Different Partners, Same Benefits
There is a growing body of evidence which questions the necessity of pRB-E2F complexes in mediating the pRB-dependent G1 arrest. Studies of the naturally occurring pRB R661W mutant demonstrate that while this pRB mutant fails to bind E2Fs, it can still mediate G1 arrest. Furthermore, a synthetic pRB mutant mouse model, which is defective for binding E2Fs through the A-B interface of the RB small pocket, maintains proliferative control with an intact G1 arrest despite deregulated expression of E2F target genes. This leaves us with the question: If not through E2Fs, then how?
The pRB-Skp2-p27 Axis
A key study by Peng, J. et al (2004) provides insights into an E2F-independent mechanism for pRB induced cell cycle arrest. Rb induction causes G1 arrest in parallel with accumulation of the CDK inhibitor p27Kip1 and a decline in CDK2 kinase activity. This decline in activity precedes a decrease in CDK2 protein levels. The accumulated and stabilized p27 Kip1 plays an active role in pRB-mediated G1 arrest since antisense knockdown of p27Kip1 along with pRB induction increases the % BrdU positive (i.e. S-phase) cells. Exit from this arrest coincides with rapid degredation of p27Kip1.
The above observations strongly suggest that the mechanism of p27Kip1 turnover must be implicated in mechanisms controlling pRB mediated cell cycle arrest. Degradation of p27Kip1 follows ubiquitination of the protein. p27Kip1 phosphorylated at T187 by CDK2 is recruited by the SCFSkp2 E3 ligase for ubiquitination. Does p27 stabilization during pRB mediated G1 arrest depend on pRB interfering with the underlying mechanisms of p27 turnover?
The aforementioned pRB relationship seems to be the case as Rb-/- MEFs show increased rates of p27Kip1 degradation with earlier S-phase entry compared to Rb+/+ MEFs. However, this p27Kip1 degradation precedes activation of CDK2 kinase activity, ruling out CDK2 phosphorylation of p27Kip1 at T187 as causal for p27Kip1 degradation just after cell cycle re-entry. Instead, pRB seems to attenuate p27Kip1 degradation by inhibiting p27Kip1 polyubiquitination.
The mechanism for this depends partly on pRB directly interacting with the Skp2 E3 ubiquitin ligase through a sequence ranging from 60-100 residues within the Skp2 N-terminus. IPs for Skp2 demonstrate that as pRB induction increases, there is a reduction in Skp2-p27 complexes, coinciding with a decrease in total Skp2 levels and an increase in p27Kip1 levels. pRB-Skp2 complex formation reduces the amount of polyubiquitinated p27Kip1 detected, suggesting that pRB induction allows for p27Kip1 stabilization through dissociation of the Skp2-p27Kip1 interaction, and inhibition of Skp2-dependent polyubiquitination of p27kip1.
The implications of this interaction on cell cycle arrest are striking. pRB induction results in p27Kip1 stabilization and G1 arrest even when using the naturally occurring partial penetrance pRB R661W mutant which fails to bind E2Fs (but still retains major tumor suppressive abilities). pRB mediated G1 arrest is abolished when using Skp2ΔN to which pRB cannot bind. This shifts the attribution of pRB-induced G1 arrest from pRB-E2F complexes to pRB-Skp2 complexes.
The Skp2 contact point on pRB seem to range residues 637-738 and 772-824 of the pRB large pocket. While mapping contact points, it was discovered pRB-Skp2 binding can be distinguished from pRB dissociation Skp2-p27Kip1 complexes as demonstrated by pRBΔ22 which binds Skp2, but does not dissociate Skp2-p27Kip1 complexes. This suggests pRB tethers to the Skp2 N-terminus, while a different region mediates the Skp2-p27kip1 dissociation.
G1 arrest cannot be entirely attributed to p27Kip1 accumulation in absence of Skp2 since Skp2-/- mice grow to adulthood. Infact, p27Kip1 dependent cell cycle arrest seems to be cell type specific.
The pRB-Cdh1-Skp2 Triple Complex
Following discovery of the pRB-Skp2-p27 axis, holes were left in the mechanism of E2F-independent cell cycle arrest. How does pRB actually inhibit Skp2 activity on p27Kip1? It has been established that during the pRB-mediated G1 arrest, pRB associates with Skp2, the F-box protein of the Skp1-Cullin-F-box protein (SCF) E3 ubiquitin ligase complex. This association with Skp2 prevents SCF-Skp2-dependent ubiquitination of p27Kip1 to allow for stabilization of p27Kip1 protein levels.
It turns out pRB itself does not inhibit Skp2 activity. Rather another pRB binding partner is responsible for this task. The anaphase-promoting complex/cyclosome (APC/C) controls p27Kip1 stability by targeting Skp2 for ubiquitin-mediated degradation. One specific activator of APC/C, Cdh1, interacts with pRB and is required for pRB induced cell cycle arrest.
Using a GST-pRB affinity column followed by mass spectrometry, it was determined that 10 subunits of APC/C bind pRB. The APC/C core complex associates with two WD-40-repeat-containing activators in a cell cycle dependent manner: Cdc20, and the G1 aactivator Cdh1. pRB preferentially binds the APC-Cdh1 co mplex over the APC-Cdc20 complex. Cdh1 binds the pRB small pocket in a manner partially dependent on the LxCxE binding cleft, through a region distinct from that used by Skp2 to bind pRB.
APC-Cdh1 targets several proteins, such as cyclin B1, for ubiquitin-mediated proteosomal degradation. Although APC-Cdh1 binds pRB, pRB is not a target for APC/C-mediated degredation as determined from an in vitro ubiquination assay using affinity-purified APC-Cdh1. Instead, APC-Cdh1 seems to associate with pRB to substantiate pRB-mediated G1 arrest. pRB induction in SaOS-2 cells induces G1 arrest in accordance with Skp2 ubiquitination and degradation, and p27Kip1 accumulation, all of which are dependent on Cdh1 as determined using shCdh1. Co-transfection of tagged pRB, APC-Cdh1, and Skp2, followed by two rounds of immunoprecipitation demonstrate the presence of pRB-APC-Cdh1-Skp2 triple complexes during G1 arrest.
In contrast to asynchronous conditions, contact inhibited (G0) IMR90 cells enrich for the pRB-APC complex. This suggests APC-Cdh1 preferentially associates with hypophosphorylated pRB, and may dissociate in a cell cycle-independent manner in response to phosphorylation of pRB at the critical contact points within the small pocket.
In summary, pRB can regulate cyclin/CDK activity directly by using APC-Cdh1 to antagonize Skp2's ability to degrade p27
The pRB-Skp2-p27 Axis
A key study by Peng, J. et al (2004) provides insights into an E2F-independent mechanism for pRB induced cell cycle arrest. Rb induction causes G1 arrest in parallel with accumulation of the CDK inhibitor p27Kip1 and a decline in CDK2 kinase activity. This decline in activity precedes a decrease in CDK2 protein levels. The accumulated and stabilized p27 Kip1 plays an active role in pRB-mediated G1 arrest since antisense knockdown of p27Kip1 along with pRB induction increases the % BrdU positive (i.e. S-phase) cells. Exit from this arrest coincides with rapid degredation of p27Kip1.
The above observations strongly suggest that the mechanism of p27Kip1 turnover must be implicated in mechanisms controlling pRB mediated cell cycle arrest. Degradation of p27Kip1 follows ubiquitination of the protein. p27Kip1 phosphorylated at T187 by CDK2 is recruited by the SCFSkp2 E3 ligase for ubiquitination. Does p27 stabilization during pRB mediated G1 arrest depend on pRB interfering with the underlying mechanisms of p27 turnover?
The aforementioned pRB relationship seems to be the case as Rb-/- MEFs show increased rates of p27Kip1 degradation with earlier S-phase entry compared to Rb+/+ MEFs. However, this p27Kip1 degradation precedes activation of CDK2 kinase activity, ruling out CDK2 phosphorylation of p27Kip1 at T187 as causal for p27Kip1 degradation just after cell cycle re-entry. Instead, pRB seems to attenuate p27Kip1 degradation by inhibiting p27Kip1 polyubiquitination.
The mechanism for this depends partly on pRB directly interacting with the Skp2 E3 ubiquitin ligase through a sequence ranging from 60-100 residues within the Skp2 N-terminus. IPs for Skp2 demonstrate that as pRB induction increases, there is a reduction in Skp2-p27 complexes, coinciding with a decrease in total Skp2 levels and an increase in p27Kip1 levels. pRB-Skp2 complex formation reduces the amount of polyubiquitinated p27Kip1 detected, suggesting that pRB induction allows for p27Kip1 stabilization through dissociation of the Skp2-p27Kip1 interaction, and inhibition of Skp2-dependent polyubiquitination of p27kip1.
The implications of this interaction on cell cycle arrest are striking. pRB induction results in p27Kip1 stabilization and G1 arrest even when using the naturally occurring partial penetrance pRB R661W mutant which fails to bind E2Fs (but still retains major tumor suppressive abilities). pRB mediated G1 arrest is abolished when using Skp2ΔN to which pRB cannot bind. This shifts the attribution of pRB-induced G1 arrest from pRB-E2F complexes to pRB-Skp2 complexes.
The Skp2 contact point on pRB seem to range residues 637-738 and 772-824 of the pRB large pocket. While mapping contact points, it was discovered pRB-Skp2 binding can be distinguished from pRB dissociation Skp2-p27Kip1 complexes as demonstrated by pRBΔ22 which binds Skp2, but does not dissociate Skp2-p27Kip1 complexes. This suggests pRB tethers to the Skp2 N-terminus, while a different region mediates the Skp2-p27kip1 dissociation.
G1 arrest cannot be entirely attributed to p27Kip1 accumulation in absence of Skp2 since Skp2-/- mice grow to adulthood. Infact, p27Kip1 dependent cell cycle arrest seems to be cell type specific.
The pRB-Cdh1-Skp2 Triple Complex
Following discovery of the pRB-Skp2-p27 axis, holes were left in the mechanism of E2F-independent cell cycle arrest. How does pRB actually inhibit Skp2 activity on p27Kip1? It has been established that during the pRB-mediated G1 arrest, pRB associates with Skp2, the F-box protein of the Skp1-Cullin-F-box protein (SCF) E3 ubiquitin ligase complex. This association with Skp2 prevents SCF-Skp2-dependent ubiquitination of p27Kip1 to allow for stabilization of p27Kip1 protein levels.
It turns out pRB itself does not inhibit Skp2 activity. Rather another pRB binding partner is responsible for this task. The anaphase-promoting complex/cyclosome (APC/C) controls p27Kip1 stability by targeting Skp2 for ubiquitin-mediated degradation. One specific activator of APC/C, Cdh1, interacts with pRB and is required for pRB induced cell cycle arrest.
Using a GST-pRB affinity column followed by mass spectrometry, it was determined that 10 subunits of APC/C bind pRB. The APC/C core complex associates with two WD-40-repeat-containing activators in a cell cycle dependent manner: Cdc20, and the G1 aactivator Cdh1. pRB preferentially binds the APC-Cdh1 co mplex over the APC-Cdc20 complex. Cdh1 binds the pRB small pocket in a manner partially dependent on the LxCxE binding cleft, through a region distinct from that used by Skp2 to bind pRB.
APC-Cdh1 targets several proteins, such as cyclin B1, for ubiquitin-mediated proteosomal degradation. Although APC-Cdh1 binds pRB, pRB is not a target for APC/C-mediated degredation as determined from an in vitro ubiquination assay using affinity-purified APC-Cdh1. Instead, APC-Cdh1 seems to associate with pRB to substantiate pRB-mediated G1 arrest. pRB induction in SaOS-2 cells induces G1 arrest in accordance with Skp2 ubiquitination and degradation, and p27Kip1 accumulation, all of which are dependent on Cdh1 as determined using shCdh1. Co-transfection of tagged pRB, APC-Cdh1, and Skp2, followed by two rounds of immunoprecipitation demonstrate the presence of pRB-APC-Cdh1-Skp2 triple complexes during G1 arrest.
In contrast to asynchronous conditions, contact inhibited (G0) IMR90 cells enrich for the pRB-APC complex. This suggests APC-Cdh1 preferentially associates with hypophosphorylated pRB, and may dissociate in a cell cycle-independent manner in response to phosphorylation of pRB at the critical contact points within the small pocket.
In summary, pRB can regulate cyclin/CDK activity directly by using APC-Cdh1 to antagonize Skp2's ability to degrade p27