Cyclin E-induced S phase without activation of the pRb/E2F pathway.

Abstract

In cells of higher eukaryotes, cyclin D-dependent kinases Cdk4 and Cdk6 and, possibly, cyclin E-dependentCdk2 positively regulate the G 1-to S-phase transition, by phosphorylating the retinoblastoma protein (pRb), thereby releasing E2F transcription factors that control S-phase genes. Here we performed microinjection and transfection experiments using rat R12 fibroblasts, their derivatives conditionally overexpressing cyclins D1 or E, and human U-2-OS cells, to explore the action of G~ cyclins and the relationship of E2F and cyclin E in S-phase induction. We demonstrate that ectopic expression of cyclin E, but not cyclin D1, can override G~ arrest imposed by either the p16 INK4a Cdk inhibitor specific for Cdk4 and Cdk6 or a novel phosphorylation-deficient mutant pRb. Several complementary approaches to assess E2F activation, including quantitative reporter assays in live cells, showed that the cyclin E-induced S phase and completion of the cell division cycle can occur in the absence of E2F-mediated transactivation. Together with the ability of cyclin E to overcome a G~ block induced by expression of dominant-negative mutant DP-1, a heterodimeric partner of E2Fs, these results provide evidence for a cyclin E-controlled S phase-promoting event in somatic cells downstream of or parallel to phosphorylation of pRb and independent of E2F activation. They furthermore indicate that a lack of E2F-mediated transactivation can be compensated by hyperactivation of this cyclin E-controlled event.[Key Words: Cyclin E; cyclin D1; p16; pRb phosphorylation; E2F; S phase] Received November 11, 1996; revised version accepted April 9, 1997.Progression from G~ to S phase of the mammalian cell cycle is regulated by cyclin D-dependent kinases Cdk4 and Cdk6 complexed to D-type cyclins, and by Cdk2 bound to cyclins E or A

Conserved region 2 of adenovirus E1A has a function distinct from pRb binding required to prevent cell cycle arrest by p16INK4a or p27Kip1

“…However, E2F is not su cient to prevent growth inhibition by p27, implying that cyclin E/CDK2 complexes have an additional role(s) at the G1-S transition, distinct from pRb phosphorylation (Alevizopoulos et al, 1997;Mann and Jones, 1996). Similar conclusions were reached by other groups (Chew et al, 1998;Hofmann and Livingston, 1996;Knudsen et al, 1998;Leng et al, 1997;Lukas et al, 1997;Ohtsubo et al, 1995;Resnitzky and Reed, 1995). In summary, the ability of the CDK4 inhibitor p16 to arrest the cell cycle in G1 seems to depend essentially on activation of pRb and sequestration of E2F, whereas p27 not only activates pRb, but also impinges on other CDK2-regulated events (discussed in Dyson, 1998).…”

Nature of the Critical Labile Event That Controls RB Phosphorylation in the Cyclic AMP-Dependent Cell Cycle of Thyrocytes in Primary Culture

“…Alternatively, a modulation of RB binding to cyclin D has not been considered and the stimulation of type 1 phosphatase dephosphorylates RB at mitosis but is not instrumental in cell cycle arrest induced by serum starvation [39]. Finally, an independent regulation through cAMP of downstream G1 events, such as cyclin E-CDK2 activity [11], is not formally excluded by the present experiments. Therefore, though subtle modifications of cyclin D3 or CDK4 conformation or localization may have escaped our analysis, the present results indicate that a key labile event(s) responsible for the last control of the activity of assembled cyclin D-CDK4 complexes and/or RB phosphorylation at restriction point remains to be uncovered, in dog thyrocytes stimulated through cAMP elevation, and possibly in other systems.…”

Deregulated cyclin E promotes p53 loss of heterozygosity and tumorigenesis in the mouse mammary gland

“…Cyclin D expression appears to be responsive to mitogenic stimuli and therefore gain-of-function mutations, including gene amplification, may represent a bypass of various receptor/ligand-dependent mitogenic stimuli. Cyclin E functions downstream of cyclin D and appears to contribute to entry into S-phase both through the hyperphosphorylation of Rb and by Rb-independent mechanisms (Lukas et al, 1997;Seghezzi et al, 1998;Geisen and Moroy, 2002;Wei et al, 2003).…”