SignificanceThe 20S proteasome is a key actor of the control of protein levels and integrity in cells. To perform its multiple functions, it works with a series of regulators, among which is a nuclear complex called PA28γ. In particular, PA28γ participates in the regulation of cell proliferation and nuclear dynamics. We describe here the characterization of a protein, PIP30/FAM192A, which binds tightly to PA28γ and favors its interaction with the 20S proteasome while inhibiting its association with coilin, a central component of nuclear Cajal bodies. Thus, PIP30/FAM192A critically controls the interactome and, consequently, the functions of PA28γ, and appears to be a previously unidentified player in the fine regulation of intracellular proteostasis in the cell nucleus.
Arpp19 is a potent PP2A-B55 inhibitor that regulates this phosphatase to ensure the stable phosphorylation of mitotic/meiotic substrates. At G2-M, Arpp19 is phosphorylated by the Greatwall kinase on S67. This phosphorylated Arpp19 form displays a high affinity to PP2A-B55 and a slow dephosphorylation rate, acting as a competitor of PP2A-B55 substrates. The molecular determinants conferring slow dephosphorylation kinetics to S67 are unknown. PKA also phosphorylates Arpp19. This phosphorylation performed on S109 is essential to maintain prophase I-arrest in Xenopus oocytes although the underlying signalling mechanism is elusive. Here, we characterize the molecular determinants conferring high affinity and slow dephosphorylation to S67 and controlling PP2A-B55 inhibitory activity of Arpp19. Moreover, we show that phospho-S109 restricts S67 phosphorylation by increasing its catalysis by PP2A-B55. Finally, we discover a double feed-back loop between these two phospho-sites essential to coordinate the temporal pattern of Arpp19-dependent PP2A-B55 inhibition and Cyclin B/Cdk1 activation during cell division.
The papillomavirus E1 protein is essential for the initiation of viral replication. We previously showed that the bovine papillomavirus E1 protein is unstable and becomes resistant to ubiquitin-mediated degradation when tightly bound to cyclin E-cyclin-dependent kinase 2 (Cdk2) before the start of DNA synthesis. However, neither the protection nor the targeted degradation of E1 appears to depend on its phosphorylation by Cdk. Here, we report that Cdk phosphorylation of E1 is also not a prerequisite for the initiation of viral DNA replication either in vitro or in vivo. Nevertheless, we found that phosphorylation of one Cdk site, Ser283, abrogates E1 replicative activity only in a cellular context. We show that this site-specific phosphorylation of E1 drives its export from the nucleus and promotes its continuous nucleocytoplasmic shuttling. In addition, we find that E1 shuttling occurs in S phase, when cyclin A-Cdk2 is activated. E1 interacts with the active cyclin A-Cdk2 complex and is phosphorylated on Ser283 by this kinase. These data suggest that the phosphorylation of E1 on Ser283 is a negative regulatory event that is involved in preventing the amplification of viral DNA during S phase. This finding reveals a novel facet of E1 regulation that could account for the variations of the viral replication capacity during different cell cycle phases, as well as in different stages of the viral cycle.Papillomaviruses are small DNA tumor viruses that induce persistent epithelial lesions in higher vertebrates, including humans. The viral genome of papillomaviruses is initially replicated and maintained as extrachromosomal plasmids in the basal cells of the infected host epithelium. In these proliferating cells, replication of the viral genomes is tightly regulated in order to occur only in S phase within the host cell nuclei. Furthermore, the maintenance of a fairly constant low copy number of viral genomes indicates that the viral DNA synthesis also must be controlled to prevent amplification, as an increasing viral copy number is likely to be incompatible with viral persistence in the basal epithelial layer. However, in differentiated, suprabasal cells, the viral genome can be amplified, suggesting that replication controls are then abrogated to allow the accumulation of viral genomes associated with the production of new virions (5,12,16,17).Like other small DNA viruses, papillomaviruses do not encode all the proteins required for their replication and so need to use the replication machinery of the host cell. Two viral proteins, E1 and E2, are needed for replication and stable plasmid maintenance (6, 34-36). However, by using the bovine papillomavirus (BPV) as a model system, we found that E1 is the only viral protein required for the initiation of viral DNA synthesis in vitro (3, 9). E1 possesses all the functions required for the initiation of replication. It is an ATP-dependent helicase that specifically binds to and unwinds the virus replication origin, and it recruits the cellular replication machinery thro...
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