Ablation of Goα Overrides G1Restriction Point Control through Ras/ERK/Cyclin D1-CDK Activities

Abstract

We have generated stable IIC9 cell lines, Goa1 and Goa2, that overexpress full-length antisense G o ␣ RNA. As shown previously, expression of antisense G o ␣ RNA ablated the ␣ subunit of the heterotrimeric G protein, G o , resulting in growth in the absence of mitogen. To better understand this change in IIC9 phenotype, we have characterized the signaling pathway and cell cycle events previously shown to be important in control of IIC9 G 1 /S phase progression. In this paper we clearly demonstrate that ablation of G o ␣ results in growth, constitutively active Ras/ERK, elevated expression of cyclin D1, and constitutively active cyclin D1-CDK complexes, all in the absence of mitogen. Furthermore, these characteristics were abolished by the transient overexpression of the transducin heterotrimeric G protein ␣ subunit strongly suggesting the transformation of G o ␣-ablated cells involves G o ␤␥ subunits. This is the first study to implicate a heterotrimeric G protein in tumor suppression.In IIC9 cells, a subclone of Chinese hamster embryo fibroblasts, platelet-derived growth factor (PDGF) 1 is a potent mitogen (1). PDGF stimulates an increase in cyclin D1 expression concomitant with an increase in cyclin D1-CDK activity (1). Cyclin D1 is an important G 1 protein in that its delayed early induction in response to mitogen is required for G 1 progression (2). Microinjection of cyclin D1 antibodies or antisense cyclin D1 plasmids into normal fibroblasts arrests them in G 1 but has no effect on cells already beyond the G 1 /S boundary (3, 4). Thus, accumulation of cyclin D1 in G 1 in response to mitogen is required for progression through the restriction point and entrance into S phase (2, 5, 6). Cyclin D1 preferentially binds to its catalytic partner, cyclin-dependent kinase 4 (CDK4), to form a holoenzyme (7-10). The activated cyclin D1-CDK4 complex preferentially binds to and phosphorylates the retinoblastoma gene product (pRb) (11-16). Hence, the mitogen-induced activation of cyclin D1-CDK4 complexes allows for progression through the restriction point in vivo presumably through the hyperphorylation and inactivation of pRb in concert with other G 1 cyclin-CDK complexes.We and others previously have shown that PDGF stimulates ERK1 activity in IIC9 and CCL39 cells and that inhibition of this activity is sufficient to cause the loss of PDGF-induced cyclin D1 expression, as well as a loss of cyclin D1-CDK activity (1). The loss of PDGF-induced cyclin D1-CDK activity was correlated with G 1 growth arrest. Attention has focused on the mitogen-induced signals involved in cell growth and more recently those signals regulating mitogen-dependent induction cyclin D1 (1, 6). Expression of constitutively active mutant Ras has been shown to transform several cell types and elevate cyclin D1 expression (3,(17)(18)(19). The role of Ras proteins in the mitogenesis and transformation of cells is mediated, in part, by a downstream cascade of serine-threonine kinases that terminates with the activation of p42 and p44 MAPKs (ERK1, ERK2...

α-Thrombin-mediated Phosphatidylinositol 3-Kinase Activation through Release of Gβγ Dimers from Gαq and Gαi2

“…Transient Transfection-The cDNA encoding pcDNA3.1 (Invitrogen), ␤-arrestin1 (a kind gift from Marc Caron), ␣-transducin (25), MAS-GRK3-ct (a kind gift from Stephen R. Ikeda), G o ␣C351G, G i2 ␣C352G, and G i3 ␣C351G (generated), carboxyl terminus G␣ peptides (Biotech, Chicago, IL), or xanthine nucleotide-binding G␣ mutants (Guthrie Research Institute, Sayre, PA) were transfected into subconfluent (60 -80%) IIC9 cells using LiptofectAMINE TM (Invitrogen) as previously described (23). The following day, cells were growth-arrested by washing once with ␣-minimal essential medium followed by a 48-h incubation in the same medium prior to agonist stimulation.…”

Thyrotropin-dependent proliferation of in vitro rat thyroid cell systems

“…Kip1 degradation to ensure the proper activation state of cyclin D1±CDK4 complexes following mitogenic stimulation (265). Interestingly, in thyroid cells where the cAMP is mitogenic, TSH stimulation down-regulates p27…”

“…Kip1 degradation have not been de®ned clearly, in Chinese hamster embryo ®broblast cells (IIC9 cells) PDGF stimulation causes the rapid activation of Ras and subsequent downstream activation of ERK (227,265). In addition, Ras also stimulates the downstream activation of the RhoA that regulates p27…”

Expression of Cyclin E Renders Cyclin D-CDK4 Dispensable for Inactivation of the Retinoblastoma Tumor Suppressor Protein, Activation of E2F, and G1-S Phase Progression

“…Light produced was measured and normalized to transfection efficiency by dividing relative light units by optical density units obtained from ␤-galactosidase activity. ␤-Galactosidase activity was measure as previously described (5).…”

“…The sequential activation of early G 1 CDK activity, CDK4 or CDK6 together with cyclin D1, D2, or D3, and the late G 1 CDK activity, CDK2 together with cyclin E1 or E2, is believed to be required for progression through G 1 and into S phase. Because IIC9 cells contain CDK4, but not CDK6, and cyclin D1, but not D2 or D3, CDK4-cyclin D1 activation in early G 1 is required for the expression of cyclin E, CDK2 activity, and G 1 -S phase progression (3)(4)(5).…”