A cascade of events is triggered upon the addition of growth factor to quiescent mammalian cells, which ultimately restarts proliferation by inducing the transition from Go/G 1 to S-phase. We have studied cyclin D1, a putative G1 cyclin, in normal diploid human fibroblasts. Cyclin D1 accumulated and reached a maximum level before S-phase upon the addition of serum to quiescent cells. The protein was localized to the nucleus, and it disappeared from the nucleus as cells proceeded into S-phase. Microinjection of anti-cyclin D1 antibodies or antisense plasmid prevented cells from entering S-phase, and the kinetics of inhibition showed that cyclin D1 is required at a point in the cell cycle earlier than cyclin A. These results demonstrate that cyclin D1 is a critical target of proliferative signals in Gx.

The cellular action of growth factors, among them basic fibroblast growth factor (bFGF), is mediated by their interaction with a cell surface receptor, but the mechanism of transfer of mitogenic (or other) signals to the nucleus has not been identified. In this work, we show that bFGF is translocated to and accumulated in the nucleolus. Furthermore, the nucleolar localization of bFGF is correlated with a stimulation of transcription of ribosomal genes during Gq-.Gl transition induced by bFGF alone in adult bovine aortic endothelial cells (ABAE cells). Stimulation of ribosomal gene transcription is preceded by a significant increase of the major nonhistone nucleolar protein, nucleolin. In vitro, the growth factor has a direct effect on the enhancement of RNA polymerase I activity in isolated nuclei from quiescent sparse (GO) ABAE cells. The direct action of bFGF on the level of ribosomal gene transcription could correspond to an additional growthsignaling pathway, mediated by this growth factor.The family of fibroblast growth factors (FGFs) includes the factors described as endothelial cell growth factor, chondrosarcoma growth factor, and heparin-binding growth factors (1). Preliminary physical analysis of some of these mitogens has suggested their classification in two groups: acidic fibroblast growth factors (aFGFs) (2) and basic fibroblast growth factors (bFGFs) (3). In vitro, aFGFs and bFGFs are potent mitogens for a wide variety of mesoderm-and neuroectoderm-derived cells, including vascular and capillary endothelial cells (4) and, as in vivo (5), they induce the angiogenic response (6).The cellular action of FGFs is exerted through its interaction with specific cell surface receptors (7,8), but the intervening steps and the mechanism of transfer of mitogenic (or other) signals to the nucleus, leading to the "ple-otropic response" required to bring quiescent cells into full proliferation, are at present unknown.The proliferation state and ribosome biogenesis, which involve a series of coordinated nucleolar events, among them the transcription of ribosomal genes (rDNA), are closely related. The level of transcription of rDNA is modulated by cell growth conditions, growth-promoting hormones (9), and growth factors (10). A specific nucleolar protein, nucleolin, was shown in different eukaryotic cells to play a direct role in the control of the synthesis of the precursor to ribosomal RNA (pre-rRNA) and assembly of ribosomes (11,12). Barely detectable in resting cells, nucleolin represents up to 5% of nucleolar proteins in exponentially growing cells. In vitro, run-off experiments with rDNA as template have shown that endoproteolytic cleavage of phosphorylated nucleolin controls rDNA transcription (13).In this report, we have focused on the effects of bFGF on the reinitiation of ribosome biogenesis in cells undergoing the Gy-3G1 transition. We show by immunocytochemistry using a monospecific polyclonal anti-bFGF antibody that the reinitiation of pre-rRNA synthesis is preceded by the accumulation of nucleo...

Primary cultures of adult bovine aortic endothelial (ABAE) cells require bFGF to grow. G1‐arrested cells, obtained after 48 h without serum and bFGF, were found to enter S phase and grow synchronously for at least two generations on addition of bFGF. In growing cells bFGF was detected both in the cytoplasm (90%) and in the nucleus (10%) where it accumulates in the nucleolus. It was not detected in the nucleus of confluent cells. bFGF uptake was continuous in the cytoplasm throughout the cell cycle with a maximum in G2, while nuclear uptake occurred only in late G1. Cytoplasmic bFGF (18.4 kd) is cleaved into a 16.5 kd peptide in G1 (t1/2 = 30 min). In the nucleus the 18.4 kd form was the only one detected 2 h following bFGF addition and was then cleaved into the 16.5 kd in early S phase. These results are consistent with the possibility that in addition to the classical pathway of signal transduction, bFGF is directly translocated to the nucleus in late G1, and could play a role in replication and/or in transcription of rDNA.

Abstract. In mammalian cells inhibition of the cdc2function results in arrest in the G2-phase of the cell cycle. Several cdc2-related gene products have been identified recently and it has been hypothesized that they control earlier cell cycle events. Here we have studied the relationship between activation of one of these cdc2 homologs, the cdk2 protein kinase, and the progression through the cell cycle in cultured human fibroblasts. We found that cdk2 was activated and specifically localized to the nucleus during S phase and G2. Microinjection of afffinity-purified anti-cdk2 antibodies but not of affinity-purified anti-cdc2 antibodies, during G1, inhibited entry into S phase. The specificity of these effects was demonstrated by the fact that a plasmid-driven cdk2 overexpression counteracted the inhibition. These results demonstrate that the cdk2 protein kinase is involved in the activation of DNA synthesis.

The pEg3 protein is a member of the evolutionarily conserved KIN1/PAR-1/MARK kinase family which is involved in cell polarity and microtubule dynamics. In Xenopus, pEg3 has been shown to be a cell cycle dependent kinase whose activity increases to a maximum level during mitosis of the first embryonic cell division. CDC25B is one of the three CDC25 phosphatase genes identified in human. It is thought to regulate the G2/M progression by dephosphorylating and activating the CDK/cyclin complexes. In the present study we show that the human pEg3 kinase is able to specifically phosphorylate CDC25B in vitro. One phosphorylation site was identified and corresponded to serine 323. This residue is equivalent to serine 216 in human CDC25C which plays an important role in the regulation of phosphatase during the cell cycle and at the G2 checkpoint. pEg3 is also able to specifically associate with CDC25B in vitro and in vivo. We show that the ectopic expression of active pEg3 in human U2OS cells induces an accumulation of cells in G2. This effect is counteracted by overexpression of CDC25B. Taken together these results suggest that pEg3 is a potential regulator of the G2/M progression and may act antagonistically to the CDC25B phosphatase.

The CDC25B dual specificity phosphatase is involved in the control of the G2/M transition of the cell cycle. Subcellular localization might represent an important aspect of the regulation of its activity. We have examined in transiently transfected asynchronous HeLa cells the localization of HA-tagged CDC25B proteins and found that they are nuclear or cytoplasmic suggesting the existence of an active shuttling. Accordingly, localization analysis of deletion and truncation proteins indicates that CDC25B contains a putative nuclear localization signal located between residues 335 and 354. We also demonstrated that a short 58 residues deletion of the amino-terminus end of CDC25B is sufficient to retain it to the nucleus. Mutational analysis indicates that a nuclear export sequence is located between residues 28 and 40. In addition, treatment of the cells with the exportin inhibitor, Leptomycin B, has the same effect. The mutation of Ser-323, a residue that is essential for the interaction with 14-3-3 proteins, also abolishes cytoplasmic staining. The subcellular localization of CDC25B is therefore dependent on the combined effects of a nuclear localization signal, a nuclear export signal and on the interaction with 14-3-3 proteins.

CDC25B2, a protein tyrosine phosphatase closely related to the putative CDC25B oncogene, was identi®ed in a Burkitt lymphoma cDNA library. CDC25B2 di ers from CDC25B by a 14 residue insertion and a 41 residue deletion, which are both located in the amino-terminal region of the protein, upstream of the catalytic domain. Examination of the genomic sequence revealed that CDC25B1 (formerly B) and CDC25B2 are splice variants of the same gene. A third variant, CDC25B3, that carries both the 14 and the 41 residue sequences was also identi®ed in the same cDNA library. All three variants were detected in a panel of human primary culture and cell lines, although at di erent levels. In primary ®broblasts and in HeLa cells the CDC25B expression is cell cycle regulated, reaching a maximum in G 2 -phase. In vitro, CDC25B1 phosphatase is slightly more active than CDC25B2 and B3. However, episomal overexpression of the three CDC25B variants in ®ssion yeast reveals that in vivo, CDC25B2 is largely more active than either B1 or B3 (B24B34B1) both to complement a thermosensitive S pombe CDC25 activity and to act as a mitotic inducer. Alternative splicing of CDC25B may therefore contribute to the control of cell proliferation.

CDC25 dual-speci®city phosphatases are essential regulators that activate cyclin-dependent kinases (CDKs) at critical stages of the cell cycle. In human cells, CDC25A and C are involved in the control of G1/S and G2/M respectively, whereas CDC25B is proposed to act both in S phase and G2/M. Evidence for an interaction between CDC25 phosphatases and members of the 14-3-3 protein family has been obtained in vitro and in vivo in several organisms. On the basis of the work performed with CDC25C, it has been proposed that phosphorylation is required to mediate the interaction with 14-3-3. Here we have examined the molecular basis of the interaction between CDC25B phosphatases and 14-3-3 proteins. We show that in the two-hybrid assay all three splice variants of CDC25B interact similarly and strongly with 14-3-3Z, b and z proteins, but poorly with e and y. In vitro, CDC25B interacts at a low level with 14-3-3b, e, z, Z, and y isoforms. This interaction is not increased upon phosphorylation of CDC25B by CHK1 and is not abolished by dephosphorylation. In contrast, a speci®c, strong interaction between CDC25B and 14-3-3z and Z isoforms is revealed by a deletion of 288 residues in the amino-terminal region of CDC25B. This interaction requires the integrity of Ser 323, although it is independent of phosphorylation. Thus, interaction between 14-3-3 proteins and CDC25B is regulated in a manner that is dierent from that with CDC25C. We propose that, in addition to a low anity binding site that is available for all 14-3-3 isoforms, post-translational modi®cation of CDC25B in vivo exposes a highanity binding site that is speci®c for the z and Z14-3-3 isoforms.