Emerging biological functions of the vaccinia-related kinase (VRK) family.

The vaccinia-related kinases (VRKs) branched off early from the family of casein kinase (CK) I and compose a relatively uncharacterized family of the kinome. The VRKs were discovered due to their close sequence relation to the vaccinia virus B1R serine/threonine kinase. They were first described in phosphorylation of transcription factors that led to the discovery of an autoregulatory mechanism between VRK and the tumor suppressor transcription factor p53. The relevance of VRKs has broadened recently by introduction of its members as essential regulators in cell signaling, nuclear envelope dynamics, chromatin modifications, apoptosis and cellular stress response. Several phosphorylation substrates have been described, as well as the first positive and negative regulators of VRK. We provide an overview of the VRKs across species and discuss the wide diversity of cellular and organismal requirements for this kinase family.

A role for the basic patch and the C terminus of RanGTP in regulating the dynamic interactions with importin beta, CRM1 and RanBP1.

Transport of macromolecules between the nucleus and cytoplasm involves the recognition of intrinsic localization signals by either import or export receptors. The interaction of the receptors with their cargo is regulated by the small GTPase Ran in its GTP bound state. We have investigated the interaction of RanGTP with the import factor, importin beta, the export factor, CRM1, and the Ran binding protein, RanBP1, in solution. Importin beta specifically protected residues in the switch regions and basic patch region of Ran against proteolytic cleavage, whereas RanBP1 protected the C terminus. Moreover, the binding of importin beta induced a conformational change in the structure of Ran leading to an exposure of the C terminus and stimulated the binding of RanBP1. Mutating the basic patch (HRKK(142)) of Ran resulted in an increased binding of RanBP1 and weakened importin beta binding. In contrast to wild-type Ran, the mutant Ran could be released from importin beta independently of importin alpha. These data provide experimental support for a model in which the accessibility of the C terminus of Ran is influenced by an intramolecular interaction between the basic patch and the C-terminal acidic DEDDDL(216) motif. Binding of importin beta probably disrupts this interaction causing an exposure of the C-terminal extension, which is favorable for RanBP1 binding. Interestingly, basic patch mutations abolish CRM1 interaction, indicating that the determinants for RanGTP binding to the export factor, CRM1, is different from the import factor, importin beta.

Nuclear export of the DEAD box An3 protein by CRM1 is coupled to An3 helicase activity.

We have recently identified the Xenopus laevis An3 protein as a bona fide substrate for the nuclear export receptor CRM1 (Exportin 1). An3 binds directly to CRM1 with high affinity via a leucine-rich nuclear export signal located in the extreme N terminus. An3 is a member of the DEAD box family of RNA helicases, which unwind RNA duplexes. RNA unwinding is coupled to hydrolysis of nucleoside triphosphates by the helicase, and the ATPase activity of several helicases is greatly stimulated by various polynucleotides. Here we report that dATP hydrolysis by An3 is stimulated approximately 6-fold by total RNA from X. laevis oocytes, whereas poly(U) RNA fails to enhance hydrolysis, suggesting the existence of a specific RNA activator for An3. Kinetic analysis reveals that a mutation within the conserved DEAD box motif reduces the rate of dATP hydrolysis by approximately 6-fold. In accordance with this, the DEAD box mutant is unable to unwind double-stranded RNA. Microinjection of the An3 DEAD box mutant into X. laevis oocytes nuclei reveals a significantly lower export rate as compared with wild-type An3 protein. This is not because the mutant has lower affinity toward CRM1, nor is it due to altered RNA binding capacity. This suggests that nuclear export of An3 protein by CRM1 is coupled to An3 helicase activity.

RanGTP-regulated interactions of CRM1 with nucleoporins and a shuttling DEAD-box helicase.

CRM1 is an export receptor mediating rapid nuclear exit of proteins and RNAs to the cytoplasm. CRM1 export cargoes include proteins with a leucine-rich nuclear export signal (NES) that bind directly to CRM1 in a trimeric complex with RanGTP. Using a quantitative CRM1-NES cargo binding assay, significant differences in affinity for CRM1 among natural NESs are demonstrated, suggesting that the steady-state nucleocytoplasmic distribution of shuttling proteins could be determined by the relative strengths of their NESs. We also show that a trimeric CRM1-NES-RanGTP complex is disassembled by RanBP1 in the presence of RanGAP, even though RanBP1 itself contains a leucine-rich NES. Selection of CRM1-binding proteins from Xenopus egg extract leads to the identification of an NES-containing DEAD-box helicase, An3, that continuously shuttles between the nucleus and the cytoplasm. In addition, we identify the Xenopus homologue of the nucleoporin CAN/Nup214 as a RanGTP- and NES cargo-specific binding site for CRM1, suggesting that this nucleoporin plays a role in export complex disassembly and/or CRM1 recycling.

The specificity of the CRM1-Rev nuclear export signal interaction is mediated by RanGTP.

Nuclear export of intron-containing human immunodeficiency virus type 1 (HIV-1) RNA is mediated by the viral Rev protein that contains both an RNA binding domain specific for the viral Rev response element (RRE) and a nuclear export signal (NES). The cellular CRM1 (Exportin1) protein functions as a nuclear export receptor for proteins carrying a Rev-like NES in a process that also requires the GTP bound form of the Ran GTPase. Using purified recombinant factors, we show by co-precipitation, gel mobility shift and protein footprinting assays that full-length Rev protein interacts directly with CRM1 in vitro independently of both the integrity of the characteristic leucine residues of the NES and the presence of the cytotoxin leptomycin B (LMB). Addition of RanGTP induces the formation of an RRE-Rev-CRM1-RanGTP complex that is sensitive to LMB, NES mutations, and Ran being charged with GTP. Within this complex, CRM1 is readily cross-linked to Cys89 near the NES of Rev. By protein footprinting, we demonstrate that the NES of Rev and two regions in CRM1 become inaccessible to endoproteinases upon binding suggesting that these regions are involved in protein-protein interactions. Our data are consistent with a model in which CRM1 is the nuclear export receptor for the Rev-RRE ribonucleoprotein complex and that RanGTP binds to a preformed Rev-CRM1 complex and specifies a functional interaction with the NES.

Mapping of multiple RNA binding sites of human T-cell lymphotropic virus type I rex protein within 5'- and 3'-Rex response elements.

Interaction between the human T-cell lymphotropic virus type I Rex protein and viral transcripts in the nucleus is essential to the cytoplasmic appearance of unspliced and singly spliced viral RNA. Rex has been shown to mediate its function through direct interaction with a highly ordered secondary structure in the 3'-untranslated region of all human T-cell lymphotropic virus type I mRNAs termed the Rex response element (3'-RxRE). Part of the 3'-RxRE sequence is also present in the 5'-end of viral transcripts (5'-RxRE), and we demonstrate that Rex binds to this RNA with essentially the same affinity and specificity as to the 3'-RxRE. We have analyzed the secondary structures and binding sites of Rex within the 5'- and 3'-RxREs by enzymatic probing and chemical modification interference and show that multiple Rex molecules bind within a stem-loop, which is similarly structured in the two RxREs. Our experiments confirm the presence of a previously characterized Rex binding site but also identify a common motif within an extended region that comprises an additional Rex binding site. This suggests that Rex oligomerizes on the RxREs similarly to what has been observed for binding of the human immunodeficiency virus type 1 Rev protein to the Rev response element.