Nuclear export and centrosome targeting of the protein phosphatase 2A subunit B56alpha: role of B56alpha in nuclear export of the catalytic subunit.

Protein phosphatase (PP) 2A is a heterotrimeric enzyme regulated by specific subunits. The B56 (or B'/PR61/PPP2R5) class of B-subunits direct PP2A or its substrates to different cellular locations, and the B56alpha, -beta, and -epsilon isoforms are known to localize primarily in the cytoplasm. Here we studied the pathways that regulate B56alpha subcellular localization. We detected B56alpha in the cytoplasm and nucleus, and at the nuclear envelope and centrosomes, and show that cytoplasmic localization is dependent on CRM1-mediated nuclear export. The inactivation of CRM1 by leptomycin B or by siRNA knockdown caused nuclear accumulation of ectopic and endogenous B56alpha. Conversely, CRM1 overexpression shifted B56alpha to the cytoplasm. We identified a functional nuclear export signal at the C terminus (NES; amino acids 451-469), and site-directed mutagenesis of the NES (L461A) caused nuclear retention of full-length B56alpha. Active NESs were identified at similar positions in the cytoplasmic B56-beta and epsilon isoforms, but not in the nuclear-localized B56-delta or gamma isoforms. The transient expression of B56alpha induced nuclear export of the PP2A catalytic (C) subunit, and this was blocked by the L461A NES mutation. In addition, B56alpha co-located with the PP2A active (A) subunit at centrosomes, and its centrosome targeting involved sequences that bind to the A-subunit. Fluorescence Recovery after Photobleaching (FRAP) assays revealed dynamic and immobile pools of B56alpha-GFP, which was rapidly exported from the nucleus and subject to retention at centrosomes. We propose that B56alpha can act as a PP2A C-subunit chaperone and regulates PP2A activity at diverse subcellular locations.

Translation of intronless RNAs is strongly stimulated by the Epstein-Barr virus mRNA export factor EB2.

The Epstein-Barr virus protein (EB2) allows the nuclear export of a particular subset of early and late viral RNAs derived from intronless genes. EB2 is conserved among most herpesvirus members and its presence is essential for the production of infectious particles. Here we show that, besides its role as a nuclear export factor, EB2 strongly stimulates translation of unspliced mRNAs without affecting overall cellular translation. Interestingly, this effect can be reversed by the addition of an intron within the gene. The spliced mRNA is then efficiently exported and translated even in the absence of EB2. Moreover, we show that EB2 associates with translating ribosomes and increases the proportion of its target RNA in the polyribosomal fraction. Finally, testing of EB2 homolog proteins derived from EBV-related herpesviruses, shows that, even if they play similar roles within the replication cycle of their respective virus, their mechanisms of action are different.

Varicella-zoster virus IE4 protein interacts with SR proteins and exports mRNAs through the TAP/NXF1 pathway.

Available data suggest that the Varicella-Zoster virus (VZV) IE4 protein acts as an important regulator on VZV and cellular genes expression and could exert its functions at post-transcriptional level. However, the molecular mechanisms supported by this protein are not yet fully characterized. In the present study, we have attempted to clarify this IE4-mediated gene regulation and identify some cellular partners of IE4. By yeast two-hybrid and immunoprecipitation analysis, we showed that IE4 interacts with three shuttling SR proteins, namely ASF/SF2, 9G8 and SRp20. We positioned the binding domain in the IE4 RbRc region and we showed that these interactions are not bridged by RNA. We demonstrated also that IE4 strongly interacts with the main SR protein kinase, SRPK1, and is phosphorylated in in vitro kinase assay on residue Ser-136 contained in the Rb domain. By Northwestern analysis, we showed that IE4 is able to bind RNA through its arginine-rich region and in immunoprecipitation experiments the presence of RNA stabilizes complexes containing IE4 and the cellular export factors TAP/NXF1 and Aly/REF since the interactions are RNase-sensitive. Finally, we determined that IE4 influences the export of reporter mRNAs and clearly showed, by TAP/NXF1 knockdown, that VZV infection requires the TAP/NXF1 export pathway to express some viral transcripts. We thus highlighted a new example of viral mRNA export factor and proposed a model of IE4-mediated viral mRNAs export.

Cell cycle-dependent caspase-like activity that cleaves p27(KIP1) is the beta(1) subunit of the 20S proteasome.

We previously described a caspase-like activity, which we termed KIPase that is implicated in the turnover of the mammalian cell cycle regulator p27(KIP1). KIPase cleaves a tetra-peptide substrate, Ac-DPSD-AMC, which mimics the target site in p27(KIP1), and inhibitors based on this tetra-peptide are ineffective against other known caspases. Here we describe the purification and characterization of KIPase, and trace its activity to the beta(1) subunit of the 20S proteasome. Further analyses revealed that the activity of the beta(1) subunit is up-regulated as cells enter the cell cycle without concomitant change in the levels of the proteasome beta(1), beta(2) or beta(5) subunits. To our knowledge, this is the first description of cell cycle regulation of the caspase-like activity of the 20S proteasome.

Protein kinase CK2 phosphorylation of EB2 regulates its function in the production of Epstein-Barr virus infectious viral particles.

The Epstein-Barr Virus (EBV) early protein EB2 (also called BMLF1, Mta, or SM) promotes the nuclear export of a subset of early and late viral mRNAs and is essential for the production of infectious virions. We show here that in vitro, protein kinase CK2alpha and -beta subunits bind both individually and, more efficiently, as a complex to the EB2 N terminus and that the CK2beta regulatory subunit also interacts with the EB2 C terminus. Immunoprecipitated EB2 has CK2 activity that phosphorylates several sites within the 80 N-terminal amino acids of EB2, including Ser-55, -56, and -57, which are localized next to the nuclear export signal. EB2S3E, the phosphorylation-mimicking mutant of EB2 at these three serines, but not the phosphorylation ablation mutant EB2S3A, efficiently rescued the production of infectious EBV particles by HEK293(BMLF1-KO) cells harboring an EB2-defective EBV genome. The defect of EB2S3A in transcomplementing 293(BMLF1-KO) cells was not due to impaired nucleocytoplasmic shuttling of the mutated protein but was associated with a decrease in the cytoplasmic accumulation of several late viral mRNAs. Thus, EB2-mediated production of infectious EBV virions is regulated by CK2 phosphorylation at one or more of the serine residues Ser-55, -56, and -57.

KIPase activity is a novel caspase-like activity associated with cell proliferation.

A novel caspase-like activity, which is directly regulated with cell proliferation is a candidate to regulate the abundance of the cyclin-dependent kinase inhibitor, p27(KIP1), in human lymphoid cells. This activity, which we term KIPase activity, can also cleave a subset of caspase substrates. Here we demonstrate that KIPase is a novel enzyme distinct from any of the previously characterized human caspases. We show that KIPase is active in a variety of cell lineages, its activity is associated with the proliferation of the human T-cell line, Jurkat, and is not inhibited by the broad spectrum caspase inhibitor z-VAD-fmk. Gel filtration analysis revealed that KIPase has a native molecular mass of approximately 100-200 kDa. Furthermore, the activity of KIPase does not change during apoptosis induced by either ligation of FAS or exposure of cells to etoposide. The uniqueness of KIPase is demonstrated by the fact that none of the human caspases tested (1-10) are able to cleave a specific KIPase substrate (Ac-DPSD-AMC) and that an aldehyde modified derivative of the DPSD tetra peptide is unable to inhibit caspases, but is a good inhibitor of KIPase activity. This supports a hypothesis whereby KIPase is a currently unidentified caspase-like enzyme which regulates the abundance of p27(KIP1) in a proliferation-dependent manner.