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A 42 kb region on human chromosome 9p21 encodes for three distinct tumor suppressors, p16INK4A, p14ARF and p15INK4B, and is altered in an estimated 30-40% of human tumors. The expression of the INK4A-ARF-INK4B gene cluster is silenced by polycomb during normal cell growth and is activated by oncogenic insults and during aging. How the polycomb is recruited to repress this gene cluster is unclear. Here, we show that expression of oncogenic Ras, which stimulates the expression of p15INK4B and p16INK4A, but not p14ARF, inhibits the expression of ANRIL (antisense non-coding RNA in the INK4 locus), a 3.8 kb-long non-coding RNA expressed in the opposite direction from INK4A-ARF-INK4B. We show that the p15INK4B locus is bound by SUZ12, a component of polycomb repression complex 2 (PRC2), and is H3K27-trimethylated. Notably, depletion of ANRIL disrupts the SUZ12 binding to the p15INK4B locus, increases the expression of p15INK4B, but not p16INK4A or p14ARF, and inhibits cellular proliferation. Finally, RNA immunoprecipitation demonstrates that ANRIL binds to SUZ12 in vivo. Collectively, these results suggest a model in which ANRIL binds to and recruits PRC2 to repress the expression of p15INK4B locus. [ABSTRACT FROM AUTHOR]

It has been postulated that the product (pRB) of the retinoblastoma gene dissociates from the E2F-pRB complex upon phosphorylation by cyclin-dependent kinase(s) (cdk). However, there is no direct evident for the regulation of formation of the E2F-pRB complex via phosphorylation by purified cdk. Therefore, we investigated the regulation of formation of this complex by phosphorylation using pRB and purified cyclin A-cdk2, cyclin E-cdk2 or cyclin D1-cdk4. Purified pRB was incubated with nuclear extracts prepared from pRB-defective cells and then subjected to gel mobility shift assays. We confirmed that unphosphorylated pRB associated with various types of E2F but pRB has been phosphorylated by cyclin A-cdk2 did not. We found that E2F-pRB complexes were disrupted as a consequence of phosphorylation by cyclin A-cdk2, and the levels of the free forms of E2Fs increased. We also found that not only the E2F-pRB complexes but also the E2F-p107 complexes were disrupted upon phosphorylation by cyclin A-cdk2. Furthermore, E2F-pRB complexes were disrupted through phosphorylation by cyclin D1-cdk4 and cyclin E-cdk2, as well as by cyclin A-cdk2. These results clearly demonstrate that the phosphorylation of pRB and p107 by cdks regulates the formation of complexes between E2F and pRB or p107.

Cyclin-dependent kinase 2 (Cdk2) controls the transition from the G1 to the S phase in the mammalian cell cycle. We found by immunoblotting that anti-Cdk2 antibodies recognize three Cdk2 proteins (of 33, 34 and 39 kDa) in FRTL-5 and FRTL-Tc cells (malignantly transformed FRTL cells). Although 33 kDa protein is a phosphorylated form of 34 kDa protein previously reported, the nature of 39 kDa protein is unknown. In order to determine the nature of this protein, we screened a FRTL-5 cDNA library. Two cDNA clones of the rat homologue (rat Cdk2-alpha and -beta) of human Cdk2 were isolated. The open reading frame of rat Cdk2-alpha cDNA encoded a protein with 428 amino acids and has a high degree of conservation with human Cdk2. The calculated molecular weight of Cdk2-alpha protein is 33892 Da. The rat Cdk2-beta cDNA was identical to Cdk2-alpha cDNA except that it had extra 144 bp; this coincided with insertion of 48 amino acids into Cdk2-alpha protein between Met 196 and Val 197. The calculated molecular weight of Cdk2-beta protein is 39087 Da. Northern blot analysis indicated that the sizes of rat Cdk2-alpha and -beta mRNAs are approximately 2.5 kb and 3.0 kb, respectively. Partial proteolytic mapping showed that Cdk2-beta gene product is 39 kDa Cdk2 in the immunoblotting. We also found that Cdk2-beta protein binds to cyclin A and suc1 proteins. During G1-S phase in FRTL-Tc cells, Cdk2-alpha protein level is constant, but is gradually phosphorylated. In contrast, the level of Cdk2-beta protein increases through the S phase and decreases at the early G2 phase. These results suggest that a variant form of Cdk2 protein might be required for entry into the S phase of the cell cycle in FRTL-Tc cells.

Transcription factor E2F-1 has a putative consensus sequence for phosphorylation by cyclin dependent kinase (Ser-Pro-X-Lys/Arg). Therefore, we studied the phosphorylation of E2F-1 in vivo and in vitro and its biological functions. E2F-1 was prepared by immunoprecipitation with anti-E2F-1 antibody from IMR32 lysates and was effectively phosphorylated by human cyclin A-cdk2 which was expressed in insect cells using baculovirus system. GST-E2F-1 was phosphorylated by cyclin A-cdk2 more efficiently than by cyclin E-cdk2. Cyclin D1-cdk4 phosphorylated pRB but scarcely phosphorylated GST-E2F-1 or H1 histone. The 60 kd protein precipitated with anti-E2F-1 antibody was phosphorylated in vivo. Phospho-peptide mapping indicated that its cleavage profile was identical with that of E2F-1 phosphorylated by cyclin A-cdk2 in vitro. This 60 kd protein, which is likely to be E2F-1, was not phosphorylated during the G0 and early G1 phase. Phosphorylation of E2F-1 began from the S phase while phosphorylation of pRB started nearly at G1/S. The in vivo phosphorylation of E2F-1 was inhibited by butyrolactone I, a cyclin-dependent kinase inhibitor (Kitagawa et al., 1993, Oncogene, 8, 2425-2432). The binding of E2F-1 to E2 promoter was found to be reduced by phosphorylation of E2F-1 by cyclin A-cdk2, suggesting that phosphorylation of E2F-1 may induce shut off of gene expression at the transcriptional level. These results suggest that E2F-1 is phosphorylated by cyclin A-cdk2 in the S phase in vivo as well as in vitro and that its phosphorylation by cyclin A-cdk2 may modulate its activity.

The transcription factor IRF-1 has been shown to function as a tumor suppressor. Here we report that a significant proportion of the IRF-1 mRNA detected in normal human hematopoietic cells and cultured cell lines lacks exon 2 (containing the AUG initiation codon) and 3 as a result of exon skipping. Surprisingly, when we examined the bone marrow and peripheral mononuclear cells from patients with myelodysplastic syndrome (MDS) or leukemia secondary to MDS, we could still detect the exon-skipped form but little or none of the intact IRF-1 mRNA. This appears to be the result of accelerated exon skipping since we could find no mutations within the exons and splicing junctions from these patients. The exon-skipped form of IRF-1 lacking exons 2 and 3 displayed neither DNA binding nor tumor suppressive activities. Thus this accelerated exon skipping may cause the inactivation of IRF-1 and thereby contribute to the development of human hematopoietic malignancies.

Butyrolactone I is a selective inhibitor of the cyclin-dependent kinase (cdk) family. It inhibits both cdk2 and cdc2 kinase, but scarcely affects C-kinase, A-kinase, casein kinases, MAP kinase or EGF receptor-tyrosine kinase (Kitagawa et al., 1993, Oncogene, 8, 2425-2432). We studied the effects of butyrolactone I on the cell cycle as well as on phosphorylation of retinoblastoma protein (pRB). Butyrolactone I inhibited phosphorylation of pRB catalyzed by cyclin A-cdk2 produced by baculovirus in vitro. Furthermore, it inhibited phosphorylation of pRB and cell cycle progression from G1 to S phase in WI38 cell cultures. WI38 cells arrested at the G0 phase by serum starvation progressed in the cell cycle after serum stimulation. pRB was phosphorylated after 10 h serum stimulation. Incorporation of [3H]thymidine into the cells began to increase after 16 h serum stimulation. These processes were inhibited by butyrolactone I. Flow cytometric analysis showed that exposure to butyrolactone I inhibited progression of the cell cycle from G1 to S phase. These data suggested that initiation of DNA synthesis was inhibited by butyrolactone I and that the cell cycle was arrested in the G1 phase. Butyrolactone I also inhibited H1 histone phosphorylation in human WI38 cells and their G2/M progression. tsFT210 cells, a temperature-sensitive cdc2 mutant cell line, were synchronized at G2/M at a nonpermissive temperature, butyrolactone I inhibited the cell cycle progression of these cells at G2/M at the permissive temperature. Thus butyrolactone I, a cyclin-dependent kinase family inhibitor, which prevented the phosphorylations of the cell cycle-regulating proteins pRB and H1 histone, inhibited the cell cycle at G1/S and G2/M, respectively. These results suggest that the phosphorylations of pRB and H1 histone may play crucial roles in G1/S and G2/M progression, respectively, although it is possible that phosphorylations of other proteins by cdks are involved in G1/S and G2/M progression.

We screened cdc2 kinase inhibitors from cultured mediums of micro organisms using purified mouse cyclin B-cdc2 kinase and a specific substrate peptide for cdc2 kinase. A selective inhibitor of cdc2 kinase was isolated from the cultured medium of Aspergillus species F-25799, and identified as butyrolactone I. Butyrolactone I inhibited cdc2 and cdk2 kinases but it had little effect on mitogen-activated protein kinase, protein kinase C, cyclic-AMP dependent kinase, casein kinase II, casein kinase I or epidermal growth factor-receptor tyrosine kinase. Its inhibitory effect was found to be due to competition with ATP. Butyrolactone I selectively inhibited the H1 histone phosphorylation in nuclear extracts. It also inhibited the phosphorylation of the product of retinoblastoma susceptibility gene in nuclear extracts and intact cells. Thus butyrolactone I should be very useful for elucidating the function of cdc2 and cdk2 kinases in cell cycle regulation.

The growth-suppressive activity of the retinoblastoma (RB) protein is suggested to be regulated by phosphorylation. In studies on the kinase that phosphorylates the RB proteins, we have previously found that RB proteins can be phosphorylated by purified cdc2 kinase. In this study, we noted that RB proteins immunoprecipitated from human cell lysates are weakly phosphorylated in the absence of purified cdc2 kinase. Immunoblot analysis showed the presence of p34cdc2 in the immunoprecipitates with anti-RB monoclonal antibody. In addition, the coprecipitated kinase was found to have the same substrate specificity as cdc2 kinase. The associated kinase activity was particularly high in cells arrested in G1/S and S phase by aphidicolin. Furthermore, RB proteins were shown to be phosphorylated in nuclear extracts by some endogenous cdc2-like kinase(s). These results suggest that cdc2-like kinase is the main kinase for phosphorylation of RB proteins in vivo.