scholarly journals Subcellular Targeting of p33ING1b by Phosphorylation-Dependent 14-3-3 Binding Regulates p21WAF1 Expression

2006 ◽  
Vol 26 (8) ◽  
pp. 2947-2954 ◽  
Author(s):  
Wei Gong ◽  
Michael Russell ◽  
Keiko Suzuki ◽  
Karl Riabowol
Keyword(s):  
Kinase Inhibitor ◽  
Biological Activities ◽  
Ectopic Expression ◽  
Growth Stress ◽  
Nuclear Localization Sequence ◽  
Binding Motif ◽  
Subcellular Targeting ◽  
Cyclin Dependent Kinase Inhibitor ◽  
Cargo Proteins ◽  
Localization Sequence

ABSTRACT ING1 is a type II tumor suppressor that affects cell growth, stress signaling, apoptosis, and DNA repair by altering chromatin structure and regulating transcription. Decreased ING1 expression is seen in several human cancers, and mislocalization has been noted in diverse types of cancer cells. Aberrant targeting may, therefore, functionally inactivate ING1. Bioinformatics analysis identified a sequence between the nuclear localization sequence and plant homeodomain domains of ING1 that closely matched the binding motif of 14-3-3 proteins that target cargo proteins to specific subcellular locales. We find that the widely expressed p33ING1b splicing isoform of ING1 interacts with members of the 14-3-3 family of proteins and that this interaction is regulated by the phosphorylation status of ING1. 14-3-3 binding resulted in significant amounts of p33ING1b protein being tethered in the cytoplasm. As shown previously, ectopic expression of p33ING1b increased levels of the p21Waf1 cyclin-dependent kinase inhibitor upon UV-induced DNA damage. Overexpression of 14-3-3 inhibited the up-regulation of p21Waf1 by p33ING1b, consistent with the idea that mislocalization blocks at least one of ING1's biological activities. These data support the idea that the 14-3-3 proteins play a crucial role in regulating the activity of p33ING1b by directing its subcellular localization.

The COOH-terminal nuclear localization sequence of interferon gamma regulates STAT1 alpha nuclear translocation at an intracellular site

2000 ◽  
Vol 113 (15) ◽  
pp. 2771-2781
Author(s):  
P.S. Subramaniam ◽  
J. Larkin ◽  
M.G. Mujtaba ◽  
M.R. Walter ◽  
H.M. Johnson
Keyword(s):  
Nuclear Localization ◽  
Nuclear Import ◽  
Nuclear Translocation ◽  
Biological Activities ◽  
Nuclear Localization Sequence ◽  
Receptor Complex ◽  
Ifn Gamma ◽  
High Affinity ◽  
Gamma Receptor ◽  
Localization Sequence

We have recently shown that the nuclear localization of IFN gamma is mediated by a polybasic nuclear localization sequence (NLS) in its C terminus. This NLS is required for the full expression of biological activity of IFN gamma, both extracellularly and intracellularly. We now show that this NLS plays an integral intracellular role in the nuclear translocation of the transcription factor STAT1 alpha activated by IFN gamma. Treatment of IFN gamma with antibodies to the C-terminal region (95–133) containing the NLS blocked the induction of STAT1 alpha nuclear translocation. The antibodies had no effect on nuclear translocation of STAT1 alpha in IFN gamma treated cells. A deletion mutant of human IFN gamma, IFN gamma (1–123), which is devoid of the C-terminal NLS region was found to be biologically inactive, but was still able to bind to the IFN gamma receptor complex on cells with a K(d) similar to that of the wild-type protein. Deletion of the NLS specifically abolished the ability of IFN gamma(1–123) to initiate the nuclear translocation of STAT1 alpha, which is required for the biological activities of IFN gamma following binding to the IFN gamma receptor complex. Thus, the NLS region appears to contribute minimally to extracellular high-affinity receptor-ligand binding, yet exerts a strong functional role in STAT1 alpha nuclear localization. A high-affinity site for the interaction of the C-terminal NLS domain of IFN gamma with a K(d) approx. 3 × 10(−8) M(−1) has been described by previous studies on the intracellular cytoplasmic domain of the IFN gamma receptor alpha-chain. To examine the role of the NLS at the intracellular level, we microinjected neutralizing antibodies raised against the C-terminal NLS domain of IFN gamma into the cytoplasm of cells before treatment of cells with IFN gamma. These intracellular antibodies specifically blocked the nuclear translocation of STAT1 alpha following the subsequent treatment of these cells extracellularly with IFN gamma. These data show that the NLS domain of IFN gamma interacts at an intracellular site to regulate STAT1 alpha nuclear import. A C-terminal peptide of murine IFN gamma, IFN gamma(95–133), that contains the NLS motif, induced nuclear translocation of STAT1 alpha when taken up intracellularly by a murine macrophage cell line. Deletion of the NLS motif specifically abrogated the ability of this intracellular peptide to cause STAT1 alpha nuclear translocation. In cells activated with IFN gamma, IFN gamma was found to as part of a complex that contained STAT1 alpha and the importin-alpha analog Npi-1, which mediates STAT1 alpha nuclear import. The tyrosine phosphorylation of STAT1 alpha, the formation of the complex IFN gamma/Npi-1/STAT1 alpha complex and the subsequent nuclear translocation of STAT1 alpha were all found to be dependent on the presence of the IFN gamma NLS. Thus, the NLS of IFN gamma functions intracellularly to directly regulate the activation and ultimate nuclear translocation STAT1 alpha.

scholarly journals Functional Analysis of Tpr: Identification of Nuclear Pore Complex Association and Nuclear Localization Domains and a Role in mRNA Export

1998 ◽  
Vol 143 (7) ◽  
pp. 1801-1812 ◽  
Author(s):  
Peter Bangs ◽  
Brian Burke ◽  
Christine Powers ◽  
Roger Craig ◽  
Aruna Purohit ◽  
...  
Keyword(s):  
Nuclear Localization ◽  
Nuclear Pore Complex ◽  
Ectopic Expression ◽  
Coiled Coil ◽  
Full Length ◽  
Nuclear Localization Sequence ◽  
Nuclear Pore ◽  
Nuclear Interior ◽  
Mammalian Cell Lines ◽  
Localization Sequence

Tpr is a 270-kD coiled-coil protein localized to intranuclear filaments of the nuclear pore complex (NPC). The mechanism by which Tpr contributes to the structure and function of the nuclear pore is currently unknown. To gain insight into Tpr function, we expressed the full-length protein and several subdomains in mammalian cell lines and examined their effects on nuclear pore function. Through this analysis, we identified an NH2-terminal domain that was sufficient for association with the nucleoplasmic aspect of the NPC. In addition, we unexpectedly found that the acidic COOH terminus was efficiently transported into the nuclear interior, an event that was apparently mediated by a putative nuclear localization sequence. Ectopic expression of the full-length Tpr caused a dramatic accumulation of poly(A)+ RNA within the nucleus. Similar results were observed with domains that localized to the NPC and the nuclear interior. In contrast, expression of these proteins did not appear to affect nuclear import. These data are consistent with a model in which Tpr is tethered to intranuclear filaments of the NPC by its coiled coil domain leaving the acidic COOH terminus free to interact with soluble transport factors and mediate export of macromolecules from the nucleus.

scholarly journals Roles of the Tumor Suppressor p53 and the Cyclin-dependent Kinase Inhibitor p21WAF1/CIP1 in Receptor-mediated Apoptosis of WEHI 231 B Lymphoma Cells

1998 ◽  
Vol 187 (10) ◽  
pp. 1671-1679 ◽  
Author(s):  
Min Wu ◽  
Robert E. Bellas ◽  
Jian Shen ◽  
Gail E. Sonenshein
Keyword(s):  
Tumor Suppressor ◽  
B Cell ◽  
Kinase Inhibitor ◽  
Ectopic Expression ◽  
Cyclin Dependent Kinase ◽  
Lymphoma Cells ◽  
B Lymphoma ◽  
Cyclin Dependent Kinase Inhibitor ◽  
B Lymphoma Cells ◽  
Wehi 231

Treatment of WEHI 231 immature B lymphoma cells with an antibody against their surface immunoglobulin M (anti-IgM) induces apoptosis and has been studied extensively as a model of self-induced B cell tolerance. Since the tumor suppressor protein p53 has been implicated in apoptosis in a large number of cell types and has been found to be mutated in a variety of B cell tumors, here we sought to determine whether p53 and the p53 target gene cyclin-dependent kinase inhibitor p21WAF1/CIP1 were involved in anti-IgM–induced cell death. Anti-IgM treatment of WEHI 231 cells increased expression of p53 and p21 protein levels. Ectopic expression of wild-type p53 in WEHI 231 cells induced both p21 expression and apoptosis. Ectopic expression of p21 similarly induced apoptosis. Rescue of WEHI 231 cells from apoptosis by costimulation with CD40 ligand ablated the increase in p21 expression. Lastly, a significant decrease in anti-IgM–mediated apoptosis was seen upon downregulation of endogenous p53 activity by expression of a dominant-negative p53 protein or upon microinjection of an antisense p21 expression vector or antibody. Taken together, the above data demonstrate important roles for p53 and p21 proteins in receptor-mediated apoptosis of WEHI 231 B cells.

scholarly journals The Role of the Cyclin Dependent Kinase Inhibitor p21cip1/waf1 in Targeting Cancer: Molecular Mechanisms and Novel Therapeutics

Cancers ◽  
2019 ◽  
Vol 11 (10) ◽  
pp. 1475 ◽  
Author(s):  
Al Bitar ◽  
Gali-Muhtasib
Keyword(s):  
Cell Cycle Progression ◽  
Molecular Mechanisms ◽  
Kinase Inhibitor ◽  
Biological Activities ◽  
Cyclin Dependent Kinase ◽  
Cycle Progression ◽  
Cyclin Dependent Kinase Inhibitor ◽  
Multiple Signaling ◽  
Made In

p21cip1/waf1 mediates various biological activities by sensing and responding to multiple stimuli, via p53-dependent and independent pathways. p21 is known to act as a tumor suppressor mainly by inhibiting cell cycle progression and allowing DNA repair. Significant advances have been made in elucidating the potential role of p21 in promoting tumorigenesis. Here, we discuss the involvement of p21 in multiple signaling pathways, its dual role in cancer, and the importance of understanding its paradoxical functions for effectively designing therapeutic strategies that could selectively inhibit its oncogenic activities, override resistance to therapy and yet preserve its tumor suppressive functions.

scholarly journals The CDK inhibitor SIAMESE targets both CDKA;1 and CDKB1 complexes to establish endoreplication in trichomes

2020 ◽  
Author(s):  
Kai Wang ◽  
Ruth Ndathe ◽  
Narender Kumar ◽  
Elizabeth A. Zeringue ◽  
Naohiro Kato ◽  
...  
Keyword(s):  
Stress Responses ◽  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
Binding Motif ◽  
Cdk Inhibitor ◽  
Cyclin Dependent Kinase ◽  
Binding Partner ◽  
Cyclin Dependent Kinase Inhibitor ◽  
Conflicting Evidence

AbstractEndoreplication, also known as endoreduplication, is a modified cell cycle in which DNA is replicated without subsequent cell division. Endoreplication plays important roles in both normal plant development and in stress responses. The SIAMESE (SIM) gene of Arabidopsis (Arabidopsis thaliana) encodes a cyclin-dependent kinase inhibitor that plays an central role in establishing endoreplication, and is the founding member of the SIAMESE-RELATED (SMR) family of plant-specific cyclin-dependent kinase inhibitors genes. However, there has been conflicting evidence regarding which specific cyclin/CDK complexes are inhibited by SIM in vivo. In this work, we use genetic evidence to show that SIM likely inhibits both CDKA;1- and CDKB1-containing CDK complexes in vivo to promote endoreplication in developing Arabidopsis trichomes. We also show that SIM interacts with CYCA2;3, a binding partner of CDKB1;1, via SIM Motif A, which we previously identified as a CDK-binding motif. In contrast, SIM Motif C, which has been indicated as a cyclin binding motif in other contexts, appears to be relatively unimportant for interaction between SIM and CYCA2;3. Together with earlier results, our work suggests that SIM and other SMRs likely have a multivalent interaction with CYC/CDK complexes.One sentence summaryThe cyclin-dependent kinase inhibitor SIAMESE (SIM) targets both CDKA;1 and CDKB1 complexes to establish endoreplication, and that SIM interacts with the cyclin CYCA2;3 via SIM Motif A.

scholarly journals Autocrine TGFβ1 Opposes Exogenous TGFβ1-Induced Cell Migration and Growth Arrest through Sustainment of a Feed-Forward Loop Involving MEK-ERK Signaling

Cancers ◽  
2021 ◽  
Vol 13 (6) ◽  
pp. 1357
Author(s):  
Hendrik Ungefroren ◽  
Jessica Christl ◽  
Caroline Eiden ◽  
Ulrich F. Wellner ◽  
Hendrik Lehnert ◽  
...  
Keyword(s):  
Cell Motility ◽  
Transforming Growth Factor ◽  
Kinase Inhibitor ◽  
Ectopic Expression ◽  
Growth Arrest ◽  
Transforming Growth Factor Β ◽  
Erk Signaling ◽  
Ductal Adenocarcinoma ◽  
Type I ◽  
Cyclin Dependent Kinase Inhibitor

Autocrine transforming growth factor β (aTGFβ) has been implicated in the regulation of cell invasion and growth of several malignant cancers such as pancreatic ductal adenocarcinoma (PDAC) or triple-negative breast cancer (TNBC). Recently, we observed that endogenous TGFB1 can inhibit rather than stimulate cell motility in cell lines with high aTGFβ production and mutant KRAS, i.e., Panc1 (PDAC) and MDA-MB-231 (TNBC). The unexpected anti-migratory role prompted us to evaluate if aTGFβ1 may be able to antagonize the action of exogenous (recombinant human) TGFβ (rhTGFβ), a well-known promoter of cell motility and growth arrest in these cells. Surprisingly, RNA interference-mediated knockdown of the endogenous TGFB1 sensitized genes involved in EMT and cell motility (i.e., SNAI1) to up-regulation by rhTGFβ1, which was associated with a more pronounced migratory response following rhTGFβ1 treatment. Ectopic expression of TGFB1 decreased both basal and rhTGFβ1-induced migratory activities in MDA-MB-231 cells but had the opposite effect in Panc1 cells. Moreover, silencing TGFB1 reduced basal proliferation and enhanced growth inhibition by rhTGFβ1 and induction of cyclin-dependent kinase inhibitor, p21WAF1. Finally, we show that aTGFβ1 promotes MEK-ERK signaling and vice versa to form a self-perpetuating feedforward loop that is sensitive to SB431542, an inhibitor of the TGFβ type I receptor, ALK5. Together, these data suggest that in transformed cells an ALK5-MEK-ERK-aTGFβ1 pathway opposes the promigratory and growth-arresting function of rhTGFβ1. This observation has profound translational implications for TGFβ signaling in cancer.

scholarly journals Coordinate Control of Muscle Cell Survival by Distinct Insulin-like Growth Factor Activated Signaling Pathways

2000 ◽  
Vol 151 (6) ◽  
pp. 1131-1140 ◽  
Author(s):  
Margaret A. Lawlor ◽  
Peter Rotwein
Keyword(s):  
Growth Factors ◽  
Muscle Cell ◽  
Kinase Inhibitor ◽  
Ectopic Expression ◽  
Pi3 Kinase ◽  
Cellular Functions ◽  
Cyclin Dependent Kinase Inhibitor ◽  
Rapid Induction ◽  
Peptide Growth Factors ◽  
Igf I

Peptide growth factors control diverse cellular functions by regulating distinct signal transduction pathways. In cultured myoblasts, insulin-like growth factors (IGFs) stimulate differentiation and promote hypertrophy. IGFs also maintain muscle cell viability. We previously described C2 skeletal muscle lines lacking expression of IGF-II. These cells did not differentiate, but underwent progressive apoptotic death when incubated in differentiation medium. Viability could be sustained and differentiation enabled by IGF analogues that activated the IGF-I receptor; survival was dependent on stimulation of phosphatidylinositol 3-kinase (PI3-kinase). We now find that IGF action promotes myoblast survival through two distinguishable PI3-kinase–regulated pathways that culminate in expression of the cyclin-dependent kinase inhibitor, p21. Incubation with IGF-I or transfection with active PI3-kinase led to rapid induction of MyoD and p21, and forced expression of either protein maintained viability in the absence of growth factors. Ectopic expression of MyoD induced p21, and inhibition of p21 blocked MyoD-mediated survival, thus defining one PI3-kinase–dependent pathway as leading first to MyoD, and then to p21 and survival. Unexpectedly, loss of MyoD expression did not impede IGF-mediated survival, revealing a second pathway involving activation by PI3-kinase of Akt, and subsequent induction of p21. Since inhibition of p21 caused death even in the presence of IGF-I, these results establish a central role for p21 as a survival factor for muscle cells. Our observations also define a MyoD-independent pathway for regulating p21 in muscle, and demonstrate that distinct mechanisms help ensure appropriate expression of this key protein during differentiation.

scholarly journals Identification, Molecular Characterization, and Tissue Expression Profiles of Three Smad Genes from Water Buffalo (Bubalus bubalis)

Genes ◽  
2021 ◽  
Vol 12 (10) ◽  
pp. 1536
Author(s):  
Jie Zhang ◽  
Guangle Zhang ◽  
Yongwang Miao
Keyword(s):  
Biological Activities ◽  
Expression Profiles ◽  
Three Dimensional ◽  
In Silico Analysis ◽  
Physicochemical Characteristics ◽  
Tissue Expression ◽  
Bubalus Bubalis ◽  
Amino Acid Sequences ◽  
Nuclear Localization Sequence ◽  
Localization Sequence

Smads are involved in a variety of biological activities by mediating bone morphogenetic protein (BMP) signals. The full-length coding sequences (CDSs) of buffalo Smads 1, 4, and 5 were isolated and identified through RT-PCR in this study. Their lengths are 1398 bp, 1662 bp, and 1398 bp, respectively. In silico analysis showed that their transcriptional region structures, as well as their amino acid sequences, physicochemical characteristics, motifs, conserved domains, and three-dimensional structures of their encoded proteins are highly consistent with their counterparts in the species of Bovidae. The three Smad proteins are all hydrophilic without the signal peptides and transmembrane regions. Each of them has an MH1 domain and an MH2 domain. A nuclear localization sequence was found in the MH1 domain of buffalo Smads 1 and 5. Prediction showed that the function of the three Smads is mainly protein binding, and they can interact with BMPs and their receptors. The three genes were expressed in all 10 buffalo tissues assayed, and their expression in the mammary gland, gonad, and spleen was relatively high. The results here indicate that the three buffalo Smads may be involved in the transcriptional regulation of genes in a variety of tissues.

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