[Lys61]N-Ras is able to induce full activation and nuclear accumulation of Cdk4 in NIH3T3 cells

Abstract

The elements of the cell cycle regulatory machinery activated by the oncogenic form of Ras, [Lys 61 ]N-Ras, have been analysed in NIH3T3 cells. We demonstrate that [Lys 61 ]N-Ras expression is able to induce full cdk4 activation. As already reported, oncogenic Ras expression was su cient to induce cyclin D1 and p21 cip1 expression and their association with cdk4. Furthermore, serum-starved [Lys 61 ]N-Ras NIH3T3 cells showed nuclear accumulation of cyclin D1 and cdk4 not observed in serum-starved NIH3T3 cells. This accumulation of cdk4 into the cell nucleus observed in serum-starved [Lys 61 ]NRas NIH3T3 cells was inhibited by a microinjection of neutralizing anti-Ras antibodies. Thus, active [Lys 61 ]NRas was a su cient signal to induce nuclear accumulation of cyclin D1/cdk4, leading to its full activation. Transfection of [Lys 61 ]N-Ras NIH3T3 cells with an inactive form of MEK or their treatment with PD 98059, showed that nuclear translocation of cdk4 was MEK dependent. Interestingly, cells constitutively expressing [Lys 61 ]N-Ras did not inactivate pRb and did not proliferate in the absence of serum. This may be due to the fact that although association of cdk2 with cyclin E and the translocation of those complexes to the nucleus were achieved, [Lys 61 ]N-Ras expression was not su cient to induce cdk2 activation. The high levels of p27 kip1 that were found in cyclin E/cdk2 complexes may be responsible for the inability of oncogenic Ras to activate this kinase. In consequence, oncogenic alterations that lead to a decrease in p27 kip1 bound to cyclin E may cooperate with Ras to induce full cdk2 activation, pRb inactivation and thus cell proliferation. Oncogene (2000) 19, 690 ± 699.

Regulation of CDK4

“…In NIH 3T3 cells, p21 promotes the nuclear accumulation of cyclin D1 by preventing its association with CRM1 [135]. Like its assembly [79], the nuclear accumulation of cyclin D1-CDK4 appears to depend on MEK activity in NIH 3T3 cells [136]. …”

Ras-inducible immortalized fibroblasts: focus formation without cell cycle deregulation

“…In support of this serum-mimicking phenotype of the ras-oncogene, activated ras, in serum starved cells, leads to induction of serum responsive genes and to activation of cell cycle regulators crucial to cycling. The cyclin D1, fos and other immediate early/delayed early genes are Ras-inducible (SchoÈ nthal et al, 1988;Albanese et al, 1995), and cdk4-cyclin D1 kinase (Villalonga et al, 2000) and the E2F transcription factors (Fan and Bertino, 1997;Gjoerup et al, 1998) are active in Rastransformed, but not parental, serum starved cells. This ®nding is in accord with many reports (Kaplan et al, 1982;Durkin and Whit®eld, 1986;Zhan and Goldfarb, 1986;Pironin et al, 1992;Winston et al, 1996;Villalonga et al, 2000), the results of which argue that activated Ras can induce DNA synthesis, but that cell division in the absence of serum may require Rasinduced autocrine growth-factor production.…”