Transport of protein kinase C alpha into the nucleus requires intact cytoskeleton while the transport of a protein containing a canonical nuclear localization signal does not

1996 ◽  
Vol 109 (9) ◽  
pp. 2401-2406 ◽  
Author(s):  
D. Schmalz ◽  
F. Kalkbrenner ◽  
F. Hucho ◽  
K. Buchner
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Nuclear Localization ◽  
Nuclear Localization Signal ◽  
Cytochalasin B ◽  
Nuclear Accumulation ◽  
Reporter Protein ◽  
Kinase C ◽  
Localization Signal ◽  
Protein Kinase C Alpha

Protein kinase C undergoes a redistribution from the cytosol into the nucleus upon various stimuli. Since protein kinase C does not contain any known nuclear localization signal, the exact pathway and mechanism of the translocation into the nucleus is not known. We used immunofluorescence microscopy to investigate the role of the cytoskeleton in this process, and to detect the subcellular distribution of protein kinase C alpha in NIH 3T3 fibroblasts. In these cells protein kinase C alpha is translocated into the nucleus after stimulation with phorbol ester. We observed that cells treated with the cytoskeleton disrupting agents cytochalasin B or colchicine do not show the nuclear translocation of protein kinase C alpha after stimulation. In contrast, the nuclear accumulation of a nuclear localization signal containing reporter protein in an in vitro nuclear transport assay is not affected by these drugs. This observation has been confirmed for intact cells by microinjection experiments: cells which have been incubated with cytochalasin B or colchicine prior to microinjection of the reporter protein show the same accumulation in the nucleus as untreated cells. Our data show that intact cytoskeleton plays an important role in the translocation of protein kinase C alpha into the nucleus but not in the nuclear import of a karyophilic reporter protein.

Nuclear import of protein kinase C occurs by a mechanism distinct from the mechanism used by proteins with a classical nuclear localization signal

1998 ◽  
Vol 111 (13) ◽  
pp. 1823-1830 ◽  
Author(s):  
D. Schmalz ◽  
F. Hucho ◽  
K. Buchner
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Nuclear Localization ◽  
Phorbol Ester ◽  
Nuclear Import ◽  
Nuclear Localization Signal ◽  
Nuclear Pore ◽  
Kinase C ◽  
Localization Signal ◽  
Protein Kinase C Alpha

Protein kinase C does not have any known nuclear localization signal but, nevertheless, is redistributed from the cytoplasm to the nucleus upon various stimuli. In NIH 3T3 fibroblasts stimulation with phorbol ester leads to a translocation of protein kinase C alpha to the plasma membrane and into the cell nucleus. We compared the mechanism of protein kinase C alpha's transport into the nucleus with the transport mechanism of a protein with a classical nuclear localization signal at several steps. To this end, we co-microinjected fluorescently labeled bovine serum albumin to which a nuclear localization signal peptide was coupled, together with substances interfering with conventional nuclear protein import. Thereafter, the distribution of both the nuclear localization signal-bearing reporter protein and protein kinase C alpha was analyzed in the same cells. We can show that, in contrast to the nuclear localization signal-dependent transport, the phorbol ester-induced transport of protein kinase C alpha is not affected by microinjection of antibodies against the nuclear import factor p97/importin/karyopherin beta or microinjection of non-hydrolyzable GTP-analogs. This suggests that nuclear import of protein kinase C alpha is independent of p97/importin/karyopherin beta and independent of GTP. At the nuclear pore there are differences between the mechanisms too, since nuclear transport of protein kinase C alpha cannot be inhibited by wheat germ agglutinin or an antibody against nuclear pore complex proteins. Together these findings demonstrate that the nuclear import of protein kinase C alpha occurs by a mechanism distinct from the one used by classical nuclear localization signal-bearing proteins at several stages.

scholarly journals Protein Kinase C Phosphorylates Ribosomal Protein S6 Kinase βII and Regulates Its Subcellular Localization

2003 ◽  
Vol 23 (3) ◽  
pp. 852-863 ◽  
Author(s):  
Taras Valovka ◽  
Frederique Verdier ◽  
Rainer Cramer ◽  
Alexander Zhyvoloup ◽  
Timothy Fenton ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Subcellular Localization ◽  
Nuclear Localization ◽  
Nuclear Localization Signal ◽  
Nucleocytoplasmic Shuttling ◽  
S6 Kinase ◽  
Ribosomal Protein S6 ◽  
Kinase C ◽  
Localization Signal

ABSTRACT The ribosomal protein S6 kinase (S6K) belongs to the AGC family of Ser/Thr kinases and is known to be involved in the regulation of protein synthesis and the G1/S transition of the cell cycle. There are two forms of S6K, termed S6Kα and S6Kβ, which have cytoplasmic and nuclear splice variants. Nucleocytoplasmic shuttling has been recently proposed for S6Kα, based on the use of the nuclear export inhibitor, leptomycin B. However, the molecular mechanisms regulating subcellular localization of S6Ks in response to mitogenic stimuli remain to be elucidated. Here we present data on the in vitro and in vivo phosphorylation of S6Kβ, but not S6Kα, by protein kinase C (PKC). The site of phosphorylation was identified as S486, which is located within the C-terminal nuclear localization signal. Mutational analysis and the use of phosphospecific antibodies provided evidence that PKC-mediated phosphorylation at S486 does not affect S6K activity but eliminates the function of its nuclear localization signal and causes retention of an activated form of the kinase in the cytoplasm. Taken together, this study uncovers a novel mechanism for the regulation of nucleocytoplasmic shuttling of S6KβII by PKC-mediated phosphorylation.

Protein kinase C regulates the nuclear localization of diacylglycerol kinase-ζ

Nature ◽  
10.1038/29337 ◽  
1998 ◽  
Vol 394 (6694) ◽  
pp. 697-700 ◽  
Author(s):  
Matthew K. Topham ◽  
Michaeline Bunting ◽  
Guy A. Zimmerman ◽  
Thomas M. McIntyre ◽  
Perry J. Blackshear ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Nuclear Localization ◽  
Diacylglycerol Kinase ◽  
Kinase C

scholarly journals Atypical protein kinase C induces cell transformation by disrupting Hippo/Yap signaling

2015 ◽  
Vol 26 (20) ◽  
pp. 3578-3595 ◽  
Author(s):  
Andrew Archibald ◽  
Maia Al-Masri ◽  
Alyson Liew-Spilger ◽  
Luke McCaffrey
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Epithelial Cells ◽  
3D Culture ◽  
Cell Junctions ◽  
Nuclear Accumulation ◽  
Cell Phenotype ◽  
Atypical Protein Kinase C ◽  
Atypical Protein Kinase ◽  
Kinase C

Epithelial cells are major sites of malignant transformation. Atypical protein kinase C (aPKC) isoforms are overexpressed and activated in many cancer types. Using normal, highly polarized epithelial cells (MDCK and NMuMG), we report that aPKC gain of function overcomes contact inhibited growth and is sufficient for a transformed epithelial phenotype. In 2D cultures, aPKC induced cells to grow as stratified epithelia, whereas cells grew as solid spheres of nonpolarized cells in 3D culture. aPKC associated with Mst1/2, which uncoupled Mst1/2 from Lats1/2 and promoted nuclear accumulation of Yap1. Of importance, Yap1 was necessary for aPKC-mediated overgrowth but did not restore cell polarity defects, indicating that the two are separable events. In MDCK cells, Yap1 was sequestered to cell–cell junctions by Amot, and aPKC overexpression resulted in loss of Amot expression and a spindle-like cell phenotype. Reexpression of Amot was sufficient to restore an epithelial cobblestone appearance, Yap1 localization, and growth control. In contrast, the effect of aPKC on Hippo/Yap signaling and overgrowth in NMuMG cells was independent of Amot. Finally, increased expression of aPKC in human cancers strongly correlated with increased nuclear accumulation of Yap1, indicating that the effect of aPKC on transformed growth by deregulating Hippo/Yap1 signaling may be clinically relevant.

scholarly journals Ebolavirus VP24 Binding to Karyopherins Is Required for Inhibition of Interferon Signaling

2009 ◽  
Vol 84 (2) ◽  
pp. 1169-1175 ◽  
Author(s):  
Mathieu Mateo ◽  
St. Patrick Reid ◽  
Lawrence W. Leung ◽  
Christopher F. Basler ◽  
Viktor E. Volchkov
Keyword(s):  
Nuclear Localization ◽  
Nuclear Localization Signal ◽  
Nuclear Accumulation ◽  
Interferon Signaling ◽  
Loss Of Function ◽  
Proposed Model ◽  
Localization Signal ◽  
Ifn Γ ◽  
Alpha Beta ◽  
Signal Receptors

ABSTRACT The Ebolavirus VP24 protein counteracts alpha/beta interferon (IFN-α/β) and IFN-γ signaling by blocking the nuclear accumulation of tyrosine-phosphorylated STAT1 (PY-STAT1). According to the proposed model, VP24 binding to members of the NPI-1 subfamily of karyopherin alpha (KPNα) nuclear localization signal receptors prevents their binding to PY-STAT1, thereby preventing PY-STAT1 nuclear accumulation. This study now identifies two domains of VP24 required for inhibition of IFN-β-induced gene expression and PY-STAT1 nuclear accumulation. We demonstrate that loss of function correlates with loss of binding to KPNα proteins. Thus, the VP24 IFN antagonist function requires the ability of VP24 to interact with KPNα.

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