Subcellular Relocalization Research Articles

Self-association of Class II Transactivator Correlates with Its Intracellular Localization and Transactivation

Class II transactivator (CIITA) is the master regulator of major histocompatibility complex class II genes that regulates both B lymphocyte-specific and interferon gamma-inducible expression. Here we identify protein regions and examine mechanisms that determine the intracellular distribution of CIITA. We show that two separate regions of CIITA mediate nuclear export: amino acids 1-114 and 408-550. Both regions interact with the export receptor CRM-1. The CIITA region spanning amino acids 408-550 of CIITA also determines its ability for homotypic self-association as well as heterotypic interactions with other regions residing at the amino and carboxyl termini of the protein. These observations are in line with data demonstrating that co-expression of amino- and carboxyl-terminal parts of CIITA promote subcellular relocalization and, remarkably, rescue transcriptional activation by individually inert molecules. CIITA point mutations that impair nuclear import and abolish its activation function show reduced self-association. We propose that the concerted action of homo- and heterotypic interactions of CIITA determine proper protein configuration that in turn controls its nucleocytoplasmic trafficking.

Ultraviolet B-Mediated Phosphorylation of the Small Heat Shock Protein HSP27 in Human Keratinocytes

Exposure of human keratinocytes to environmental stress is known to induce changes in the expression, phosphorylation, and subcellular relocalization of the 27 kDa heat shock protein. This study demonstrates that ultraviolet B (280-320 nM) irradiation with physiologic doses induces a dose-dependent phosphorylation of 27 kDa heat shock protein, generating the more acidic 27 kDa heat shock protein B, C, and D isoforms. Ultraviolet B also induces perinuclear cytoplasmic relocation and nuclear translocation of 27 kDa heat shock protein and caused aggregation of cytoplasmic actin filaments into a broad perinuclear distribution. The ultraviolet B-induced phosphorylation is reversible, returning to baseline levels 4 h after exposure, and this coincides with the reversal of ultraviolet B-induced actin reorganization. The ultraviolet B-induced phosphorylation is not affected by the protein kinase C inhibitor, GF 109203X, is partially inhibited by epidermal growth factor receptor tyrosine kinase inhibitor, PD 153035, and is substantially inhibited by the specific p38 mitogen-activated protein kinase inhibitor, SB 203580. In addition, pretreatment of cells with the anti-oxidant N-acetyl cysteine partially inhibits ultraviolet B-and oxidant-induced 27 kDa heat shock protein phosphorylation. The p38 mitogen-activated protein kinase cascade is thus the major transduction pathway for ultraviolet B-induced 27 kDa heat shock protein phosphorylation, and reactive oxygen species generated in response to ultraviolet B also contribute to this phosphorylation. As 27 kDa heat shock protein phosphorylation and relocalization has been associated with increased cell survival after environmental insult, our data suggest that ultraviolet B, in addition to initiating recognized cytotoxic events in keratinocytes, also initiates a signaling pathway that may provide cellular protection against this ubiquitous environmental insult.

Nuclear Localization of Mitogen-activated Protein Kinase Kinase 1 (MKK1) Is Promoted by Serum Stimulation and G2-M Progression: REQUIREMENT FOR PHOSPHORYLATION AT THE ACTIVATION LIP AND SIGNALING DOWNSTREAM OF MKK

Stimulation of mammalian cells results in subcellular relocalization of Ras pathway enzymes, in which extracellular signal-regulated protein kinases rapidly translocate to nuclei. In this study, we define conditions for nuclear localization of mitogen-activated protein kinase kinase 1 (MKK1) by examining effects of perturbing the nuclear export signal (NES), the regulatory phosphorylation sites Ser218 and Ser222, and a regulatory domain at the N terminus. After disrupting the NES (Delta32-37), nuclear uptake of MKK was enhanced when quiescent cells were activated with serum-phorbol 12-myristate 13-acetate or BXB-Raf-1 cotransfection. Uptake was enhanced by mutation of Ser218 and Ser222 to Glu and Asp, respectively, and blocked by mutation of these residues to Ala, although mutation of Lys97 to Met, which renders MKK catalytically inactive, did not interfere with uptake. Therefore, nuclear uptake of MKK requires incorporation of phosphate or negatively charged residues at the activation lip but not enzyme activity. On the other hand, uptake of an active MKK mutant with disrupted NES (Delta32-51) was elevated in quiescent as well as stimulated cells, and pretreatment of cells with the MKK inhibitor 1,4-diamino-2, 3-dicyano-1,4-bis[2-aminophenylthio]butadiene blocked nuclear uptake. Thus, signaling downstream of MKK is also necessary for translocation. Finally, wild type MKK containing an intact NES translocates to nuclei during mitosis before envelope breakdown. Comparison of mutants with Ser to Glu and Asp or Ala substitutions indicates that Ser phosphorylation is also required for mitotic nuclear uptake of MKK.

Two Regions in the CD80 Cytoplasmic Tail Regulate CD80 Redistribution and T Cell Costimulation

CD28 is a major T cell costimulatory molecule, delivering signals distinct from those of the CD3/TCR complex, which regulate cytokine and cytokine receptor expression, cell proliferation, and cell viability. CD28 needs to be cross-linked to initiate signals, yet both of its ligands, CD80 and CD86, are expressed as monomers. Previously, we determined the cytoplasmic tail of CD80 is required for CD28-mediated costimulation and subcellular relocalization of CD80 in lymphocytes. In this study, we report that Reh B cell transfectants expressing CD80 with mutations in the cytoplasmic tail region either at 275-278 (RRNE-->AAAA, CD80/4A) or serine 284 (S-->A, CD80/SA) can bind ligand similar to transfectants expressing wild-type CD80, yet are unable to costimulate T cell proliferation. These mutant CD80 molecules are expressed on the surface of the Reh cells in small clusters or foci indistinguishable from those of wild-type CD80 molecules. However, mutant CD80 molecules unlike wild-type CD80 cannot be readily induced by ligand into caps. Thus, small clusters of CD80 found on APC are insufficient to initiate CD28-mediated signals, and the formation of CD80 caps appears to be a critical factor regulating the initiation of T cell costimulation. A 30-kDa phosphoprotein that associates with the cytoplasmic tail of CD80 in activated cells may play a role in CD80 redistribution and thus CD28-mediated costimulation. These results indicate two distinct regions of the CD80 cytoplasmic tail regulate its costimulatory function, and both regions are required for CD80 function.

Dynamic fluorescence changes during photodynamic therapy in vivo and in vitro of hydrophilic A1 (III) phthalocyanine tetrasulphonate and lipophilic ZN(II) phthalocyanine administered in liposomes

The fluorescence emission of hydrophilic tetrasulphonated aluminium phthalocyanine (AlPcS 4) and hydrophobic zinc phthalocyanine (ZnPc), bound to the membrane of liposomes, was investigated in vivo in an appropriate tumour model of the rat bladder and in RR 1022 epithelial cells of the rat. The sensitizers were administered systemically to the rats and photodynamic therapy (PDT) was performed 24 h later. During PDT treatment, the fluorescence was measured every 30 s. The fluorescence was excited with 633 nm light from an HeNe laser and the fluorescence spectra were detected with an optical multichannel analyser system. PDT was performed for both sensitizers using 672 nm light from an Ar + dye laser. The fluorescence changes during PDT were significantly different for the two phthalocyanines. For AlPcS 4, an initial fluorescence intensity increase, followed by subsequent photobleaching, was observed. In contrast, ZnPc fluorescence showed an exponential decrease and no increase at the start of treatment. Tumour necrosis 24 h after PDT was significant only for ZnPc. RR 1022 cells incubated for 24 h with AlPcS 4 revealed a granular fluorescence pattern, whereas ZnPc was localized diffusely in the cytoplasm of the cells. In agreement with the in vivo measurements, subcellular relocalization and a fluorescence intensity increase were detected exclusively in the case of AlPcS 4. Morphological changes at this time were significant only for ZnPc. The subcellular localization and fluorescence kinetics were obtained using a confocal laser scanning microscope.

Determination of specific protein kinase activities using phosphorus-33

The immune complex kinase assay is the most widely applied method to assess the catalytic activity of protein tyrosine kinases. It offers the advantage that the activity of a single selected enzyme can be determined, and that the enzyme activity can be normalized for the amount of enzyme in a parallel immunoblotting experiment. Here, we describe the use of the recently introduced isotope phosphorus-33 for the protein kinase assay. The lower energy of 33P, compared with the traditionally applied 32P, allows the simultaneous examination of the amount of enzyme with 125I-labeled antibodies. By analysing one and the same sample for both kinase activity and protein amount, the variation between parallel processed samples is avoided. Using this method, specific kinase activities can be calculated with high precision. The assay is particularly useful for the detection of cytokine and growth factor-induced activation of kinases, as changes in enzyme amounts by subcellular relocalization can be distinguished.

Subcellular Redistribution of HTLV-I Tax Protein by NF-κB/Rel Transcription Factors

The viral oncogene Tax derived from human T cell leukemia virus type I (HTLV-I) is a positive transcriptional activator of HTLV-I gene expression. Tax is also able to indirectly stimulate transcription of several growth regulatory genes by an indirect mechanism via association with host transcription factors. One of the cellular targets of the trans-activating effects of Tax is the NF-κB/Rel family of transcription factors, pleiotropic regulators of immunoregulatory, cytokine, and viral gene expression. Recent studies demonstrated that specific subunits of NF-κB (NFKB2(p100) and c-Rel) were overexpressed in HTLV-I-infected and Tax-expressing cells. Furthermore, Tax physically associated with NFKB2(p100). Monospecific antibodies directed against individual NF-κB subunits were generated and used to investigate the consequences of the interactions between Tax and NF-κB in a cotransfection-immunofluorescence assay. These studies demonstrate: (1) distinct compartmentalization of NF-κB precursors and products, (2) differential induction of the endogenous IκBα protein by transfected NF-κB subunits, (3) subcellular relocalization of Tax to the cytoplasm or nucleus depending on the coexpressed NF-κB subunit, and (4) Tax interaction with the Rel homology domain region of NFκB2. These studies indicate that the transcription modulatory influence of HTLV-I Tax may be significantly influenced by cytoplasmic-nuclear partitioning associated with the NF-κB proteins.

Transformation by middle T antigens

The polyomaviruses, members of the papovavirus group of DNA tumor viruses, encode in the 'early' region of the genome, three proteins involved in transformation, the tumor (or T) antigens, called large (LT), middle (mT) and small (sT) T. LT is required for establishment of primary fibroblasts, and sT promotes the efficiency of transformation both in vivo and in vitro, but it is mT that carries the transforming ability. The mTs of the two known polyomaviruses, from mouse and hamster, possess no intrinsic catalytic activity, but rather interact with and change the activity of several cellular proteins, including Src family protein tyrosine kinases, protein phosphatase 2A, phosphatidylinositol 3-kinase and Shc. Some of these proteins are also involved in signal transduction events elicited by growth factors. Like activated growth factor receptors, mT brings its associated proteins to a membranous environment. Transformation by mT might result not only from allosteric effects of mT on its interacting proteins but also as a result of their subcellular relocalization.