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PubMed2003

Cyclin L2, a novel RNA polymerase II-associated cyclin, is involved in pre-mRNA splicing and induces apoptosis of human hepatocellular carcinoma cells.

We report the cloning and functional characterization of human cyclin L2, a novel member of the cyclin family. Human cyclin L2 shares significant homology to cyclin L1, K, T1, T2, and C, which are involved in transcriptional regulation via phosphorylation of the C-terminal domain of RNA polymerase II. The cyclin L2 protein contains an N-terminal "cyclin box" and C-terminal dipeptide repeats of alternating arginines and serines, a hallmark of the SR family of splicing factors. A new isoform and the mouse homologue of human cyclin L2 have also been cloned in this study. Human cyclin L2 is expressed ubiquitously in normal human tissues and tumor cells. We show here that cyclin L2 co-localizes with splicing factors SC-35 and 9G8 within nuclear speckles and that it associates with hyperphosphorylated, but not hypophosphorylated, RNA polymerase II and CDK p110 PITSLRE kinase via its N-terminal cyclin domains. It can also associate with the SC-35 and 9G8 through its RS repeat region. Recombinant cyclin L2 protein can stimulate in vitro mRNA splicing. Overexpression of human cyclin L2 suppresses the growth of human hepatocellular carcinoma SMMC 7721 cells both in vitro and in vivo, inducing cellular apoptosis. This process involves up-regulation of p53 and Bax and decreased expression of Bcl-2. The data suggest that cyclin L2 represents a new member of the cyclin family, which might regulate the transcription and RNA processing of certain apoptosis-related factors, resulting in tumor cell growth inhibition and apoptosis.

Amino Acid Sequence↗
PubMed1997

Cyclin ET, a new splice variant of human cyclin E with a unique expression pattern during cell cycle progression and differentiation.

Cyclin E is the regulatory subunit of the cdc2-related protein kinase cdk2 and is a rate limiting factor for the entry into S phase. To date, cyclin E is the only cyclin for which alternative splicing has been described. We report here the isolation of a new splice variant of cyclin E, termed cyclin ET, which has an internal deletion of 45 amino acids compared with the full-length cyclin E protein. Even though cyclin ETcontains an intact cyclin box, it is unable to complement a triple cln mutant strain of Saccharomyces cerevisiae or to interfere with rescue by cyclin E, indicating that an intact cyclin box is functionally insufficient. The expression pattern of cyclin ET during cell cycle entry, progression and differentiation differs from that of cyclin E. Thus, ET expression precedes that of the other isoforms during the G0-->S progression; it shows a sharp peak in early G1 in cells released from a mitotic block and is strongly down-regulated in terminally differentiated myeloid cells. These observations point to different functions for cyclin ET and E and show for the first time that the alternative splicing of cyclin E is a regulated mechanism governed by the cell cycle and differentiation.

Alternative Splicing↗
PubMed2000

Down-regulation of cyclin D1 expression by prostaglandin A(2) is mediated by enhanced cyclin D1 mRNA turnover.

Prostaglandin A(2) (PGA(2)), an experimental chemotherapeutic agent, causes growth arrest associated with decreased cyclin D1 expression in several cancer cell lines. Here, using human non-small-cell lung carcinoma H1299 cells, we investigated the mechanisms whereby PGA(2) down-regulates cyclin D1 expression. Transcription rates of the cyclin D1 gene, studied using a cyclin D1 promoter-luciferase construct and nuclear run-on assays, were not affected by PGA(2) treatment. Instead, the cyclin D1 mRNA was rendered unstable after exposure to PGA(2). Since the stability of labile mRNA is modulated through binding of proteins to specific mRNA sequences, we sought to identify protein(s) recognizing the cyclin D1 mRNA. In electrophoretic mobility-shift assays using radiolabeled RNA probes derived from different regions of cyclin D1 mRNA, we observed that (i) lysates prepared from PGA(2)-treated cells exhibited enhanced protein-cyclin D1 RNA complex formation; (ii) the kinetics of complex formation correlated closely with that of cyclin D1 mRNA loss; and (iii) binding occurred within a 390-base cyclin D1 3' untranslated region (UTR) (K12). This binding activity could be cross-linked, revealing proteins ranging from 30 to 47 kDa. The RNA-binding protein AUF1, previously associated with the degradation of target mRNAs, bound cyclin D1 mRNA, because anti-AUF1 antibodies were capable of supershifting or immunoprecipitating cyclin D1 mRNA-protein complexes. Finally, insertion of K12 in the 3'UTR of reporter genes markedly reduced the expression and half-life of the resulting chimeric mRNAs in transfected, PGA(2)-treated cells. Our data demonstrate that PGA(2) down-regulates cyclin D1 expression by decreasing cyclin D1 mRNA stability and implicates a 390-base element in the 3'UTR in this regulation.

3' Untranslated Regions↗
PubMed1997

Estrogen-induced cyclin D1 and D3 gene expressions during mouse uterine cell proliferation in vivo: differential induction mechanism of cyclin D1 and D3.

D-type cyclins are involved in the regulation of the G1/S transition of the cell cycle in various cell types cultured in vitro. Little is, however, known about the expression pattern and functional role of D-type cyclins in physiological processes in vivo. In this report, we studied whether the expression of murine D-type cyclins correlates with the states of mouse uterine cell proliferation in vivo. Time-course changes in cyclin D1 and D3 mRNA levels in the uterine tissues of immature mice primed with 17 beta-estradiol (E2) were examined by Northern blot hybridization. c-fos and thymidine kinase (TK) mRNA levels were also examined as markers for the transition from G0 to G1 and the onset of S phase, respectively. Cyclin D1 and D3 mRNAs were induced 2.5-fold between c-fos and TK mRNA peaks. The E2-induced cyclin D1 and D3 gene expressions were blocked by antiestrogens tamoxifen and ICI 182,780. We also investigated the effects of cycloheximide (CHX), a protein synthesis inhibitor, on cyclin D1 and D3 gene expressions. When CHX was treated alone, cyclin D3, but not cyclin D1, mRNA was immediately superinduced. The E2-induced cyclin D3 gene expression was shifted by approximately 6 h when CHX was pretreated 1 hr before E2 administration. Interestingly, the 3H-thymidine incorporation experiment showed that the mouse uterine cell cycle progression also shifted by 6 hr with pretreatment of CHX. The overall results suggest that both cyclin D1 and D3 mRNAs are constitutively expressed in uterine tissues and induced by E2 at G1 phase of the mouse uterine cell cycle. However, the superinducibility and temporal shift of cyclin D3 by CHX suggest that there is a different regulatory mechanism underlying cyclin D1 and D3 gene expressions in the mouse uterine cell cycle progression.

Animals↗
PubMed1999

Flow cytometry study of human cyclin B1 and cyclin E expression in leukemic cell lines: cell cycle kinetics and cell localization.

Experiments by flow cytometry (FCM) after nuclei isolation have never been done to investigate cyclins. We have conducted different experiments by FCM using whole cells and isolated nuclei to study the immunolocalization and kinetic patterns of cyclin B1 and cyclin E in various leukemic cell lines. During asynchronous growth, all whole cells had a scheduled, cell cycle phase-restricted expression of cyclin B1. By using a washless immunostaining of unfixed nuclei, cyclin B1 was detected in all cell cycle phases, including G1, although to a lesser extent than in G2/M, suggesting that in whole cells the cyclin B1 epitope is masked and accessible only in isolated nuclei. When the cells were synchronized at the G1/S boundary by thymidine or in the G1 phase by sodium n-butyrate, an identical accumulation of cyclin B1 was observed. As for cyclin E, its expression was higher with thymidine treatment than with sodium n-butyrate, particularly in nuclei. The elevated cyclin B1 level in the cells arrested at the G1/S boundary may reflect the increased half-life of this protein stabilized as the result of cyclin E overexpression. However, our FCM data also support the notion that accumulation of human cyclin B1 in leukemic cell lines begins during the G1 phase of the cell cycle, probably in the nucleus. The detection of cyclin B1 by Western blot in cells sorted in the G1 phase of the cell cycle confirms this finding. It is possible, therefore, that tumor transformation or leukemic phenotype may invariably be associated with altered cyclin B1 expression.

Blotting, Western↗
PubMed1996

Functional analysis of the human cyclin D2 and cyclin D3 promoters.

The D-type cyclins promote progression through the G1 phase of the cell cycle and may provide a link between growth factors and the cell cycle machinery. We determined the nucleotide sequence of the 5'-flanking region of the human cyclin D2 and cyclin D3 genes and identified the transcription start sites. Analysis of the upstream sequences required for transcription of the cyclin D2 and cyclin D3 genes in continuously dividing cells revealed marked differences in their regulatory elements. In the cyclin D2 gene positive elements were localized between positions -306 and -114 relative to the ATG codon at +1. Additional positive elements were localized between -444 and -345, whereas sequences that reduced transcription were identified between nucleotides -1624 and -892. In the cyclin D3 gene all of the positive elements required for maximal transcription were localized between nucleotides -366 and -167, and no negative elements were found. The activities of a reporter gene linked to the upstream regulatory sequences of the cyclin D2 gene but not the cyclin D3 gene were induced when starved cells were serum stimulated. This suggests that although the abundance of both the cyclin D2 and cyclin D3 mRNAs is increased by serum stimulation, only the cyclin D2 gene is up-regulated at the transcriptional level. Sequences between nucleotides -306 and -1624 of the cyclin D2 gene were necessary for serum inducibility.

Animals↗
PubMed1997

Transforming growth factor-alpha enhances cyclin D1 transcription through the binding of early growth response protein to a cis-regulatory element in the cyclin D1 promoter.

Cyclin D1 is a critical oncogene involved in the regulation of progression through the G1 phase of the cell cycle, thereby contributing to cell proliferation. This is mediated through interaction of cyclin D1 with its catalytic partners, the cyclin-dependent kinases, and the subsequent phosphorylation of the retinoblastoma protein. Cyclin D1, in turn, is regulated by mitogenic stimuli. We demonstrate that transforming growth factor-alpha (TGFalpha) induces cyclin D1 mRNA in esophageal squamous epithelial cells, and this appears to correlate with increased cyclin D1 protein expression and cyclin-dependent kinase 6 activity. The induction of cyclin D1 transcription by TGFalpha is mediated in part through the induction of the early growth response protein (Egr-1) and its subsequent binding of Egr-1 to a cis-regulatory region spanning nucleotides -144 to -104 of the cyclin D1 promoter. The Egr-1 binding activity to the cyclin D1 promoter appears to require de novo protein synthesis and is not influenced by Sp1 binding to overlapping Sp1 motifs. Taken together, these data provide evidence that TGFalpha enhances cyclin D1 transcription through the induction of Egr-1 binding to a cis-regulatory region in the cyclin D1 promoter. This has important mechanistic implications into the transcriptional regulation of cyclin D1 by an essential proproliferative growth factor and cell cycle progression.

Cyclin D1↗
PubMed2000

Degradation of cyclin A does not require its phosphorylation by CDC2 and cyclin-dependent kinase 2.

Many cyclins are degraded by the ubiquitination/proteasome pathways involving the anaphase-promoting complex and SCF complexes. These degradations are frequently dependent on phosphorylation by cyclin-dependent kinases (CDKs), providing a self-limiting mechanism for CDK activity. Here we present evidence from in vitro and in vivo assay systems that the degradation of human cyclin A can be inhibited by kinase-inactive mutants of CDK2 and CDC2. One obvious interpretation of these results is that like other cyclins, CDK-dependent phosphorylation of the cyclin A may be involved in cyclin A degradation. Our data indicated that CDK2 can phosphorylate cyclin A on Ser-154. Site-directed mutagenesis of Ser-154 abolished the phosphorylation by recombinant CDK2 in vitro and the majority of cyclin A phosphorylation in the cell. Activation of CDK2 and binding to SKP2 or p27(KIP1) were not affected by the phosphorylation of Ser-154. Surprising, in marked contrast to cyclin E, where phosphorylation of Thr-380 by CDK2 is required for proteolysis, degradation of cyclin A was not affected by Ser-154 phosphorylation. It is likely that the stabilization of cyclin A by the kinase-inactive CDKs was mainly due to a cell cycle effect. These data suggest an important difference between the regulation of cyclin A and cyclin E.

CDC2 Protein Kinase↗
PubMed1994

Differential expression and regulation of Cyclin D1 protein in normal and tumor human cells: association with Cdk4 is required for Cyclin D1 function in G1 progression.

In this study we have surveyed by immunoblotting the protein levels of Cyclin D1, D2, D3 and their catalytic partners, Cdk4 and Cdk6 in normal and transformed human cells. We found that all these proteins were differentially expressed in diploid cells derived from different tissues, in contrast to Cyclin E, Cyclin A and Cdk2 which are ubiquitously expressed. D-type Cyclins were never dramatically overexpressed and often very poorly expressed in tumor cell lines when compared to the levels in their normal counterparts. In contrast, Cdk4 was expressed at high levels in several tumor cell lines and Cdk6 was ectopically expressed in two sarcoma lines, suggesting a possible involvement of these two Cdks in oncogenesis. Interestingly, low levels of Cyclin D1 and D3 proteins always correlated with functional inactivation of the retinoblastoma gene product (pRb). In cells displaying active pRb, Cyclin D1 was found associated with Cdk4 regardless of whether the p53 gene was wild-type or mutant. Microinjection during G1 of Cyclin D1 anti-sense cDNA or anti-Cyclin D1 antibody in these cells arrested the cell cycle in G1. In cells lacking pRb function, Cyclin D1 was dissociated from Cdk4. Microinjection during G1 of Cyclin D1 antisense cDNA or anti-Cyclin D1 antibody in these cells did not affect G1 progression. These results show that (i) in the absence of pRb, Cyclin D1 is expressed at low levels, is dissociated from Cdk4 and becomes dispensable in G1; (ii) Cyclin D1 needs to be associated with its catalytic subunit, Cdk4, to function as a positive regulator of G1 progression.

CDC2 Protein Kinase↗
PubMed1993

G2 delay induced by nitrogen mustard in human cells affects cyclin A/cdk2 and cyclin B1/cdc2-kinase complexes differently.

We investigated the temporal regulation of cyclin A- and B1-dependent kinases in human lymphoma cells treated with nitrogen mustard (HN2) and pentoxifylline, to determine whether the activity of these complexes correlated with cell cycle arrest induced by DNA damage. Cells were synchronized in G1/S, treated with HN2, and then postincubated with pentoxifylline. HN2-induced a protracted delay in G2 phase. This delay correlated with suppression of cyclin B1- and cdc2-kinase activities, and stabilization of hyperphosphorylated-cdc2 in the presence of similar cyclin B1 levels to those found in mitosis. HN2 had no discernible effect on the S phase activity of cyclin A- or cdk2-immune complexes. Entry of control cells into mitosis correlated with destruction of cyclin A, disappearance of cyclin A-bound cdk2 and decreased cdk2 kinase activity. G2 delay induced by HN2 was associated with stabilization of cyclin A, increased abundance of cyclin A-bound cdk2, and increased cdk2 activity. Cyclin A was also associated with cdc2, which, contrary to complexes containing cdk2, were only activated upon entry into mitosis. Pentoxifylline abrogated cell cycle arrest induced by aphidicolin and HN2 in human lymphoma cells. Pentoxifylline also reverted the activity of cyclin A- and B1-kinases in HN2-treated cells to approximately that observed in controls. Our findings suggest that delayed entry into mitosis following DNA damage correlates with suppression of cyclin B1/cdc2 and cyclin A/cdc2 complexes, while maintaining cyclin A/cdc2 complexes in an active state. Furthermore, we found that pentoxifylline disrupts the signal transduction pathway that regulates these complexes when damaged DNA is present, resulting in abrogation of cell cycle arrest.

Antigen-Antibody Complex↗
PubMed2002

Estrogen activates cyclin-dependent kinases 4 and 6 through induction of cyclin D in rat primary osteoblasts.

Estrogen plays important roles in maintaining bone density and protecting against osteoporosis, but the underlying mechanisms of estrogen action via estrogen receptors (ERs) in bone remain to be clarified. In the present study, we isolated primary osteoblasts derived from transgenic rats harboring a dominant negative ER mutant, rat ERalpha (1-535) cDNA, and from their wild-type littermates. We observed that the rate of cell growth of osteoblasts from the transgenic rats was reduced compared to that of wild-type osteoblasts. Utilizing cDNA microarray analysis, we found that mRNA level of cyclin D2 was lower in the osteoblasts from the transgenic rats. D-type cyclins including cyclin D1, cyclin D2, and cyclin D3 are cell cycle regulators that promote progression through the early-to-mid G1 phase of the cell cycle. The protein levels of D-type cyclins including cyclin D2 and cyclin D3 but not cyclin D1 were elevated in wild-type osteoblasts with 17beta-estradiol treatment, resulting in the activation of cyclin-dependent kinases 4 and 6 (Cdk4/6) activities and the promotion of cell growth. Moreover, an anti-estrogen ICI 182,780 abolished the induction of the expression of D-type cyclins by 17beta-estradiol. Our findings indicate that estrogen and its receptors enhance Cdk4/6 activities through the induction of D-type cyclins, leading to the growth promotion of osteoblasts.

Animals↗
PubMed2004

Overexpression of G1-S cyclins and cyclin-dependent kinases during multistage human pancreatic duct cell carcinogenesis.

PURPOSE: Molecular analysis of pancreatic intraepithelial neoplasia lesions and ductal adenocarcinoma suggested a multistage paradigm for pancreatic duct cell carcinogenesis. This study investigated the molecular basis for the neoplastic duct cells in this pancreatic intraepithelial neoplasia-carcinoma sequence to acquire progressive enhancement of their proliferative potential. EXPERIMENTAL DESIGN: Using tissue microarray blocks containing 15 to 40 pancreatic intraepithelial neoplasia lesions and ductal adenocarcinoma of pancreas, we studied by immunohistochemistry the expression profiles of cyclins and cyclin dependent kinases (CDKs) that regulate the G1-S cell cycle checkpoints. The role of cyclins D3 and D1 in three pancreatic cancer cell lines was investigated using specific short interfering RNA technique. RESULTS: Cyclin D3 overexpression was noted the earliest in pancreatic intraepithelial neoplasia-1A and was prevalent in 90% to 100% of high-grade pancreatic intraepithelial neoplasias and ductal cancer. Cyclin A overexpression was also noted early and reached 50% to 100% of high-grade pancreatic intraepithelial neoplasias and cancer, but the percentage of abnormal duct cells showing overexpression of cyclin A was significantly lower than cyclin D3. Cyclin E overexpression occurred in 20% to 25% of high-grade pancreatic intraepithelial neoplasias and in 75% of ductal carcinoma. Cyclin D1 demonstrated the lowest frequency of overexpression that occurred late. CDK2 and CDK4 overexpression was also noted in early pancreatic intraepithelial neoplasias and progressively increased to reach 60% to 75% in carcinoma. The down-regulation of cyclin D3 mRNA and protein levels using specific short interfering RNA resulted in growth inhibition of pancreatic cancer cell lines. CONCLUSION: The results provide additional insight into the mechanism of G1-S cell cycle checkpoints deregulation during stepwise pancreatic duct cell carcinogenesis, and suggest a p16-independent role for cyclin D3 in deregulating the G1 cell cycle checkpoints during early stages of pancreatic duct cell carcinogenesis.

Blotting, Western↗
PubMed2001

Up-regulation of cyclin-dependent kinase 4/cyclin D2 expression but down-regulation of cyclin-dependent kinase 2/cyclin E in testicular germ cell tumors.

Testicular germ cell tumors (GCT) characteristically display two chromosome 12 abnormalities: the isochromosome i(12p) and concomitant deletions of the long arm. Some genes important in the control of the G(1)-S cell cycle checkpoint G(1)-S, i.e., cyclin-dependent kinases 2 and 4, cyclin D2 are located on this chromosomal region. Therefore, testicular GCTs were analyzed as to the expression of CDK2, CDK4, CDK6, and the expression of their catalytic partners cyclins D1, D2 and E by semiquantitative reverse transcription-PCR. Cyclin D2, located on 12p, was overexpressed in 69% (31 of 45) of the tumors by a mean factor of 8, including all histological subtypes. In addition, the cyclin D2 partner CDK4 was increased in 41% (21 of 51) of all tumors by a factor of 6, most strongly in embryonal carcinomas. Sixty-four percent of the seminomas and 23% of the non-seminomas had decreased expression of CDK6 by a mean factor of 5 (P = 0.009). Statistical analysis using configural frequency analysis and regression analysis revealed that cyclin D2 and CDK4 expression were strongly correlated (r(2) = 0.682; P = 0.000052), whereas expression of CDK6 did not correlate with either of them (r(2) = 0.382; P = 0.00085). CDK2 and its catalytic partner cyclin E were down-regulated in 40% (19 of 47) and 42% (19 of 45) of the tumors, respectively, by a factor of 7 each. Western blots and immunohistochemical experiments confirmed cyclin D2 and CDK4 overrepresentation and reduced expression of cyclin E and CDK2 tumors in the few tumors under protein study. Despite its localization on 12q13, a hot spot for loss of heterozygosity in testicular GCTs (>40%), Southern blotting revealed no gross DNA alteration of the CDK2 gene. Because up-regulation of the cyclin D2/CDK4 complex and down-regulation of cyclin E/CDK2 complex were found in seminomas as well as in non-seminomas and in all tumor stages, these findings seem to be early events during tumorigenesis of testicular GCTS: Together with previous findings that retinoblastoma mRNA and protein expression is strongly decreased in these tumors, these data suggest an unusual deregulated G(1)-S checkpoint as a decisive event for germ cell tumors.

CDC2-CDC28 Kinases↗
PubMed1995

Cyclin I: a new cyclin encoded by a gene isolated from human brain.

A new member of the cyclin family has been isolated from an equalized cDNA library derived from human forebrain cortex. This putative cyclin, designated cyclin I, contains a typical cyclin box near the N-terminus and a PEST sequence near the C-terminus. Cyclin I shows the highest sequence similarity in the cyclin box to cyclins G and E, while the similarity between cyclins I and G also extends toward the C-terminus from the cyclin box. Cyclin I mRNA was expressed at high levels in postmitotic tissues, including skeletal muscle, heart, and brain, and was expressed constantly during cell cycle progression. The expression of cyclin I mRNA does not correlate directly to the cell cycle, and therefore cyclin I may be a novel cyclin member that functions independently of the cell cycle control.

Adult↗
PubMed1998

Association of downregulation of cyclin D1 and of overexpression of cyclin E with p53 mutation, high tumor grade and poor prognosis in hepatocellular carcinoma.

BACKGROUND/AIMS: The major regulatory events leading to cell proliferation occur in the G1 phase of cell cycle, and the deranged expression of G1 cyclins is related to oncogenesis. In this study, we analyzed the aberrant expressions of cyclins D1 and E, and their role in hepatocellular carcinoma. METHODS: We examined paired hepatocellular carcinoma and liver RNAs taken from 71 patients who had been followed for more than 4 years after tumor resection, using reverse transcription-polymerase chain reaction supplemented with Northern blotting and immunohistochemistry. The genetic alterations of the p53 gene were also studied. RESULTS: Downregulation of cyclin D1 mRNA was detected in 29 hepatocellular carcinomas (40.8%), while overexpression was detected in only 4 hepatocellular carcinomas (5.6%). Downregulation of cyclin D1 was associated significantly with large hepatocellular carcinomas (p=0.0006) and poorly differentiated (grades III-IV) hepatocellular carcinoma (p=0.057), but not seen in any of 15 minute hepatocellular carcinomas (< or =2.5 cm in size). Cyclin E mRNA was overexpressed in 40 hepatocellular carcinomas, regardless of tumor size. Overexpression of cyclin E was significantly associated with poorly differentiated and invasive hepatocellular carcinoma (p=0.001 and p=0.015, respectively). Downregulation of cyclin D1 and overexpression of cyclin E were significantly associated with the p53 mutation (p=0.023 and p=0.005, respectively). Hepatocellular carcinomas expressing both downregulation of cyclin D1 and overexpression of cyclin E had the worst 4-year survival (p<0.03), and higher frequencies of the p53 mutation (p<0.001), large hepatocellular carcinoma (p<0.001), and invasive tumor (p<0.01). CONCLUSIONS: The deranged expressions of G1 cyclins correlate with the p53 mutation, tumor progression, and tumor biologic behavior of hepatocellular carcinoma. Overexpression of cyclin E occurs early, and downregulation of cyclin D1 late in hepatocellular carcinoma growth.

Carcinoma, Hepatocellular↗
PubMed1997

Temporal expressions of cyclins and cyclin dependent kinases during renal development and compensatory growth.

The family of cyclins and cyclin-dependent kinases (CDKs) are important participants in the regulation of eukaryotic cell cycle. Our purpose was to examine temporal expressions of cyclins and CDKs during renal development and compensatory growth. During embryonic development the mRNA levels of all cyclins were high, and after birth their levels decreased at different rates. G2 and M phase cyclins, cyclin A and B, decreased immediately after birth. G1 and S phase cyclins, cyclins D1, D2, D3, and E, were observed during all stages of development and maintained almost constant levels until seven days after birth. They decreased thereafter and expressed very low levels during the adult period. The protein levels of cdc2, CDK2, and proliferating cell nuclear antigen (PCNA) were high during embryonic renal development and slowly decreased after birth. Their levels were very low during the youth and adult periods. Levels of CDK4 protein were high and did not change during renal development. Compensatory hypertrophic renal growth (CHRG) induced by unilateral nephrectomy (Unx) did not increase any cyclins, CDKs or PCNA. Subtotal nephrectomy (Snx) did not increase any cyclins or CDKs in remaining viable renal tissue (RVRT). However, Snx increased PCNA in RVRT. An immunohistochemical study revealed that PCNA was induced in a limited area adjacent to ischemic areas. Interestingly, Western blot analysis of protein extracts from RVRT showed the induction of a new 40 kDa protein that cross-reacted with the cyclin D3 antibody. These findings suggest that the marked reductions in mitotic cyclins may be associated with the withdrawal of renal cell cycle after birth. In addition, expressions of cyclins and CDKs did not change in the adult kidney during active phase of compensatory hypertrophic growth.

Animals↗
PubMed1997

The cyclin box and C-terminus of cyclins A and E specify CDK activation and substrate specificity.

The cyclins and their catalytic partners, the Cyclin Dependent Kinases (CDKs), are essential for progression through the cell cycle. Cyclin/kinase complexes containing cyclins A or E are active primarily in late G1 to S phase and both have been shown to phosphorylate histone H1 and the retinoblastoma gene product (pRb) in vitro. Despite these similarities, cyclins A and E display differences in CDK activation and substrate specificity. We find that in vitro, cyclin E/CDK2 and cyclin A/CDK2 phosphorylate histone H1 similarly but only cyclin A/CDK2 phosphorylates lamin B. While both cyclin A and cyclin E bind CDK1 efficiently, only cyclin A activates CDK1 kinase activity. Using chimeric proteins between cyclins A and E we find that both the cyclin box and C-terminus of cyclins A and E are required for CDK binding, activation and targeting of substrate specificity.

Animals↗
PubMed1996

Studies on the in vitro phosphorylation of HSSB-p34 and -p107 by cyclin-dependent kinases. Cyclin-substrate interactions dictate the efficiency of phosphorylation.

Cyclin-dependent kinases (Cdks) are required for cell cycle progression. Two potentially significant Cdk substrates in human cells are the human single-stranded binding protein (HSSB or RPA), which plays an essential role in DNA replication, repair, and recombination, and the tumor suppressor p107 which acts to negatively regulate cell growth. In this report we describe the in vitro phosphorylation of these two proteins by Cdks in an attempt to understand how cyclin-substrate interactions direct phosphorylation efficiencies. We show that cyclin A-Cdk2 efficiently phosphorylates the p34 subunit of HSSB (HSSB-p34) alone or as a part of the heterotrimeric complex. In contrast, cyclin E-Cdk2 that is active in phosphorylating histone H1, does not support the phosphorylation of the p34 subunit of HSSB. We provide evidence that this differential phosphorylation results from a specific interaction between HSSB-p34 and cyclin A, but not cyclin E. Thus the observed cell cycle-dependent phosphorylation of HSSB-p34 at the G1 to S transition is most likely catalyzed by cyclin A-Cdk2 initiated by the direct interaction between cyclin A and the HSSB-p34 subunit. These studies are consistent with our previous observation that p107, which directly binds cyclin A, is efficiently phosphorylated by cyclin A-Cdk2 but not cyclin B-associated kinases. Here we further demonstrate that cyclin A only complexes with p107 in its unphosphorylated form. These data suggest a catalytic mechanism by which Cdk acts: substrate targeting by a cyclin-substrate interaction followed by dissociation of the Cdk upon phosphate incorporation allowing the Cdk to become available for the next cycle of phosphorylation.

Base Sequence↗