Programmed destruction of regulatory proteins through the ubiquitin-proteasome system is a widely used mechanism for controlling signalling pathways. Cullins are proteins that function as scaffolds for modular ubiquitin ligases typified by the SCF (Skp1-Cul1-F-box) complex. The substrate selectivity of these E3 ligases is dictated by a specificity module that binds cullins. In the SCF complex, this module is composed of Skp1, which binds directly to Cul1, and a member of the F-box family of proteins. F-box proteins bind Skp1 through the F-box motif, and substrates by means of carboxy-terminal protein interaction domains. Similarly, Cul2 and Cul5 interact with BC-box-containing specificity factors through the Skp1-like protein elongin C. Cul3 is required for embryonic development in mammals and Caenorhabditis elegans but its specificity module is unknown. Here we report the identification of a large family of BTB-domain proteins as substrate-specific adaptors for C. elegans CUL-3. Biochemical studies using the BTB protein MEL-26 and its genetic target MEI-1 (refs 12, 13) indicate that BTB proteins merge the functional properties of Skp1 and F-box proteins into a single polypeptide.

Cyclin E/Cdk2 acts at the G1/S-phase transition to promote the E2F transcriptional program and the initiation of DNA synthesis. To explore further how cyclin E/Cdk2 controls S-phase events, we examined the subcellular localization of the cyclin E/Cdk2 interacting protein p220 NPAT and its regulation by phosphorylation. p220 is localized to discrete nuclear foci. Diploid fibroblasts in Go and G1 contain two p220 foci, whereas S-and G2-phase cells contain primarily four p220 foci. Cells in metaphase and telophase have no detectable focus. p220 foci contain cyclin E and are coincident with Cajal bodies (CBs), subnuclear organelles that associate with histone gene clusters on chromosomes 1 and 6. Interestingly, p220 foci associate with chromosome 6 throughout the cell cycle and with chromosome 1 during S phase. Five cyclin E/Cdk2 phosphorylation sites in p220 were identified. Phospho-specific antibodies against two of these sites react with p220 within CBs in a cell cycle-specific manner. The timing of p220 phosphorylation correlates with the appearance of cyclin E in CBs at the G1/S boundary, and this phosphorylation is maintained until prophase. Expression of p220 activates transcription of the histone H2B promoter. Importantly, mutation of Cdk2 phosphorylation sites to alanine abrogates the ability of p220 to activate the histone H2B promoter. Collectively, these results strongly suggest that p220 NPAT links cyclical cyclin E/Cdk2 kinase activity to replication-dependent histone gene transcription.

Highly ordered hexagonal mesoporous aluminosilicates (MAS-5) with uniform pore sizes have been successfully synthesized from assembly of preformed aluminosilcate precursors with cetyltrimethylammonium bromide (CTAB) surfactant. The aluminosilicate precursors were obtained by heating, at 100--140 degrees C for 2--10 h, aluminasilica gels at the Al(2)O(3)/SiO(2)/TEAOH/H(2)O molar ratios of 1.0/7.0--350/10.0--33.0/500--2000. Mesoporous MAS-5 shows extraordinary stability both in boiling water (over 300 h) and in steam (800 degrees C for 2 h). Temperature-programmed desorption of ammonia shows that the acidic strength of MAS-5 is much higher than that of MCM-41 and is comparable to that of microporous Beta zeolite. In catalytic cracking of 1,3,5-triisopropylbenzene and alkylation of isobutane with butene, MAS-5 exhibits greater catalytic activity and selectivity, as compared with MCM-41 and HZSM-5. The MAS-5 samples were characterized with infrared, UV--Raman, and NMR spectroscopy and numerous other techniques. The results suggest that MAS-5 consists of both mesopores and micropores and that the pore walls of MAS-5 contain primary and secondary structural building units, similar to those of microporous zeolites. Such unique structural features might be responsible for the observed strong acidity and high thermal stability of the mesoporous aluminosilicates with well-ordered hexagonal symmetry.

Genome replication in eukaryotic cells necessitates the stringent coupling of histone biosynthesis with the onset of DNA replication at the G 1 /S phase transition. A fundamental question is the mechanism that links the restriction (R) point late in G 1 with histone gene expression at the onset of S phase. Here we demonstrate that HiNF-P, a transcriptional regulator of replication-dependent histone H4 genes, interacts directly with p220NPAT , a substrate of cyclin E/CDK2, to coactivate histone genes during S phase. HiNF-P and p220 are targeted to, and colocalize at, subnuclear foci (Cajal bodies) in a cell cycle-dependent manner. Genetic or biochemical disruption of the HiNF-P/p220 interaction compromises histone H4 gene activation at the G 1 /S phase transition and impedes cell cycle progression. Our results show that HiNF-P and p220 form a critical regulatory module that directly links histone H4 gene expression at the G 1 /S phase transition to the cyclin E/CDK2 signaling pathway at the R point.Fidelity of genome replication in eukaryotic cells is essential for cell division and necessitates the stringent coupling of histone biosynthesis with DNA replication to ensure that nascent DNA is immediately assembled into chromatin during DNA synthesis. Cell division requires staged expression of genes in response to growth factors, which induce cell growth from quiescence or maintain competency for cell cycle progression during periods of active proliferation. Stimulation of cell proliferation initially triggers a cyclin/cyclin-dependent kinase (CDK) cascade, which activates the cyclin E/CDK2 kinase complex at the restriction (R) point (17,19). The R point is the major cell cycle checkpoint that controls the commitment for DNA replication in late G 1 via CDK2-dependent release of E2F from Rb-related proteins. The R point is mechanistically linked through E2F to activate the gene regulatory program necessary for nucleotide metabolism and DNA replication (17,19). Passage beyond the R point permits growth factor-independent entry into S phase and subsequent cell cycle stages. However, cell cycle progression remains constrained by multiple checkpoints, including surveillance mechanisms that monitor DNA integrity and fidelity of chromatin assembly.We postulate that the induction of histone gene expression at the G 1 /S phase transition represents a second necessary cell cycle regulatory event. The coupling of DNA synthesis with histone protein production is maintained by coordinately inducing expression of the multiple core histone gene subtypes, including the 15 distinct histone H4 genes, at the onset of S phase (1,3,12,20,23,24). The cell cycle regulatory sequence of histone H4 genes lacks E2F binding sites (28). We have recently identified the key transcription factor of H4 genes, histone nuclear factor P (HiNF-P), which interacts with a highly conserved histone H4 subtype-specific element in the site II cell cycle regulatory domain (16). HiNF-P supports histone gene transcription at the G 1 /S phase transition indepe...

Cyclin E/Cdk2, a central regulator of the G 1 /S transition, coordinates multiple cell cycle events, including DNA replication, centrosome duplication, and activation of the E2F transcriptional program. Recent studies suggest a role for cyclin E/Cdk2 in activation of histone transcription during S phase via the Cajal bodyassociated protein p220NPAT , and in addition, p220 can promote S-phase entry independently of histone transcriptional activation when overexpressed. Here we have examined the requirement for p220 in histone transcription, cell cycle progression, and Cajal body function through analysis of human somatic HCT116 cells engineered to contain a conditional p220 allele. p220 is required for proliferation of HCT116 cells, as assessed after expression of Cre recombinase in p220 flox/؊ cells. This defect was due to an inability of these cells to transit from G 0 /G 1 into S phase, and cell cycle arrest occurred in the presence of elevated Cdk2 kinase activity. Expression of human papillomavirus E7, but not E6, eliminated cell cycle arrest in response to p220 depletion. Optimal expression of all four core histone genes required p220, as did optimal transcription of a histone H4 promoter-luciferase construct. Basal histone H4 expression in G 0 /G 1 , although p220 dependent, occurs in the absence of detectable phosphorylation of p220 on Cdk2 sites. Cells lacking p220 displayed defects in the localization of the Cajal body component p80 coilin as cells progressed from G 0 to S phase in response to mitogenic signals. These finding indicate that p220 is an essential downstream component of the cyclin E/Cdk2 signaling pathway and functions to coordinate multiple elements of the G 1 /S transition.