rounds of origin firing within the same cell cycle. Here we characterize a link between the two levels. Immunoprecipitation of Xenopus origin recognition complex with anti-XOrc1 or anti-XOrc2 antibodies specifically co-immunoprecipitates a histone H1 kinase activity. The kinase activity is sensitive to several inhibitors of cyclin-dependent kinases including 6-dimethylaminopurine (6-DMAP), olomoucine, and p21(Cip1). This kinase activity also copurifies with ORC over several fractionation steps and was identified as a complex of the Cdc2 catalytic subunit and cyclin A1. Neither Cdk2 nor cyclin E could be detected in ORC immunoprecipitations. Reciprocal immunoprecipitations with anti-Xenopus Cdc2 or anti-Xenopus cyclin A1 antibodies specifically co-precipitate XOrc1 and XOrc2. Our results indicate that Xenopus ORC and Cdc2 x cyclin A1 physically interact and demonstrate a physical link between an active cyclin-dependent kinase and proteins involved in the initiation of DNA replication.     

Regulation of fumonisin B(1) biosynthesis and conidiation in Fusarium verticillioides by a cyclin-like (C-type) gene, FCC1

Fumonisins are a group of mycotoxins produced in corn kernels by the plant-pathogenic fungus Fusarium verticillioides. A mutant of the fungus, FT536, carrying a disrupted gene named FCC1 (for Fusarium cyclin C1) resulting in altered fumonisin B(1) biosynthesis was generated. FCC1 contains an open reading frame of 1,018 bp, with one intron, and encodes a putative 319-amino-acid polypeptide. This protein is similar to UME3 (also called SRB11 or SSN8), a cyclin C of Saccharomyces cerevisiae, and contains three conserved motifs: a cyclin box, a PEST-rich region, and a destruction box. Also similar to the case for C-type cyclins, FCC1 was constitutively expressed during growth. When strain FT536 was grown on corn kernels or on defined minimal medium at pH 6, conidiation was reduced and FUM5, the polyketide synthase gene involved in fumonisin B(1) biosynthesis, was not expressed. However, when the mutant was grown on a defined minimal medium at pH 3, conidiation was restored, and the blocks in expression of FUM5 and fumonisin B(1) production were suppressed. Our data suggest that FCC1 plays an important role in signal transduction regulating secondary metabolism (fumonisin biosynthesis) and fungal development (conidiation) in F. verticillioides.     

Distinct roles for PP1 and PP2A in phosphorylation of the retinoblastoma protein. PP2a regulates the activities of G(1) cyclin-dependent kinases.

The function of the retinoblastoma protein (pRB) in controlling the G(1) to S transition is regulated by phosphorylation and dephosphorylation on serine and threonine residues. While the roles of cyclin-dependent kinases in phosphorylating and inactivating pRB have been characterized in detail, the roles of protein phosphatases in regulating the G(1)/S transition are not as well understood. We used cell-permeable inhibitors of protein phosphatases 1 and 2A to assess the contributions of these phosphatases in regulating cyclin-dependent kinase activity and pRB phosphorylation. Treating asynchronously growing Balb/c 3T3 cells with PP2A-selective concentrations of either okadaic acid or calyculin A caused a time- and dose-dependent decrease in pRB phosphorylation. Okadaic acid and calyculin A had no effect on pRB phosphatase activity even though PP2A was completely inhibited. The decrease in pRB phosphorylation correlated with inhibitor-induced suppression of G(1) cyclin-dependent kinases including CDK2, CDK4, and CDK6. The inhibitors also caused decreases in the levels of cyclin D2 and cyclin E, and induction of the cyclin-dependent kinase inhibitors p21(Cip1) and p27(Kip1). The decrease in cyclin-dependent kinase activities were not dependent on induction of cyclin-dependent kinase inhibitors since CDK inhibition still occurred in the presence of actinomycin D or cycloheximide. In contrast, selective inhibition of protein phosphatase 1 with tautomycin inhibited pRB phosphatase activity and maintained pRB in a highly phosphorylated state. The results show that protein phosphatase 1 and protein phosphatase 2A, or 2A-like phosphatases, play distinct roles in regulating pRB function. Protein phosphatase 1 is associated with the direct dephosphorylation of pRB while protein phosphatase 2A is involved in pathways regulating G(1) cyclin-dependent kinase activity.     

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.     

Roughex mediates G(1) arrest through a physical association with cyclin A

Differentiation in the developing Drosophila eye requires synchronization of cells in the G(1) phase of the cell cycle. The roughex gene product plays a key role in this synchronization by negatively regulating cyclin A protein levels in G(1). We show here that coexpressed Roughex and cyclin A physically interact in vivo. Roughex is a nuclear protein, while cyclin A was previously shown to be exclusively cytoplasmic during interphase in the embryo. In contrast, we demonstrate that in interphase cells in the eye imaginal disk cyclin A is present in both the nucleus and the cytoplasm. In the presence of ectopic Roughex, cyclin A becomes strictly nuclear and is later degraded. Nuclear targeting of both Roughex and cyclin A under these conditions is dependent on a C-terminal nuclear localization signal in Roughex. Disruption of this signal results in cytoplasmic localization of both Roughex and cyclin A, confirming a physical interaction between these molecules. Cyclin A interacts with both Cdc2 and Cdc2c, the Drosophila Cdk2 homolog, and Roughex inhibits the histone H1 kinase activities of both cyclin A-Cdc2 and cyclin A-Cdc2c complexes in whole-cell extracts. Two-hybrid experiments suggested that the inhibition of kinase activity by Roughex results from competition with the cyclin-dependent kinase subunit for binding to cyclin A. These findings suggest that Roughex can influence the intracellular distribution of cyclin A and define Roughex as a distinct and specialized cell cycle inhibitor for cyclin A-dependent kinase activity.     

Stoichiometry of cyclin A-cyclin-dependent kinase 2 inhibition by p21Cip1/Waf1

Progression through the eukaryotic cell cycle is regulated by phosphorylation, which is catalyzed by cyclin-dependent kinases. Cyclin-dependent kinases are regulated through several mechanisms, including negative regulation by p21 (variously called CAP20, Cip1, Sdi1, and WAF1). It has been proposed that multiple p21 molecules are required to inhibit cyclin-dependent kinases, such that p21 acts as a sensitive buffer of cyclin-dependent kinase activity or as an assembly factor for the complexes formed by the cyclins and cyclin-dependent kinases. Using purified, full-length proteins of known concentration (determined by absorbance) and cyclin A-Cdk2 of known activity (calibrated with staurosporine), we find that a 1:1 molar ratio of p21 to cyclin A-Cdk2 is able to inhibit Cdk2 activity both in the binary cyclin A-Cdk2 complex and in the presence of proliferating cell nuclear antigen (PCNA). Our results indicate that the mechanism of p21 inhibition of cyclin A-Cdk2 does not involve multiple molecules of bound p21.     

Sequences within the conserved cyclin box of human cyclin A are sufficient for binding to and activation of cdc2 kinase.

Cyclins are pivotal in the coordinate regulation of the cell cycle. By physical association, they are able to activate at least one of the cyclin-dependent kinases, cdc2. How this association between the catalytic moiety and cyclins leads to subsequent activation of the kinase remains unclear. In this report, we describe experiments to investigate this event at a physical level. Our approach was to map the regions required on the cyclin A molecule for interaction with cdc2. We have mapped the contact regions to two small noncontiguous stretches of amino acids, residues 189 to 241 and 275 to 320, both located within the conserved cyclin box domain of the protein. We have further shown that this region not only represents a contact site for cdc2 but apparently represents an intact functional domain with respect to cdc2 activation. This region alone is sufficient to stimulate maturation when injected into immature Xenopus laevis oocytes. This observation implies that events leading to the activation of cdc2 kinase can be mediated through small regions of the cyclin molecule that are located in the cyclin box. These regions contain some of the most highly conserved residues found between all the cyclin members so far identified. This suggests that the cyclin family members may have conserved a similar mechanism to bind and activate cyclin-dependent kinases.     

Characterization of cyclin L2, a novel cyclin with an arginine/serine-rich domain: phosphorylation by DYRK1A and colocalization with splicing factors

A novel method employing filter arrays of a cDNA expression library for the identification of substrates for protein kinases was developed. With this technique, we identified a new member of the cyclin family, cyclin L2, as a substrate of the nuclear protein kinase DYRK1A. Cyclin L2 contains an N-terminal cyclin domain and a C-terminal arginine/serine-rich domain (RS domain), which is a hallmark of many proteins involved in pre-mRNA processing. The gene for cyclin L2 encodes the full-length cyclin L2, which is predominantly expressed in testis, as well as a truncated splicing variant (cyclin L2S) that lacks the RS domain and is ubiquitously expressed in human tissues. Full-length cyclin L2, but not cyclin L2S, was associated with the cyclin-dependent kinase PITSLRE. Cyclin L2 interacted with splicing factor 2 in vitro and was co-localized with the splicing factor SC35 in the nuclear speckle compartment. Photobleaching experiments showed that a fusion protein of green fluorescent protein and cyclin L2 in nuclear speckles rapidly exchanged with unbleached molecules in the nucleus, similar to other RS domain-containing proteins. In striking contrast, the closely related green fluorescent protein-cyclin L1 was immobile in the speckle compartment. DYRK1A interacted with cyclin L2 in pull-down assays, and overexpression of DYRK1A stimulated phosphorylation of cyclin L2 in COS-7 cells. These data characterize cyclin L2 as a highly mobile component of nuclear speckles and suggest that DYRK1A may regulate splicing by phosphorylation of cyclin L2.     

PKR and PKR-like endoplasmic reticulum kinase induce the proteasome-dependent degradation of cyclin D1 via a mechanism requiring eukaryotic initiation factor 2alpha phosphorylation

Cyclin D1 plays a critical role in controlling the G(1)/S transition via the regulation of cyclin-dependent kinase activity. Several studies have indicated that cyclin D1 translation is decreased upon activation of the eukaryotic initiation factor 2alpha (eIF2alpha) kinases. We examined the effect of activation of the eIF2alpha kinases PKR and PKR-like endoplasmic reticulum kinase (PERK) on cyclin D1 protein levels and translation and determined that cyclin D1 protein levels decrease upon the induction of PKR and PERK catalytic activity but that this decrease is not due to translation. Inhibition of the 26 S proteasome with MG132 rescued cyclin D1 protein levels, indicating that rather than inhibiting translation, PKR and PERK act to increase cyclin D1 degradation. Interestingly, this effect still requires eIF2alpha phosphorylation at serine 51, as cyclin D1 remains unaffected in cells containing a non-phosphorylatable form of the protein. This proteasome-dependent degradation of cyclin D1 requires an intact ubiquitination pathway, although the ubiquitination of cyclin D1 is not itself affected. Furthermore, this degradation is independent of phosphorylation of cyclin D1 at threonine 286, which is mediated by the glycogen synthase kinase 3beta and mitogen-activated protein kinase pathways as described in previous studies. Our study reveals a novel functional cross-talk between eIF2alpha phosphorylation and the proteasomal degradation of cyclin D1 and that this degradation is dependent upon eIF2alpha phosphorylation during short, but not prolonged, periods of stress.     

Virus-encoded cyclin.

Herpesvirus saimiri contains an open reading frame called eclf2 with homology to the cellular type D cyclins. We now show that the eclf2 gene product is a novel virus-encoded cyclin (v-cyclin). The protein encoded by the v-cyclin gene of this oncogenic herpesvirus was found to have an apparent molecular size of 29 kDa in transformed cells. v-Cyclin protein was found to be associated with cdk6, a cellular cyclin-dependent kinase known to interact with cellular type D cyclins. cdk6/v-cyclin complexes strongly phosphorylated Rb fusion protein and histone H1 as substrates in vitro. Mutational analyses showed that highly conserved amino acids in the cyclin box of v-cyclin were important for association with cdk6 and for activation of cdk6 kinase activity. Thus, v-cyclin resembles cellular type D cyclins in primary sequence, in its association with cdk6, by its ability to activate protein kinase activity, and by the presence of functional cyclin box sequences. v-Cyclin exhibited a selective preference for association with cdk6 over other cyclin-dependent kinases and a high level of kinase activation. The properties of v-cyclin suggest a likely role in oncogenic transformation by this T-lymphotropic herpesvirus.     

Analysis of wild-type and mutant p21WAF-1 gene activities.

The p21WAF-1 gene is positively regulated by the wild-type p53 protein. p21WAF-1 has been shown to interact with several cyclin-dependent kinase complexes and block the activity of G1 cyclin-dependent kinases (cdks). Mutational analysis with the p21WAF-1 gene localized a site, at amino acid residues 21 and 24 in the amino terminus of the protein, for p21WAF-1 binding to cyclins D and E. This region of the protein is conserved (residues 21 to 26) in other p21WAF-1 family members, p27kip-1 and p57kip-2. The same p21WAF-121,24 mutant also fails to bind to cyclin D1-cdk 4 or cyclin E-cdk 2 complexes in vitro, suggesting that amino acid residues 21 and 24 are important for p21WAF-1-cdk-cyclin trimeric complex interactions. The p21WAF-1 wild-type protein will suppress tumor cell growth in culture while p21WAF-1 mutant proteins with defects in residues 21 and 24 fail to suppress tumor cell growth. The overexpression of cyclin D or E in these cells will partially overcome the growth suppression of wild-type p21WAF-1 protein in cells. These results provide evidence that p21WAF-1 acts through cyclin D1-cdk4 and cyclin E-cdk2 complexes in vivo to induce the growth suppression. The p21WAF-1 binding sites for cyclins (residues 21 to 26), cdk2 (residues 49 to 71), and proliferating-cell nuclear antigen (residues 124 to 164) have all been mapped to discrete sites on the protein.     

Cyclin D1 Promotes Cell Cycle Progression through Enhancing NDR1/2 Kinase Activity Independent of Cyclin-dependent Kinase 4

Cyclin/cyclin-dependent kinases (Cdks) are critical protein kinases in regulating cell cycle progression. Among them, cyclin D1/Cdk4 exerts its function mainly in the G₁ phase. By using the tandem affinity purification tag approach, we identified a set of proteins interacting with Cdk4, including NDR1/2. Interestingly, confirming the interactions between NDR1/2 and cyclin D1/Cdk4, we observed that NDR1/2 interacted with cyclin D1 independent of Cdk4, but NDR1/2 and cyclin D1/Cdk4 did not phosphorylate each other. In addition, we found that NDR1/2 did not affect the kinase activity of cyclin D1/Cdk4 upon phosphorylation of GST-Rb. However, cyclin D1 but not Cdk4 promoted the kinase activity of NDR1/2. We also demonstrated that cyclin D1 K112E, which could not bind Cdk4, enhanced the kinase activity of NDR1/2. To test whether cyclin D1 promotes G₁/S transition though enhancing NDR1/2 kinase activity, we performed flow cytometry analysis using cyclin D1 and cyclin D1 K112E Tet-On inducible cell lines. The data show that both cyclin D1 and cyclin D1 K112E promoted G₁/S transition. Importantly, knockdown of NDR1/2 almost completely abolished the function of cyclin D1 K112E in promoting G₁/S transition. Consistently, we found that the protein level of p21 was reduced in cells overexpressing cyclin D1 K112E but not when NDR1/2 was knocked down. Taken together, these results reveal a novel function of cyclin D1 in promoting cell cycle progression by enhancing NDR kinase activity independent of Cdk4.     

The localization of human cyclins B1 and B2 determines CDK1 substrate specificity and neither enzyme requires MEK to disassemble the Golgi apparatus

In this paper, we show that substrate specificity is primarily conferred on human mitotic cyclin-dependent kinases (CDKs) by their subcellular localization. The difference in localization of the B-type cyclin-CDKs underlies the ability of cyclin B1-CDK1 to cause chromosome condensation, reorganization of the microtubules, and disassembly of the nuclear lamina and of the Golgi apparatus, while it restricts cyclin B2-CDK1 to disassembly of the Golgi apparatus. We identify the region of cyclin B2 responsible for its localization and show that this will direct cyclin B1 to the Golgi apparatus and confer upon it the more limited properties of cyclin B2. Equally, directing cyclin B2 to the cytoplasm with the NH(2) terminus of cyclin B1 confers the broader properties of cyclin B1. Furthermore, we show that the disassembly of the Golgi apparatus initiated by either mitotic cyclin-CDK complex does not require mitogen-activated protein kinase kinase (MEK) activity.     

Disruption of the Rb--Raf-1 interaction inhibits tumor growth and angiogenesis

The retinoblastoma tumor suppressor protein (Rb) plays a vital role in regulating mammalian cell cycle progression and inactivation of Rb is necessary for entry into S phase. Rb is inactivated by phosphorylation upon growth factor stimulation of quiescent cells, facilitating the transition from G(1) phase to S phase. Although the signaling events after growth factor stimulation have been well characterized, it is not yet clear how these signals contact the cell cycle machinery. We had found previously that growth factor stimulation of quiescent cells lead to the direct binding of Raf-1 kinase to Rb, leading to its inactivation. Here we show that the Rb-Raf-1 interaction occurs prior to the activation of cyclin and/or cyclin-dependent kinases and facilitates normal cell cycle progression. Raf-1-mediated inactivation of Rb is independent of the mitogen-activated protein kinase cascade, as well as cyclin-dependent kinases. Binding of Raf-1 seemed to correlate with the dissociation of the chromatin remodeling protein Brg1 from Rb. Disruption of the Rb-Raf-1 interaction by a nine-amino-acid peptide inhibits Rb phosphorylation, cell proliferation, and vascular endothelial growth factor-mediated capillary tubule formation. Delivery of this peptide by a carrier molecule led to a 79% reduction in tumor volume and a 57% reduction in microvessel formation in nude mice. It appears that Raf-1 links mitogenic signaling to Rb and that disruption of this interaction could aid in controlling proliferative disorders.     

The p21(Cip1) and p27(Kip1) CDK 'inhibitors' are essential activators of cyclin D-dependent kinases in murine fibroblasts

The widely prevailing view that the cyclin-dependent kinase inhibitors (CKIs) are solely negative regulators of cyclin-dependent kinases (CDKs) is challenged here by observations that normal up-regulation of cyclin D- CDK4 in mitogen-stimulated fibroblasts depends redundantly upon p21(Cip1) and p27(Kip1). Primary mouse embryonic fibroblasts that lack genes encoding both p21 and p27 fail to assemble detectable amounts of cyclin D-CDK complexes, express cyclin D proteins at much reduced levels, and are unable to efficiently direct cyclin D proteins to the cell nucleus. Restoration of CKI function reverses all three defects and thereby restores cyclin D activity to normal physiological levels. In the absence of both CKIs, the severe reduction in cyclin D-dependent kinase activity was well tolerated and had no overt effects on the cell cycle.