As shown in Fig. 1A, β2SP overexpression decreased the expression of several proteins responsible for cell cycle regulation including phosphorylated Rb (pRb). Comparing protein expression from identical preparations,

the most dramatic reduction in expression was for CDK4 (33% of control), suggesting that CDK4 is a downstream effector in cell cycle regulation mediated by β2SP signaling. Then we further compared the expression levels of CDK4, cyclin D1, pRb, and Rb upon transfection of β2SP in HepG2 and SNU475 cells in three independent experiments. The most remarkable reductions of CDK4 were shown in HepG2 (39%) and SNU475 (31%) cells (Fig. 1B). However, it was not clear that the change in CDK4 due to the loss of β2SP was sufficient to disrupt the cell cycle. Thus, we tested whether the increase in Rb phosphorylation OSI906 was due to the down-regulation of β2SP or activation of CDK4. We inhibited β2SP expression in SNU-475 cells by the infection of a lentivirus containing shRNA against β2SP and then analyzed Rb phosphorylation. β2SP expression was decreased by 44% after lentiviral infection and Rb phosphorylation

was increased by 55%, whereas ICG-001 cell line the levels of Rb were unchanged (Fig. 1C). To determine whether CDK4 is responsible for Rb phosphorylation due to the down-regulation of β2SP, we inhibited CDK4 expression by siRNA in SNU-475 cells infected with β2SP shRNA. CDK4 siRNA in the presence of β2SP shRNA restored Rb phosphorylation to basal levels (Fig. 1C). These results suggest that CDK4 is a key regulator of Rb phosphorylation affected by β2SP expression. We then determined whether the induction of CDK4 expression due to the down-regulation of β2SP accelerates cell cycle progression. SNU-475

cells were infected with the β2SP shRNA lentivirus followed by treatment with a CDK4 inhibitor, and then analyzed cell cycle phases by fluorescence-activated cell sorting (FACS) with propidium iodide (PI) staining. The number of cells in G1 phase was significantly decreased from 46% to 35% upon the knockdown of β2SP (Fig. 2A,B). Additional treatment with CDK inhibitor (200 nM) in β2SP short hairpin RNA (shRNA)-treated cells returned 43% of the cells to old G1. However, we did not detect the additional accumulation of cells in G1 phase in control lentiviral-treated cells exposed to the same CDK4 inhibitor. Taken together, these data demonstrate that dysregulation of the cell cycle resulting from the disruption of β2SP expression is mediated by CDK4 activation and Rb phosphorylation. We further investigated the mechanism by which β2SP modulates CDK4 by examining interactions between these proteins. Recent reports indicate that CDK4 phosphorylates Smad3 to inhibit its transcriptional activity and antiproliferative functions.7 Thus, we sought to determine whether CDK4 phosphorylates β2SP as it does Smad3.