Chong-Yun Xiao

Nuclear targeting signal recognition: a key control point in nuclear transport?

Recent progress indicates that there are multiple pathways of nucleocytoplasmic transport which involve specific targeting sequences, such as nuclear localization sequences (NLSs), and cytosolic receptor molecules of the importin/karyopherin superfamily which recognise and dock the NLS‐containing proteins at the nuclear pore. This first step of nuclear import/export is of central importance, with the affinity of the importin‐targeting sequence interaction a critical parameter in determining transport efficiency. Different importins possess distinct NLS‐binding specificities, which allows the system to be modulated through differential expression of the importins themselves, as well as through competition between different importins for the same protein, and between different proteins for the same importin. The targeting sequence‐importin interaction can also be influenced directly by phosphorylation increasing the affinity of the interaction with importins or by targeting sequence masking through phosphorylation or specific protein binding. Targeting sequence recognition thus appears to represent a key control point in the regulation of nuclear transport. BioEssays 22:532–544, 2000.

The Protein Kinase CK2 Site (Ser111/112) Enhances Recognition of the Simian Virus 40 Large T-antigen Nuclear Localization Sequence by Importin

The mechanism by which phosphorylation regulates nuclear localization sequence (NLS)-dependent nuclear protein import is largely unclear. Whereas nuclear accumulation of SV40 large tumor antigen (T-ag) fusion proteins is completely dependent on the T-ag NLS (amino acids 126–132), the rate of nuclear import is increased 50-fold by amino acid residues 111–125 and in particular a site for the protein kinase CK2 (CK2) at serine 111/112. Because the first step of nuclear protein import involves the binding of the NLS by an NLS-receptor complex such as the importin 58/97 heterodimer, we established a novel enzyme-linked immunosorbent assay to test whether NLS recognition is influenced by amino acids amino-terminal to the NLS and the CK2 site. We found that recognition of the T-ag NLS by importin 58/97 was enhanced 10-fold in the presence of amino acid residues 111–125 and strongly dependent on importin 97. A T-ag fusion protein in which the spacer between the CK2 site and the NLS was decreased showed 30% reduced binding by importin 58/97. Maximal nuclear accumulation of this protein was reduced by more than 50%, indicating the physiological importance of the correctly positioned CK2 site. Phosphorylation by CK2 increased the T-ag NLS binding affinity for importin 58/97 by a further 40%. We conclude that flanking sequences and in particular phosphorylation at the CK2 site are mechanistically important in NLS recognition and represent the basis of their enhancement of T-ag nuclear import. This study thus represents the first elucidation of the mechanistic basis of the regulation of nuclear protein import through phosphorylation within a phosphorylation-regulated NLS.

SV40 Large Tumor Antigen Nuclear Import Is Regulated by the Double-stranded DNA-dependent Protein Kinase Site (Serine 120) Flanking the Nuclear Localization Sequence

Nuclear localization sequence (NLS)-dependent nuclear import of SV40 large tumor antigen (T-Ag) fusion proteins is regulated by phosphorylation sites for casein kinase II (CKII) and the cyclin-dependent kinase Cdc2 amino-terminal to the NLS (amino acids 126–132). Between the T-Ag CKII and Cdc2 sites is a site (Ser120) for the double-stranded DNA-dependent protein kinase (dsDNA-PK), which we show here for the first time to play a role in regulating T-Ag nuclear import. We replaced Ser120 by aspartic acid or alanine using site-directed mutagenesis and assessed the effects on nuclear transport kinetics both in vivo (microinjected cells) and in vitro (mechanically perforated cells) in HTC rat hepatoma cells. Maximal nuclear accumulation of the Asp120 and Ala120 protein derivatives was approximately 40% and 70% reduced in vivo, respectively, compared with that of the wild type protein, and similarly reducedin vitro, although to a lesser extent. This implies that the dsDNA-PK site regulates the maximal level of nuclear accumulation, normally functioning to enhance T-Ag nuclear transport; the higher accumulation of the Asp120 protein compared with the Ala120 protein indicates that negative charge at the dsDNA-PK site is mechanistically important in regulating nuclear import. The Asp120 protein accumulated in the nucleus at a faster rate than the wild type protein, implying that phosphorylation at Ser120 may also regulate the nuclear import rate. CKII phosphorylation of the Asp120 protein in cytosol or by purified CKII was approximately 30% higher than that of the Ser120 and Ala120 proteins, while negative charge at the CKII site increased dsDNA-PK phosphorylation of Ser120 by approximately 80% compared with wild type, implying physical and functional interactions between the two phosphorylation sites. Quantitation of NLS recognition by the importin 58/97 subunits using an enzyme-linked immunosorbent assay indicated that while the Ala120 protein derivative had a binding affinity very similar to that of wild type, the Asp120derivative showed 40% higher affinity. In vitro CKII phosphorylation increased importin binding by about 30% in all cases. These results imply that negative charge at the dsDNA-PK site may enhance nuclear import through increasing both NLS recognition by importin subunits, and phosphorylation at the CKII site, which itself also facilitates NLS recognition by importin 58/97.

Negative charge at the protein kinase CK2 site enhances recognition of the SV40 large T‐antigen NLS by importin: effect of conformation

SV40 large tumor‐antigen (T‐ag) nuclear import is enhanced by the protein kinase CK2 (CK2) site (Ser111Ser112) flanking the nuclear localization sequence (NLS). Here we use site‐directed mutagenesis to examine the influence of negative charge and conformation at the site on T‐ag nuclear import and recognition by the NLS‐binding importin subunits. Negative charge through aspartic acid in place of Ser111 simulated CK2 phosphorylation in enhancing nuclear accumulation to levels well above those of proteins lacking a functional CK2 site. This was shown to be through enhancement of T‐ag NLS recognition by importin using an ELISA‐based assay. Asp112‐substituted mutants containing proline at positions 109, 110 (wild‐type position) or 111 were compared to assess the role of conformation at the CK2 site. Maximal nuclear import of the protein with Pro109 was lower than that of the Pro110 derivative, with the Pro111 variant even lower, these differences also being attributable to effects on importin binding. All results indicate a correlation of the initial nuclear import rate with the importin binding affinity, demonstrating that NLS recognition by importin is a key rate‐determining step in nuclear import.

Signal- and importin-dependent nuclear targeting of the kidney anion exchanger 1-binding protein kanadaptin

Kanadaptin (kidney anion exchanger adaptor protein) has recently been identified as a protein with binding activity to the cytoplasmic domain of the kidney Na(+)-independent Cl(-)/HCO(-)(3) anion exchanger 1 (kAE1). Since it is widely expressed in tissues devoid of kAE1, however, kanadaptin is likely to have additional cellular roles. This is supported by its multidomain structure, and possession of three clusters of basic amino acids exhibiting similarity to known nuclear localization sequences (NLSs). In the present study, we use immunofluorescence and subcellular fractionation approaches to demonstrate that kanadaptin is localized within the nuclei of various epithelial and non-epithelial cultured cell types. The role of the different NLSs is examined in transfection studies using plasmids encoding full-length kanadaptin with or without green fluorescent protein (GFP) as a fusion tag, as well as truncation derivatives thereof. Strong nuclear localization of fusion proteins containing amino acids 140-230 of kanadaptin, which include the sequence AVSRKRKA(193) (NLS1) was observed. Substitution of Arg(191) with a threonine residue resulted in a cytoplasmic location of the expressed protein, while NLS1 proved sufficient to target an otherwise cytoplasmically localized beta-galactosidase-GFP fusion protein to the nucleus. Using a direct binding assay we show that a fusion protein containing kanadaptin amino acids 1-231 (but not the Thr(191) substituted derivative) is recognized with nM affinity by the conventional NLS-binding importin alpha/beta heterodimer. Nuclear import studies on microinjected and permeabilized rat hepatoma cells demonstrated functionality of the NLS in nuclear targeting, with inhibition by antibodies demonstrating the requirement of both importin alpha and beta for nuclear import of kanadaptin. That kanadaptin possesses a functional importin-alpha/beta-recognized NLS explains the nuclear localization of kanadaptin in various cultured cell types, and opens up the possibility that kanadaptin may have a signalling role in the nucleus.

An engineered site for protein kinase C flanking the SV40 large T-antigen NLS confers phorbol ester-inducible nuclear import.

Nuclear import of simian virus SV40 large tumour antigen (T‐ag) is enhanced by the protein kinase CK2 (CK2) site flanking the nuclear localisation sequence (NLS). We report here that replacement of this site with a consensus site for protein kinase C (PK‐C) can alter the regulation of T‐ag nuclear import and render it inducible by phorbol ester. Measurement of nuclear import kinetics using fluorescently labelled proteins and confocal laser scanning microscopy show that the introduced PK‐C site is functional in enhancing T‐ag nuclear import compared to a protein lacking the CK2 site. Treatment with the PK‐C activator phorbol 12‐myristate 13‐acetate (PMA) further increases the level of maximal nuclear accumulation and the initial nuclear import rate. This engineered PMA‐responsive NLS may have application in targeting of molecules of interest to the nucleus in response to agents stimulating PK‐C.