A novel human protein, ASK (activator of S phase kinase), was identified on the basis of its ability to bind to human Cdc7-related kinase (huCdc7). ASK forms an active kinase complex with huCdc7 that is capable of phosphorylating MCM2 protein. ASK appears to be the major activator of huCdc7, since immunodepletion of ASK protein from the extract is accompanied by the loss of huCdc7-dependent kinase activity. Expression of ASK is regulated by growth factor stimulation, and levels oscillate through the cell cycle, reaching a peak during S phase. Concomitantly, the huCdc7-dependent kinase activity significantly increases when cells are in S phase. Furthermore, we have demonstrated that ASK serves an essential function for entry into S phase by showing that microinjection of ASK-specific antibodies into mammalian cells inhibited DNA replication. Our data show that ASK is a novel cyclin-like regulatory subunit of the huCdc7 kinase complex and that it plays a pivotal role in G1/S transition in mammalian cells.

The D-type cyclins and their major kinase partners CDK4 and CDK6 regulate G 0 -G 1 -S progression by contributing to the phosphorylation and inactivation of the retinoblastoma gene product, pRB. Progress through the G 1 phase of the mammalian cell cycle is regulated by the ordered synthesis, assembly, and activation of distinct cyclin-CDK holoenzymes (45, 46). Cyclins D1, D2, and D3 are up-regulated as cells exit from quiescence and associate with their major kinase partners CDK4 and CDK6 (3, 29, 32, 53). These two kinase molecules are highly homologous and associate exclusively with the D-type cyclins (3). Numerous studies have implicated cyclin D-CDK4-CDK6 complexes as key regulators of the cell cycle up to a hypothetical point during late G 1 (24, 25), the restriction point, when hyperphosphorylation and inactivation of the retinoblastoma tumor suppressor gene product, pRB, occur (37,44).In contrast to mitotic cyclin-CDK complexes, the D-type cyclins do not automatically assemble into complexes with either CDK4 or CDK6. For example, when overexpressed in NIH 3T3 cells in the absence of serum, D-type cyclins and CDK4 do not interact efficiently (30). Hence, assembly of Dtype cyclins and CDK4 and CDK6 into functional complexes in vivo is likely to depend on numerous factors, in particular, synthesis rates and stability of the various components. Indeed, the D-type cyclins possess canonical PEST sequences near their C termini and have short half-lives in vivo (4,31

Progression into G(1) in B lymphocytes is regulated by cyclins D2 and D3, components of the cell cycle machinery currently believed to have overlapping and potentially redundant roles in cell cycle control. To study the specific role of cyclin D2 in B lymphocyte proliferation, we examined B cells from cyclin D2(-/-) mice and demonstrate a specific requirement for cyclin D2 in BCR- but not CD40- or lipopolysaccharide-induced proliferation. Furthermore, conventional B cell development proceeds normally in the mutant mice; however, the CD5 B cell compartment is dramatically reduced, suggesting that cyclin D2 is important in CD5 B cell development as well as antigen-dependent B cell clonal expansion.

According to the morphogen gradient concept, cells in one part of an embryo secrete diffusible molecules (morphogens) that spread to other nearby cells and activate genes at different threshold concentrations. Strong support for the operation of a morphogen gradient mechanism in vertebrate development has come from the biochemical experiments of Green and Smith, who induced different kinds of gene expression in amphibian blastula cells exposed to small changes in activin concentration. But the interpretation of these experiments has been complicated by recent reports that cells tested for gene expression 3 hours after exposure to activin fail to show the graded response previously reported at 15 hours, a result suggesting that cells recognize their position in a gradient by an indirect mechanism. Here we conclude from the in situ analysis of blastula tissue containing activin-loaded beads that cells respond directly to changing morphogen concentrations in a way that resembles a ratchet-like process.

SWI-SNF complexes have been implicated in transcriptional regulation by chromatin remodeling. We have identified an interaction between two components of the mammalian SWI-SNF complex and cyclin E, an essential cell cycle regulatory protein required for G 1 /S transition. BRG1 and BAF155, mammalian homologs of yeast SWI2 and SWI3, respectively, are found in cyclin E complexes and are phosphorylated by cyclin E-associated kinase activity. In this report, we show that overexpression of BRG1 causes growth arrest and induction of senescence-associated ␤-galactosidase activity, which can be overcome by cyclin E. Our results suggest that cyclin E may modulate the activity of the SWI-SNF apparatus to maintain the chromatin in a transcriptionally permissive state.Progression through the cell cycle is a tightly controlled process requiring many critical regulatory proteins (reviewed in reference 47). The cyclin-dependent kinases (cdks) and their regulatory cyclin subunits promote passage through each phase of the cell division cycle. The activation of cyclin-cdk complexes is strictly regulated both at the level of protein synthesis and destruction and by posttranslational modifications to dictate precisely when in the cell cycle each complex becomes active (28,36).Cyclin E is synthesized during the G 1 phase of the cell cycle and binds cdk2 to become maximally active at the G 1 /S boundary (10,26). Cyclin E-cdk2 complexes have been shown to play an essential and rate-limiting role in the transition between G 1 and S phase (40,44,52,53). The manner in which cyclin E-cdk2 promotes S-phase entry remains poorly defined, since few downstream effectors of cyclin E-cdk2 are known. One potential substrate is the protein product of the retinoblastoma tumor suppressor gene, pRb, which is also phosphorylated by the D-type cyclin-cdk complexes (9, 13, 24). However, unlike cyclin D, cyclin E remains essential in the absence of pRb, illustrating a fundamental difference between these two complexes and strongly suggesting that other key rate-limiting substrates exist for cyclin E-cdk2 (1, 33). Other targets identified more recently include SAP155, a component of the pre-mRNA splicing apparatus (46), and NPAT (58). The role of such molecules in modulating cell cycle progression has yet to be established.SWI-SNF complexes are evolutionarily conserved and have been implicated in transcriptional regulation through remodeling of chromatin structure (reviewed in references 5 and 42). Components of the SWI-SNF apparatus are believed to bind to chromatin and relieve nucleosome-mediated repression of transcription, thus providing access to transcriptional activators (7,21,27,31,42,45). While SWI-SNF complexes are nonessential in yeast, a second related complex, RSC, is required for yeast cell growth (3,4,32). In mammalian cells, SWI-SNF complexes have been implicated in hormone receptor activation and growth control (6,11,25,39,49). The ability of SWI-SNF complexes to regulate cell growth is believed to be mediated through the interaction of th...