Identification and characterization of the CDK12/cyclin L1 complex involved in alternative splicing regulation

CrkRS is a Cdc2-related protein kinase that contains an arginine- and serine-rich (SR) domain, a characteristic of the SR protein family of splicing factors, and is proposed to be involved in RNA processing. However, whether it acts together with a cyclin and at which steps it may function to regulate RNA processing are not clear. Here, we report that CrkRS interacts with cyclin L1 and cyclin L2, and thus rename it as the long form of cyclin-dependent kinase 12 (CDK12(L)). A shorter isoform of CDK12, CDK12(S), that differs from CDK12(L) only at the carboxyl end, was also identified. Both isoforms associate with cyclin L1 through interactions mediated by the kinase domain and the cyclin domain, suggesting a bona fide CDK/cyclin partnership. Furthermore, CDK12 isoforms alter the splicing pattern of an E1a minigene, and the effect is potentiated by the cyclin domain of cyclin L1. When expression of CDK12 isoforms is perturbed by small interfering RNAs, a reversal of the splicing choices is observed. The activity of CDK12 on splicing is counteracted by SF2/ASF and SC35, but not by SRp40, SRp55, and SRp75. Together, our findings indicate that CDK12 and cyclin L1/L2 are cyclin-dependent kinase and cyclin partners and regulate alternative splicing.     

NIPP1-mediated interaction of protein phosphatase-1 with CDC5L, a regulator of pre-mRNA splicing and mitotic entry

NIPP1 is a regulatory subunit of a species of protein phosphatase-1 (PP1) that co-localizes with splicing factors in nuclear speckles. We report that the N-terminal third of NIPP1 largely consists of a Forkhead-associated (FHA) protein interaction domain, a known phosphopeptide interaction module. A yeast two-hybrid screening revealed an interaction between this domain and a human homolog (CDC5L) of the fission yeast protein cdc5, which is required for G(2)/M progression and pre-mRNA splicing. CDC5L and NIPP1 co-localized in nuclear speckles in COS-1 cells. Furthermore, an interaction between CDC5L, NIPP1, and PP1 in rat liver nuclear extracts could be demonstrated by co-immunoprecipitation and/or co-purification experiments. The binding of the FHA domain of NIPP1 to CDC5L was dependent on the phosphorylation of CDC5L, e.g. by cyclin E-Cdk2. When expressed in COS-1 or HeLa cells, the FHA domain of NIPP1 did not affect the number of cells in the G(2)/M transition. However, the FHA domain blocked beta-globin pre-mRNA splicing in nuclear extracts. A mutation in the FHA domain that abolished its interaction with CDC5L also canceled its anti-splicing effects. We suggest that NIPP1 either targets CDC5L or an associated protein for dephosphorylation by PP1 or serves as an anchor for both PP1 and CDC5L.     

Epstein-Barr virus nuclear antigen 3C regulates cyclin A/p27 complexes and enhances cyclin A-dependent kinase activity

Epstein-Barr virus (EBV) nuclear antigen 3C (EBNA3C) is essential for primary B-cell transformation. In this report we show that cyclin A, an activator of S phase progression, bound tightly to EBNA3C. EBNA3C interacted with cyclin A in vitro and associated with cyclin A complexes in EBV-transformed lymphoblastoid cell lines. Importantly, EBNA3C stimulated cyclin A-dependent kinase activity and rescued p27-mediated inhibition of cyclin A/Cdk2 kinase activity by decreasing the molecular association between cyclin A and p27 in cells. Additionally, phosphorylation of the retinoblastoma protein, a major regulator of cell cycle progression, was enhanced both in vitro and in vivo in the presence of EBNA3C. Cyclin A interacted with a region of the carboxy terminus of EBNA3C, shown to be important both for stimulation of cyclin A-dependent kinase activity and for cell cycle progression. This provides the first evidence of an essential EBV latent antigen's directly targeting a cell cycle regulatory protein and suggests a novel mechanism by which EBV deregulates the mammalian cell cycle, which is of critical importance in B-cell transformation.     

Sequestration of pRb by cyclin D3 causes intranuclear reorganization of lamin A/C during muscle cell differentiation

The A-type lamins that localize in nuclear domains termed lamin speckles are reorganized and antigenically masked specifically during myoblast differentiation. This rearrangement was observed to be linked to the myogenic program as lamin speckles, stained with monoclonal antibody (mAb) LA-2H10, were reorganized in MyoD-transfected fibroblasts induced to transdifferentiate to muscle cells. In C2C12 myoblasts, speckles were reorganized early during differentiation in cyclin D3-expressing cells. Ectopic cyclin D3 induced lamin reorganization in C2C12 myoblasts but not in other cell types. Experiments with adenovirus E1A protein that can bind to and segregate the retinoblastoma protein (pRb) indicated that pRb was essential for the cyclin D3-mediated reorganization of lamin speckles. Cyclin D3-expressing myoblasts displayed site-specific reduction of pRb phosphorylation. Furthermore, disruption of lamin structures by overexpression of lamins inhibited expression of the muscle regulatory factor myogenin. Our results suggest that the reorganization of internal lamins in muscle cells is mediated by key regulators of the muscle differentiation program.     

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.     

FOXM1c is activated by cyclin E/Cdk2, cyclin A/Cdk2, and cyclin A/Cdk1, but repressed by GSK-3alpha

Two different inhibitory domains, N-terminus and central domain, keep FOXM1c almost inactive despite its strong transactivation domain. Here, we demonstrate that cyclin E/Cdk2, cyclin A/Cdk2, and cyclin A/Cdk1 activate FOXM1c. Cyclin E/Cdk2 does not target its transactivation domain or its DNA-binding domain. Instead, its activating effect strictly depends on the presence of either the central domain or the N-terminus of FOXM1c and thus is completely lost if both inhibitory domains are deleted. Cyclin E/Cdk2 activates FOXM1c by releasing its transactivation domain from the repression by these two inhibitory domains. However, it does not directly increase the transactivation potential of the TAD. The DNA-binding is not affected by cyclin E/Cdk2, neither directly nor indirectly. These two activating effects of cyclin E/Cdk2 via central domain and N-terminus are additive. Cyclin A/Cdk2 and cyclin A/Cdk1 show similar characteristics. GSK-3alpha, another proliferation-associated kinase, represses FOXM1c.     

Identification of interaction partners and substrates of the cyclin A1-CDK2 complex

The CDK2-associated cyclin A1 is essential for spermatogenesis and contributes to leukemogenesis. The detailed molecular functions of cyclin A1 remain unclear, since the molecular networks involving cyclin A1-CDK2 have not been elucidated. Here, we identified novel cyclin A1/CDK2 interaction partners in a yeast triple-hybrid approach. Several novel proteins (INCA1, KARCA1, and PROCA1) as well as the known proteins GPS2 (G-protein pathway suppressor 2), Ku70, receptor for activated protein kinase C1/guanine nucleotide-binding protein beta-2-like-1, and mRNA-binding motif protein 4 were identified as interaction partners. These proteins link the cyclin A1-CDK2 complex to diverse cellular processes such as DNA repair, signaling, and splicing. Interactions were confirmed by GST pull-down assays and co-immunoprecipitation. We cloned and characterized the most frequently isolated unknown gene, which we named INCA1 (inhibitor of CDK interacting with cyclin A1). The nuclear INCA1 protein is evolutionarily conserved and lacks homology to any known gene. This novel protein and two other interacting partners served as substrates for the cyclin A1-CDK2 kinase complex. Cyclin A1 and all interaction partners were highly expressed in testis with varying degrees of tissue specificity. The highest expression levels were observed at different time points during testis maturation, whereas expression levels in germ cell cancers and infertile testes decreased. Taken together, we identified testicular interaction partners of the cyclin A1-CDK2 complex and studied their expression pattern in normal organs, testis development, and testicular malignancies. Thereby, we establish a new basis for future functional analyses of cyclin A1. We provide evidence that the cyclin A1-CDK2 complex plays a role in several signaling pathways important for cell cycle control and meiosis.     

PACSIN 2 represses cellular migration through direct association with cyclin D1 but not its alternate splice form cyclin D1b

Cyclin D1 overexpression is a common feature of many human malignancies. Genomic deletion analysis has demonstrated a key role for cyclin D1 in cellular proliferation, angiogenesis and cellular migration. To investigate the mechanisms contributing to cyclin D1 functions, we purified cyclin D1a-associated complexes by affinity chromatography and identified the PACSIN 2 (protein kinase C and casein kinase substrate in neurons 2) protein by mass spectrometry. The PACSIN 2, but not the related PACSIN 1 and 3, directly bound wild-type cyclin D1 (cyclin D1a) at the carboxyl terminus and failed to bind cyclin D1b, the alternative splicing variant of cyclin D1. PACSIN 2 knockdown induced cellular migration and reduced cell spreading in LNCaP cells expressing cyclin D1a. In cyclin D1^−/− mouse embryonic fibroblasts (MEFs), cyclin D1a, but not cyclin D1b, reduced the cell spreading to a polarized morphology. siPACSIN 2 had no effect on cellular migration of cyclin D1^−/− MEFs. Cyclin D1a restored the migratory ability of cyclin D1^−/− MEFs, which was further enhanced by knocking down PACSIN 2 with siRNA. The cyclin D1-associated protein, PACSIN 2, regulates cell spreading and migration, which are dependent on cyclin D1 expression.Key words: PACSIN 2, cyclin D1, polymorphism, cellular migration, cell spreading, cancer     

The dynamic mobility of histone H1 is regulated by cyclin/CDK phosphorylation

The linker histone H1 is involved in maintaining higher-order chromatin structures and displays dynamic nuclear mobility, which may be regulated by posttranslational modifications. To analyze the effect of H1 tail phosphorylation on the modulation of the histone's nuclear dynamics, we generated a mutant histone H1, referred to as M1-5, in which the five cyclin-dependent kinase phosphorylation consensus sites were mutated from serine or threonine residues into alanines. Cyclin E/CDK2 or cyclin A/CDK2 cannot phosphorylate the mutant in vitro. Using the technique of fluorescence recovery after photobleaching, we observed that the mobility of a green fluorescent protein (GFP)-M1-5 fusion protein is decreased compared to that of a GFP-wild-type H1 fusion protein. In addition, recovery of H1 correlated with CDK2 activity, as GFP-H1 mobility was decreased in cells with low CDK2 activity. Blocking the activity of CDK2 by p21 expression decreased the mobility of GFP-H1 but not that of GFP-M1-5. Finally, the level and rate of recovery of cyan fluorescent protein (CFP)-M1-5 were lower than those of CFP-H1 specifically in heterochromatic regions. These data suggest that CDK2 phosphorylates histone H1 in vivo, resulting in a more open chromatin structure by destabilizing H1-chromatin interactions.     

Role of the Modular Domains of SR Proteins in Subnuclear Localization and Alternative Splicing Specificity

SR proteins are required for constitutive pre-mRNA splicing and also regulate alternative splice site selection in a concentration-dependent manner. They have a modular structure that consists of one or two RNA-recognition motifs (RRMs) and a COOH-terminal arginine/serine-rich domain (RS domain). We have analyzed the role of the individual domains of these closely related proteins in cellular distribution, subnuclear localization, and regulation of alternative splicing in vivo. We observed striking differences in the localization signals present in several human SR proteins. In contrast to earlier studies of RS domains in the Drosophila suppressor-of-white-apricot (SWAP) and Transformer (Tra) alternative splicing factors, we found that the RS domain of SF2/ASF is neither necessary nor sufficient for targeting to the nuclear speckles. Although this RS domain is a nuclear localization signal, subnuclear targeting to the speckles requires at least two of the three constituent domains of SF2/ASF, which contain additive and redundant signals. In contrast, in two SR proteins that have a single RRM (SC35 and SRp20), the RS domain is both necessary and sufficient as a targeting signal to the speckles. We also show that RRM2 of SF2/ASF plays an important role in alternative splicing specificity: deletion of this domain results in a protein that, although active in alternative splicing, has altered specificity in 5′ splice site selection. These results demonstrate the modularity of SR proteins and the importance of individual domains for their cellular localization and alternative splicing function in vivo.     

Newly assembled snRNPs associate with coiled bodies before speckles, suggesting a nuclear snRNP maturation pathway.

BACKGROUND: Small nuclear ribonucleoproteins (snRNPs), which are essential components of the mRNA splicing machinery, comprise small nuclear RNAs, each complexed with a set of proteins. An early event in the maturation of snRNPs is the binding of the core proteins - the Sm proteins - to snRNAs in the cytoplasm followed by nuclear import. Immunolabelling with antibodies against Sm proteins shows that splicing snRNPs have a complex steady-state localisation within the nucleus, the result of the association of snRNPs with several distinct subnuclear structures. These include speckles, coiled bodies and nucleoli, in addition to a diffuse nucleoplasmic compartment. The reasons for snRNP accumulation in these different structures are unclear. RESULTS: When mammalian cells were microinjected with plasmids encoding the Sm proteins B, D1 and E, each tagged with either the green fluorescent protein (GFP) or yellow-shifted GFP (YFP), a pulse of expression of the tagged proteins was observed. In each case, the newly synthesised GFP/YFP-labelled snRNPs accumulated first in coiled bodies and nucleoli, and later in nuclear speckles. Mature snRNPs localised immediately to speckles upon entering the nucleus after cell division. CONCLUSIONS: The complex nuclear localisation of splicing snRNPs results, at least in part, from a specific pathway for newly assembled snRNPs. The data demonstrate that the distribution of snRNPs between coiled bodies and speckles is directed and not random.     

Conservation and function of a potential substrate-binding domain in the yeast Clb5 B-type cyclin

Cyclin A contains a region implicated in binding to the p27 inhibitor and to substrates. There is strong evolutionary conservation of surface residues contributing to this region in many cyclins, including yeast B-type cyclins, despite the absence of a yeast p27 homolog. The yeast S-phase B-type cyclin Clb5p interacted with mammalian p27 in a two-hybrid assay. This interaction was disrupted by mutations designed to disrupt hydrophobic interactions (hpm mutation) or hydrogen bonding (Q241A mutation) based on the cyclin A-p27 crystal structure. In contrast, mutation of the Clb5p p27-binding domain only slightly reduced binding and inhibition by the Sic1p Clb-Cdc28p kinase inhibitor. Mutations disrupting the p27-binding domain strongly reduced Clb5p biological activity in diverse assays without reducing Clb5p-associated kinase activity. An analogous hpm mutation in the mitotic cyclin Clb2p reduced mitotic function, but in some assays this mutation increased the ability of Clb2p to perform functions normally restricted to Clb5p. These results support the idea of a modular, structurally conserved cyclin domain involved in substrate targeting.