Negative regulation of DNA replication by the retinoblastoma protein is mediated by its association with MCM7.

A yeast two-hybrid screen was employed to identify human proteins that specifically bind the amino-terminal 400 amino acids of the retinoblastoma (Rb) protein. Two independent cDNAs resulting from this screen were found to encode the carboxy-terminal 137 amino acids of MCM7, a member of a family of proteins that comprise replication licensing factor. Full-length Rb and MCM7 form protein complexes in vitro, and the amino termini of two Rb-related proteins, p107 and p130, also bind MCM7. Protein complexes between Rb and MCM7 were also detected in anti-Rb immunoprecipitates prepared from human cells. The amino-termini of Rb and p130 strongly inhibited DNA replication in an MCM7-dependent fashion in a Xenopus in vitro DNA replication assay system. These data provide the first evidence that Rb and Rb-related proteins can directly regulate DNA replication and that components of licensing factor are targets of the products of tumor suppressor genes.

Use of biocarrier beads and flow cytometry for single-cell studies of fibronectin gene regulation in dibutyryl cyclic AMP reverse transformed CHO-K1 cells.

The protease sensitivity of a number of cell surface or cytoskeletal components and the relationship of these to the substratum in attached cells has prevented the quantitative measurement of their expression by flow cytometry. Using traditional cell sorting techniques, cells must be treated with a protease to detach them from a substrate in order to produce a single-cell suspension. Unfortunately, proteolytic treatment alters or destroys a number of cellular proteins. Fibronectin either on the cell surface or as part of the substratum laid down by the cell is particularly sensitive to proteases, preventing its quantitative study by flow cytometry. To circumvent these problems and produce a single cell suspension necessary for flow cytometric analysis, CHO-K1, a Chinese hamster ovary cell line, were grown in suspension on specially-treated 25 microns biocarrier beads. The CHO-K1 cell line is composed of transformed epithelial-like cells that have lost the fibronectin deposit around their cell membranes. To restore the typical fibroblastic deposit of fibronectin, the cells attached to beads were induced by dibutyryl cAMP to undergo a reverse transformation reaction to restore fibroblastic morphology and the typical fibroblastic deposite of fibronectin on the cell surface and substratum. The cells attached to beads were then immunofluorescently labeled for the protease-sensitive, extracellular matrix component, fibronectin, and examined on a flow cytometer. Cell surface fibronectin heterogeneity was then examined on a cell-by-cell basis as a function of cell cycle using Hoechst 33342 dye that binds to AT base pairs of cellular DNA. Dual laser measurement and multiparameter list mode data analysis were used to study the relationship between cell surface fibronectin of biocarrier bead attached cells and cell cycle.

The amino terminus of the retinoblastoma (Rb) protein associates with a cyclin-dependent kinase-like kinase via Rb amino acids required for growth suppression.

We have shown previously that a novel cell cycle-regulated histone H1 kinase activity, retinoblastoma kinase (RbK), associates with and phosphorylates the amino terminus of the Rb protein in G2-M. We have shown also that the amino terminus of p107, a Rb-related protein, does not associate with a similar kinase in vitro or in vivo. Here, we report that a RbK-like kinase associates with the amino terminus of p130, another Rb-related protein, only marginally. Moreover, the association of RbK with Rb in vitro is shown to require a discrete portion of the Rb amino terminus, amino acids 89-202. This region has been shown previously to be subject to inactivating mutations in retinoblastoma and to be required for Rb-mediated growth suppression in vitro. Taken together, these data indicate that the formation of Rb-RbK complexes may play an important role in Rb-mediated growth suppression. We have mapped two in vitro sites of Rb phosphorylation by RbK to sites that are phosphorylated in vivo and are targets of cyclin-dependent kinase phosphorylation in vitro. As such, at least some sites of RbK phosphorylation overlap with those of other proline-directed serine and threonine kinases. Consistent with this latter observation, we report that the trans-activation domain of c-myc is phosphorylated specifically by RbK in vitro at a site (serine 62) that is phosphorylated in vivo during G2-M, cell-cycle phases in which RbK activity is maximal.

Detection of a novel cell cycle-regulated kinase activity that associates with the amino terminus of the retinoblastoma protein in G2/M phases.

Recent genetic and functional evidence suggests that the amino terminus of the retinoblastoma (Rb) protein plays an important role in Rb-mediated growth suppression. To explore the mechanism(s) by which this portion of Rb may regulate cell growth, we have sought to characterize cellular proteins that associate with the Rb amino terminus using an in vitro protein-binding assay. Here we report that at least one such protein is a cell cycle-regulated Rb/histone H1 kinase (RbK) whose enzymatic and/or Rb association activity is most prevalent in G2/M phases of cells. In contrast to previously characterized cyclin-dependent and Rb-associated kinases, such as cdk1 (cdc2) and cdk2, G2/M RbK 1) is not depleted by incubation with p13suc-beads, 2) is not detected with antisera against several Rb-associated cyclins-cdks, and 3) associated with Rb via the Rb amino terminus, a region that is dispensable for interaction with other Rb-associated kinases. RbK is clearly distinct from previously characterized mitotic cdks since cyclin A-cdc2, cyclin A-cdk2, cyclin B-cdc2, and cyclin B-cdk2 did not associate with the Rb amino terminus. Coprecipitation experiments with Rb antisera confirmed the association of Rb with a RbK-like kinase in metaphase-arrested cells in vivo. Interestingly, G2/M RbK did not appreciably associate with an analogous portion of p107, a Rb-related protein. Taken together, these data indicate that the Rb amino terminus specifically associates with a novel cell cycle-regulated kinase in late cell cycle stages.