A three-residue signal confers localization of a reporter protein in the inner nuclear membrane

By alignment to the carboxy-terminal-deduced aa sequence of human cytomegalovirus glycoprotein B (gB), conserved hexameric aa motifs with putative function for localization in the inner nuclear membrane (INM) were identified in the nucleoplasmic tails of herpes simplex virus type 1 gB and of the cellular lamin B receptor. Fusion of the respective hexamers to CD8 as a reporter redirected transport of the chimeras into the INM, suggesting their functioning as modular signal motifs. Consecutive experiments showed that the three-residue motif RxR represents a consensus sequence which is sufficient for localization of the CD8 reporter in the INM.     

Characterization of three paralogous members of the Mammalian vaccinia related kinase family

Members of the novel vaccinia related kinase (VRK) protein family are characterized by notable sequence homology to the vaccinia virus-encoded B1 kinase (vvB1). vvB1 plays an essential role in viral DNA replication, and Boyle and Traktman have demonstrated that VRK1 enzymes complement the replication defect of a temperature-sensitive viral mutant defective in vvB1 (Boyle, K., and Traktman, P. (2004) J. Virol. 78, 1992-2005). This mammalian kinase family comprises three members, VRK1, VRK2, and VRK3. We have annotated the gene structure for the members of this family and have characterized the enzyme activity and subcellular localization for the human and mouse proteins. VRK1 enzymes show robust autophosphorylation activity and will phosphorylate casein; VRK2 enzymes show modest autophosphorylation activity and will also phosphorylate casein. The VRK3 proteins have key amino acid substitutions that disrupt invariant motifs required for catalytic activity, rendering them enzymatically inert. The VRK1 and VRK2 proteins contain COOH-terminal extracatalytic sequences that mediate intracellular localization. VRK1 proteins possess a basic nuclear localization signal and are indeed nuclear; the extreme C termini of the VRK2 proteins are highly hydrophobic, and the proteins are membrane-associated and colocalize with markers of the endoplasmic reticulum. The NH(2)-terminal region of the VRK3s contains a bipartite nuclear localization signal, which directs these proteins to the nucleus. Our findings provide the basis for further studies of the structure and function of this newly discovered family of protein kinases.     

Cell cycle-dependent and DNA damage-inducible nuclear localization of FEN-1 nuclease is consistent with its dual functions in DNA replication and repair

Flap endonuclease-1 (FEN-1), a 43-kDa protein, is a structure-specific and multifunctional nuclease. It plays important roles in RNA primer removal of Okazaki fragments during DNA replication, DNA base excision repair, and maintenance of genome stability. Three functional motifs of the enzyme were proposed to be responsible for its nuclease activities, interaction with proliferating cell nuclear antigen, and nuclear localization. In this study, we demonstrate in HeLa cells that a signal located at the C terminus (the nuclear localization signal (NLS) motif) facilitates nuclear localization of the enzyme during S phase of the cell cycle and in response to DNA damage. Truncation of the NLS motif prevents migration of the protein from the cytoplasm to the nucleus, while having no effect on the nuclease activities and its proliferating cell nuclear antigen interaction capability. Site-directed mutagenesis further revealed that a mutation of the KRK cluster to three alanine residues completely blocked the localization of FEN-1 into the nucleus, whereas mutagenesis of the KKK cluster led to a partial defect of nuclear localization in HeLa cells without observable phenotype in yeast. Therefore, the KRKXXXXXXXXKKK motif may be a bipartite NLS driving the protein into nuclei. Yeast RAD27Delta cells transformed with human mutant M(krk) survived poorly upon methyl methanesulfonate treatment or when they were incubated at an elevated temperature.     

Functional characterization of nuclear localization signals in yeast Sm proteins

In mammals, nuclear localization of U-snRNP particles requires the snRNA hypermethylated cap structure and the Sm core complex. The nature of the signal located within the Sm core proteins is still unknown, both in humans and yeast. Close examination of the sequences of the yeast SmB, SmD1, and SmD3 carboxyl-terminal domains reveals the presence of basic regions that are reminiscent of nuclear localization signals (NLSs). Fluorescence microscopy studies using green fluorescent protein (GFP)-fusion proteins indicate that both yeast SmB and SmD1 basic amino acid stretches exhibit nuclear localization properties. Accordingly, deletions or mutations in the NLS-like motifs of SmB and SmD1 dramatically reduce nuclear fluorescence of the GFP-Sm mutant fusion alleles. Phenotypic analyses indicate that the NLS-like motifs of SmB and SmD1 are functionally redundant: each NLS-like motif can be deleted without affecting yeast viability whereas a simultaneous deletion of both NLS-like motifs is lethal. Taken together, these findings suggest that, in the doughnut-like structure formed by the Sm core complex, the carboxyl-terminal extensions of Sm proteins may form an evolutionarily conserved basic amino acid-rich protuberance that functions as a nuclear localization determinant.     

Structure-based design of a pathway-specific nuclear import inhibitor

Kapbeta2 (also called transportin) recognizes PY nuclear localization signal (NLS), a new class of NLS with a R/H/Kx((2-5))PY motif. Here we show that Kapbeta2 complexes containing hydrophobic and basic PY-NLSs, as classified by the composition of an additional N-terminal motif, converge in structure only at consensus motifs, which explains ligand diversity. On the basis of these data and complementary biochemical analyses, we designed a Kapbeta2-specific nuclear import inhibitor, M9M.