Distinct Action of the Retinoblastoma Pathway on the DNA Replication Machinery Defines Specific Roles for Cyclin-Dependent Kinase Complexes in Prereplication Complex Assembly and S-Phase Progression

Abstract

The retinoblastoma (RB) and p16ink4a tumor suppressors are believed to function in a linear pathway that is functionally inactivated in a large fraction of human cancers. Recent studies have shown that RB plays a critical role in regulating S phase as a means for suppressing aberrant proliferation and controlling genome stability. Here, we demonstrate a novel role for p16ink4a in replication control that is distinct from that of RB. Specifically, p16ink4a disrupts prereplication complex assembly by inhibiting mini-chromosome maintenance (MCM) protein loading in G 1 , while RB was found to disrupt replication in S phase through attenuation of PCNA function. This influence of p16ink4a on the prereplication complex was dependent on the presence of RB and the downregulation of cyclin-dependent kinase (CDK) activity. Strikingly, the inhibition of CDK2 activity was not sufficient to prevent the loading of MCM proteins onto chromatin, which supports a model wherein the composite action of multiple G 1 CDK complexes regulates prereplication complex assembly. Additionally, p16ink4a attenuated the levels of the assembly factors Cdt1 and Cdc6. The enforced expression of these two licensing factors was sufficient to restore the assembly of the prereplication complex yet failed to promote S-phase progression due to the continued absence of PCNA function. Combined, these data reveal that RB and p16ink4a function through distinct pathways to inhibit the replication machinery and provide evidence that stepwise regulation of CDK activity interfaces with the replication machinery at two discrete execution points.Due to their profound influence on tumorigenesis, substantial effort has been directed at determining the function of the p16ink4a and retinoblastoma (RB) tumor suppressors in cell cycle control. Both tumor suppressors are inactivated in human tumors, as achieved by genetic mutation, gene silencing, or functional inactivation (6,53,63,64). Biochemical and genetic evidence demonstrate a functional connection between the two tumor suppressors as part of a growth inhibitory network that is disrupted in the majority of cancers.p16ink4a is a cyclin-dependent kinase (CDK) inhibitor, which arrests cells in G 1 (38,48) and is dependent on inhibition of CDK4 and CDK2-associated activity. The influence of p16ink4a on CDK4 is mediated by direct binding and involves the disruption of CDK4/cyclin D interactions (57,63). This function of p16ink4a is required for its ability to inhibit cell cycle progression and is disrupted in tumors which express elevated levels of CDK4 (thus titrating p16ink4a) or in tumors that harbor specific CDK4 mutations that compromise p16ink4a association (28,70). In addition, p16ink4a-mediated disruption of the CDK4/cyclin D complex releases p27Kip1 and p21Cip1, leading to inhibition of CDK2 complexes (31,65). This action is similarly required for p16ink4a-mediated cell cycle inhibition and therefore differentiates the function of p16ink4a from the genetic loss of CDK4 alone. A principle target of CDK4 and CD...

RETRACTED ARTICLE: Cyclin-dependent kinase 4/6 activity is a critical determinant of pre-replication complex assembly

“…These studies have suggested an important role for this kinase in stimulating the initiation of S phase and replication origin firing. Consistent with previous studies from our laboratory and others, we find that inhibition of CDK2 activity does not influence the loading of MCM proteins on chromatin, nor the expression of cdt1 and cdc6, which are required for pre-RC assembly (Braden et al, 2006;Savio et al, 2006). However, inhibition of CDK2 activity does inhibit ongoing replication in S phase as determined by retention of PCNA on chromatin (Sever-Chroneos et al, 2001;Angus et al, 2004;Braden et al, 2006;Savio et al, 2006).…”

“…Much of the emphasis regarding CDK activity and positive regulation of replication has focused on roles for CDK2 in modulating the firing of origins in S phase. However, we and others have recently found that CDK/cyclin activity is important in establishing the pre-RC in G 1 (Mailand and Diffley, 2005;Braden et al, 2006;Geng et al, 2007). Specifically, the accumulation of both cdt1 and cdc6, which mediate the assembly of the MCM complex on chromatin, are influenced by the activity of CDK/ cyclins.…”

“…The accumulation of cyclin E contributes to the activation of CDK2, which further phosphorylates RB and promotes S phase entry (Hwang and Clurman, 2005). As cells progress into S phase, high levels of cyclin A are involved in DNA replication control (Angus et al, 2004;Braden et al, 2006). DNA replication is a highly ordered process that ensures cells replicate their genome only once per cell cycle (Diffley, 1994;Bell and Dutta, 2002;Blow and Hodgson, 2002;Diffley and Labib, 2002;Dimitrova et al, 2002).…”

Cyclin D3 action in androgen receptor regulation and prostate cancer

“…These data indicate that cyclin D3 alters ligand-induced AR residence at a prostate-specific target, and suggest that cyclin D3 elicits effects on AR activity that are distinct from cyclin D1. Cyclin D3 exerts its activity through CDK4-independent mechanisms that parallel cyclin D1 function To assess the requirement of CDK4 for cyclin D3-mediated AR modulation, cells deficient in retinoblastoma expression (Huang et al, 1988) and those that lack substantive CDK4 expression were utilized (Saos-2) (Braden et al, 2006). Residual cyclin D3/CDK4 is inactive, and due to the lack of retinoblastoma, Saos-2 are refractory to the pro-proliferative activity of CDK/ cyclin D3 (Serrano et al, 1993).…”

“…Cell culture, transfections and reporter assays Cell lines were obtained and cultured as previously described (Petre et al, 2002;Petre-Draviam et al, 2003Braden et al, 2006;Burd et al, 2006). Transient assays of AR function were previously described (Knudsen et al, 1999;Petre et al, 2002;Petre-Draviam et al, 2003;Burd et al, 2005Burd et al, , 2006.…”