Regulation of cell cycle molecules by the Ras effector system

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Abstract

Eukaryotic cell cycle progression is driven by an ordered array of phosphorylation events that are specifically catalyzed by members of CDK (cyclin-dependent kinase) family serine/threonine protein kinases, each consisting of a catalytic subunit CDK and a positive regulatory subunit cyclin. In mammalian somatic cells extracellular cues act mainly during the G1 phase to regulate the activity of D type cyclin-dependent CDKs, which, in turn, serve as key regulators of G1–S phase progression by phosphorylating and functionally inactivating the tumor suppressor retinoblastoma (Rb) protein. The small molecular weight G protein Ras has been implicated as a crucial molecule that transduces extracellular growth stimuli into intracellular signals. Recent studies, including our own, have demonstrated that maintained cellular Ras activity is required until late in the G1 phase for inactivation of the Rb protein and the G1/S transition and mediates both upregulation of cyclin D1 and downregulation of p27kip1 CDK inhibitor.

Introduction

To elucidate molecular mechanisms by which receptor activation induces cellular responses is the subject of investigation in the field of signal transduction. Recent identification of cyclin-dependent kinases as direct mediators of cell cycle progression, together with advances in our understanding of the effector pathways for the small molecular weight G protein Ras, has provided a new field of investigation into the molecular link between growth factor receptors and the cell cycle molecules (Marshall, 1999). In this review, we provide a brief overview of the molecular basis for cell cycle progression, and recently identified signaling pathways through which the Ras effector system activates the cell cycle machinery.

Section snippets

Cell cycle progression is driven by CDK family protein kinases

During the past decade the basic architecture of the molecular mechanism for eukaryote cell cycle progression has rapidly been elucidated (Takuwa and Takuwa, 1996). Thus, transitions from one stage of the cell cycle to the next are directly driven by phosphorylation events mediated by a family of serine/threonine protein kinases, termed CDKs (for cyclin-dependent kinases), which are composed of the catalytic subunit CDK and the positive regulatory subunit cyclin (Pines, 1993). Cyclins were

Phosphorylation of Rb protein by the G1 cyclin-CDKs at the late G1 restriction (R) point is required for commitment for S phase entry

Rb protein is the product of the retinoblastoma-susceptibility gene, the first tumor suppressor gene to be identified whose germ line mutation causes susceptibility to familial retinoblastoma (Knudson, 1971). In the G0 and the early G1 phases Rb protein is hypophosphorylated and is functionally active. Then, Rb becomes gradually phosphorylated by cyclin D-CDK4/CDK6 during the mid G1 phase. Later in the G1 phase another type of G1-cyclin-dependent kinase, cyclin E-CDK2, becomes rapidly

The Ras effector system is turned on upon stimulation of quiescent cells

It has been well established that stimulation of quiescent cells with mitogenic ligands rapidly activates cellular Ras activity. The ligands that activate Ras include peptide growth factors that act on receptor/protein tyrosine kinases (Gibbs et al., 1990), G protein-coupled receptor agonists such as bombesin, endothelin, and lysophosphatidic acid (van Corven et al., 1993) and hemopoietins and interleukins acting on cytokine receptors (Satoh et al., 1991). The activation of Ras usually depends

Cellular Ras activity is required not only for re-entry into the cell cycle but also late in the G1 phase for passage through the R point

Several lines of evidence strongly suggest that cellular Ras activity is required at multiple points as cells traverse the G1 phase. Thus, microinjection of a neutralizing anti-Ras antibody into NIH3T3 fibroblasts resulted in potent inhibition of DNA synthesis whether the antibody was injected into quiescent cells before serum stimulation or 6 h after the addition of serum (Mulcahy et al., 1985). We have demonstrated that cellular Ras activity is required for both re-entry into the cell cycle

The Ras effector system is linked to Rb inactivation at the R point

We found in NIH(M17) cells that the expression of Ras(Asn17), either in the quiescent state before EGF stimulation or late in the G1 phase in EGF-stimulated cells, similarly and completely blocked phosphorylation of the Rb protein (Takuwa and Takuwa, 1997). By contrast, in Rb-null mouse embryo-derived fibroblasts, in which inactivation of Rb is no longer required, inhibition of Ras is reported to exert much less inhibition of proliferation (Mittnacht et al., 1997, Peeper et al., 1997). These

Multiple steps are required for activation of the G1 cyclin-dependent kinases

The activation process for G1 cyclin-dependent kinases includes multiple steps as follows: (1) an upregulation of G1 cyclin proteins; (2) the assembly of cyclin and CDK molecules; (3) a downregulation of CDK inhibitor proteins; (4) an activating phosphorylation of CDK by CAK (CDK activating kinase) which enables access of the catalytic core to the substrate; and (5) an activating dephosphorylation of CDK at the ATP binding domain by the phosphatase Cdc25A (Morgan, 1995). Among these,

Ras activation early in the G1 phase is required for upregulation of cyclin D1

By taking advantage of the temporally well controlled, inducible expression of the dominant negative Ras mutant in NIH(M17) cells, we investigated which component of the G1 cyclin-CDKs was actually regulated by the Ras effector system (Takuwa and Takuwa, 1997). In this cell type, all three D-type cyclins were expressed in the quiescent state at very low levels, among which only cyclin D1 became strongly upregulated by serum or EGF stimulation. The increase in the cyclin D1 protein level was

Ras activity during the late protion of the G1 phase is required for downregulation of p27kip1 CDK inhibitor

We further examined how endogenous Ras function is related to the cell cycle machinery late in the G1 phase. In contrast to its early induction, the delayed expression of Ras(Asn17) exclusively during the late portion of the G1 phase did not considerably inhibit the upregulation of cyclin D1. We found, however, that the CDK inhibitor p27kip1, which was present at high levels in quiescent cells and became gradually downregulated late in the G1 phase in EGF-stimulated cells, persisted at high

Concluding remarks

In summary, recent studies have disclosed key molecules of the cell cycle machinery, as well as signal transduction pathways that connect cell surface receptors to this cell cycle machinery. Future work should unveil a more precise map of these pathways and also enable development of useful strategies for controlling disorders that are based on dysregulated cellular proliferation, including malignancies and proliferative vascular disorders, which represent major causes of death in developed

Acknowledgements

This work was supported by a grant-in-aid from the Japanese Ministry of Science, Education and Art, and by Hoh-Ansha Foundation for Basic Medical Researches. We thank N. Yamaguchi and Y. Hiratsuka for secretarial assistance.

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