The retinoblastoma (RB) tumor suppressor is a nuclear phosphoprotein important for cell growth control and able to bind specifically to viral oncoproteins such as the SV40 large tumor antigen (T-ag). Human RB possesses a bipartite nuclear localization sequence (NLS) consisting of two clusters of basic amino acids within amino acids 860 -877, also present in mouse and Xenopus homologs, which resembles that of nucleoplasmin. The T-ag NLS represents a different type of NLS, consisting of only one stretch of basic amino acids. To compare the nuclear import kinetics conferred by the bipartite NLS of RB to those conferred by the T-ag NLS, we used ␤-galactosidase fusion proteins containing the NLSs of either RB or T-ag. The RB NLS was able to target ␤-galactosidase to the nucleus both in vivo (in microinjected cells of the HTC rat hepatoma line) and in vitro (in mechanically perforated HTC cells). Mutational substitution of the proximal basic residues of the NLS abolished nuclear targeting activity, confirming its bipartite character. Nuclear accumulation of the RB fusion protein was half-maximal within about 8 min in vivo, maximal levels being between 3-4-fold those in the cytoplasm, which was less than 50% of the maximal levels attained by the T-ag fusion protein, while the initial rate of nuclear import of the RB protein was also less than half that of T-ag. Nuclear import conferred by both NLSs in vitro was dependent on cytosol and ATP and inhibited by the nonhydrolyzable GTP analog GTP␥S. Using an ELISA-based binding assay, we determined that the RB bipartite NLS had severely reduced affinity, compared with the T-ag NLS, for the high affinity heterodimeric NLS-binding protein complex importin 58/97, this difference presumably representing the basis of the reduced maximal nuclear accumulation and import rate in vivo. The results support the hypothesis that the affinity of NLS recognition by NLS-binding proteins is critical in determining the kinetics of nuclear protein import.All passive and active transport into and out of the nucleus occurs through the nuclear pore complex (NPC) 1 (1-3). Proteins larger than 45 kDa require a nuclear localization sequence (NLS) (4) to be targeted to the nucleus. NLSs are defined as the sequences sufficient and necessary for nuclear import of their respective proteins (5-7) and are generally functional in targeting heterologous, normally cytoplasmic proteins to the nucleus. NLS-dependent protein transport can be divided into two steps. The first is energy-independent and involves recognition and targeting of the NLS-containing protein to the NPC by a heterodimeric protein complex consisting of importin 58/97 (8, 9) or ␣/␤ (10, 11). The second, energy-dependent step is the translocation of the NLS-bearing protein through the NPC into the nucleus (12, 13) and requires GTP hydrolysis mediated by the GTP-binding protein Ran/TC4 (14 -16) and the interacting factor p10/NTF2 (17-19). We were interested in the nuclear import kinetics of tumor suppressor proteins, one of which is the retino...

The different classes of conventional nuclear localization sequences (NLSs) resemble one another in that NLS-dependent nuclear protein import is energy-dependent and mediated by the cytosolic NLS-binding importin/karyopherin subunits and monomeric GTP-binding protein Ran/TC4. Based on analysis of the nuclear import kinetics mediated by the NLS of the human immunodeficiency virus accessory protein Tat using in vivo and in vitro nuclear transport assays and confocal laser scanning microscopy, we report a novel nuclear import pathway. We demonstrate that the Tat-NLS, not recognized by importin 58/97 subunits as shown using an enzyme-linked immunosorbent assay-based binding assay, is sufficient to target the 476-kDa heterologous beta-galactosidase protein to the nucleus in ATP-dependent but cytosolic factor-independent fashion. Excess SV40 large tumor antigen (T-ag) NLS-containing peptide had no significant effect on the nuclear import kinetics implying that the Tat-NLS was able to confer nuclear accumulation through a pathway distinct from conventional NLS-dependent pathways. Nucleoplasmic accumulation of the Tat-NLS-beta-galactosidase fusion protein, in contrast to that of a T-ag-NLS-containing fusion protein, also occurred in the absence of an intact nuclear envelope, implying that the Tat-NLS conferred binding to nuclear components. This is in stark contrast to known NLSs such as those of T-ag which confer nuclear entry rather than retention. Significantly, the ability to accumulate in the nucleus in the absence of an intact nuclear envelope was blocked in the absence of ATP, as well as by nonhydrolyzable ATP and GTP analogs, demonstrating that ATP is required to effect release from a complex with insoluble cytoplasmic components. Taken together, the results demonstrate that, dependent on ATP for release from cytoplasmic retention, the Tat-NLS is able to confer nuclear entry and binding to nuclear components. These unique properties indicate that Tat accumulates in the nucleus through a novel import pathway.

Desmocollin (Dsc) 1-3 and desmoglein (Dsg) 1-4, transmembrane proteins of the cadherin family, form the adhesive core of desmosomes. Here we provide evidence that Dsc3 homo-and heterophilic trans-interaction is crucial for epidermal integrity. Single molecule atomic force microscopy (AFM) revealed homophilic trans-interaction of Dsc3. Dsc3 displayed heterophilic interaction with Dsg1 but not with Dsg3. A monoclonal antibody targeted against the extracellular domain reduced homophilic and heterophilic binding as measured by AFM, caused intraepidermal blistering in a model of human skin, and a loss of intercellular adhesion in cultured keratinocytes. Because autoantibodies against Dsg1 are associated with skin blistering in pemphigus, we characterized the role of Dsc3 binding for pemphigus pathogenesis. In contrast to AFM experiments, laser tweezer trapping revealed that pemphigus autoantibodies reduced binding of Dsc3-coated beads to the keratinocyte cell surface. These data indicate that loss of heterophilic Dsc3/Dsg1 binding may contribute to pemphigus skin blistering.

Control over the nuclear import of transcription factors (TFs) represents a level of gene regulation integral to cellular processes such as differentiation and transformation. The Drosophila TF Dorsal shares with other rel TF family members the fact that it contains a phosphorylation site for the cAMP-dependent protein kinase (PKA) 22 amino acids N-terminal to the nuclear localization signal (NLS) at amino acids 335-340. This study examines for the first time the nuclear import kinetics of Dorsal fusion proteins in rat hepatoma cells in vivo and in vitro. Nuclear uptake was found to be not only NLS-dependent, but also strongly dependent on the PKA site, whereby substitution of Ser 312 by either Ala or Glu using site-directed mutagenesis severely reduced nuclear accumulation. Exogenous cAMP or PKA catalytic subunit significantly enhanced the nuclear import of wild-type proteins both in vivo and in vitro. Using a direct binding assay, the molecular basis of PKA site enhancement of Dorsal fusion protein nuclear import was determined to be PKA site-mediated modulation of NLS recognition by the importin 58/97 complex. The physiological relevance of these results is supported by the observation that Drosophila embryos expressing PKA site Dorsal mutant variants were impaired in development. We conclude that the Dorsal NLS and PKA site constitute a phosphorylation-regulated NLS essential to Dorsal function and able to function in heterologous mammalian cell systems, where phosphorylation modulates the affinity of NLS recognition by importin.Precisely scheduled nuclear import of transcription factors (TFs) 1 is a key factor in eukaryotic cell function (1-3). While proteins such as histones appear to be constitutively targeted to the nucleus, TFs such as those of the rel family (1, 4 -7), the nuclear factor of activated T-cells (8), SWI5 from yeast (9, 10), and the cytokine responsive signal transducers and activators of transcription (STATs) (11, 12) are translocated to the nucleus only under specific conditions, being otherwise cytoplasmic and thereby directly accessible to cytoplasmic signal-transducing systems (1). The fact that nuclear translocation of many TFs and oncogene products accompanies changes in the differentiation or metabolic state of eukaryotic cells underlines the fact that nuclear protein import is a key control point in the regulation of gene expression (2, 3).Proteins larger than 45 kDa require a nuclear localization sequence (NLS) (2, 3) in order to be targeted to the nucleus. We and others have shown that phosphorylation in the vicinity of NLSs plays a central role in regulating NLS-dependent nuclear protein import (7-21). The modular sequence motifs able to confer regulated nuclear protein import on heterologous proteins, called phosphorylation-regulated NLSs (prNLSs) (2, 3), have been identified for a number of proteins, including the CcN motif of the simian virus SV40 large tumor antigen (T-ag) where transport is regulated by dual phosphorylation by protein kinase CK2 (CK2) and the cyclin-depe...

Male infertility affects at least 5% of reproductive age males. The most common pathology is a complex presentation of decreased sperm output and abnormal sperm shape and motility referred to as oligoasthenoteratospermia (OAT). For the majority of OAT men a precise diagnosis cannot be provided. Here we demonstrate that leucine-rich repeats and guanylate kinase-domain containing isoform 1 (LRGUK-1) is required for multiple aspects of sperm assembly, including acrosome attachment, sperm head shaping and the initiation of the axoneme growth to form the core of the sperm tail. Specifically, LRGUK-1 is required for basal body attachment to the plasma membrane, the appropriate formation of the sub-distal appendages, the extension of axoneme microtubules and for microtubule movement and organisation within the manchette. Manchette dysfunction leads to abnormal sperm head shaping. Several of these functions may be achieved in association with the LRGUK-1 binding partner HOOK2. Collectively, these data establish LRGUK-1 as a major determinant of microtubule structure within the male germ line.

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Pemphigus vulgaris (PV) autoantibodies directly inhibit desmoglein (Dsg) 3-mediated transinteraction. Because cellular signaling also seems to be required for PV pathogenesis, it is important to characterize the role of direct inhibition in pemphigus acantholysis to allow establishment of new therapeutic approaches. Therefore, we modeled the Dsg1 and Dsg3 sequences into resolved cadherin structures and predicted peptides targeting the adhesive interface of both Dsg3 and Dsg1. In atomic force microscopy single molecule experiments, the selfdesigned cyclic single peptide specifically blocked homophilic Dsg3 and Dsg1 transinteraction, whereas a tandem peptide (TP) consisting of two combined single peptides did not. TP did not directly block binding of pemphigus IgG to their target Dsg antigens but prevented PV-IgG-induced inhibition of Dsg3 transinteraction in cell-free (atomic force microscopy) and cell-based (laser tweezer) experiments, indicating stabilization of Dsg3 bonds. Similarly, PV-IgG-mediated acantholysis and disruption of Dsg3 localization in HaCaT keratinocytes was partially blocked by TP. This is the first evidence that direct inhibition of Dsg3 binding is important for PV pathogenesis and that peptidomimetics stabilizing Dsg transinteraction may provide a novel approach for PV treatment. Autoantibodies in pemphigus vulgaris (PV)4 are mainly directed against the adhesion molecules desmoglein (Dsg) 3 and Dsg1 (1, 2). Recently, we provided direct evidence that PV autoantibodies directly block Dsg3 transinteraction (3). In contrast, Dsg1 autoantibodies in pemphigus foliaceus (PF) were found to disrupt Dsg1 transinteraction most likely via cellular signaling events rather than by direct inhibition (3, 4). However, it still remained unanswered whether direct inhibition of Dsg3 transinteraction is the sole pathogenic mechanism in mucosaldominant PV where autoantibodies against Dsg3 but not against Dsg1 are present (1). Alternatively, direct inhibition could act in concert with autoantibody-induced cellular signaling or may just be an epiphenomenon secondary to skin blistering (1, 5-7).The idea of direct interference with Dsg transinteraction by pemphigus autoantibodies has been proposed when Dsg3 was discovered to be a cadherin-type cell adhesion molecule (8, 9). From structural and mutational analyses, it was concluded that classical cadherins are able to form adhesive dimers through their N-terminal cadherin extracellular domain (EC1) via different interaction schemes (10 -13). Investigations of desmosomes using electron tomography from tissue sections also indicated various interaction models for desmosomal cadherins (14). Some of these interaction models resembled the interaction schemes found in the high resolution crystal structure analyses of N-and E-cadherin.This study was designed to modulate Dsg3 and Dsg1 transinteraction by using peptides fitting the most likely Dsg adhesive interface revealed by three-dimensional modeling. In an earlier study, it was reported that peptides targeting the putativ...

transinteraction resistant against an inhibitory monoclonal antibody. Moreover, TP reduced VE-cadherin lateral mobility and enhanced binding of VE-cadherin-coated microbeads to cultured endothelial cells, but acted independently of the actin cytoskeleton. TP also stabilized endothelial barrier properties against the Ca 2+ ionophore A23187 and the inhibitory antibody. Finally, TP abolished endothelial permeability increase induced by tumour necrosis factor-α in microperfused venules in vivo. Stabilization of VE-cadherin adhesion by cross-bridging peptides may therefore be a novel therapeutic approach for the treatment of vascular hyperpermeability.