scholarly journals Regulation of Gli1 Localization by the cAMP/Protein Kinase A Signaling Axis through a Site Near the Nuclear Localization Signal

2005 ◽  
Vol 281 (1) ◽  
pp. 9-12 ◽  
Author(s):  
Tao Sheng ◽  
Sumin Chi ◽  
Xiaoli Zhang ◽  
Jingwu Xie
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Nuclear Localization ◽  
Nuclear Localization Signal ◽  
Signaling Axis ◽  
Localization Signal ◽  
A Site ◽  
Kinase A

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 Regulation of gene expression by cyclic GMP-dependent protein kinase requires nuclear translocation of the kinase: identification of a nuclear localization signal.

1997 ◽  
Vol 17 (9) ◽  
pp. 5244-5254 ◽  
Author(s):  
T Gudi ◽  
S M Lohmann ◽  
R B Pilz
Keyword(s):  
Protein Kinase ◽  
Nuclear Localization ◽  
Cyclic Gmp ◽  
Nuclear Localization Signal ◽  
Single Amino Acid ◽  
Immunofluorescent Staining ◽  
Dependent Protein Kinase ◽  
Localization Signal ◽  
Dependent Protein

We recently demonstrated that cyclic GMP (cGMP)-dependent protein kinase (G-kinase) activates the human fos promoter in a strictly cGMP-dependent manner (T. Gudi et al., J. Biol. Chem. 271:4597-4600, 1996). Here, we demonstrate that G-kinase translocates to the nucleus by an active transport mechanism which requires a nuclear localization signal (NLS) and is regulated by cGMP. Immunofluorescent staining of G-kinase was predominantly cytoplasmic in untreated cells, but intense nuclear staining appeared in 8-bromo (Br)-cGMP-treated cells. We identified a putative NLS in the G-kinase ATP binding domain which resembles the NLS of the interleukin-1alpha precursor. Fusion of the G-kinase NLS to the N terminus of beta-galactosidase produced a chimeric protein which localized to the nucleus. Mutation of a single amino acid residue (K407-->E) within the G-kinase NLS produced an enzyme with normal cGMP-dependent activity in vitro which did not translocate to the nucleus and did not transactivate the fos promoter in the presence of 8-Br-cGMP in vivo. In contrast, N-terminally truncated versions of G-kinase with constitutive, cGMP-independent activity in vitro localized to the nucleus and transactivated the fos promoter in the absence of 8-Br-cGMP. These results indicate that nuclear localization of G-kinase is required for transcriptional activation of the fos promoter and suggest that a conformational change of the kinase, induced by cGMP binding or by removal of the N-terminal autoinhibitory domain, functionally activates an otherwise cryptic NLS.

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.

scholarly journals Protein Kinase A Site-specific Phosphorylation Regulates ATP-binding Cassette A1 (ABCA1)-mediated Phospholipid Efflux

2002 ◽  
Vol 277 (44) ◽  
pp. 41835-41842 ◽  
Author(s):  
Raymond H. See ◽  
Rosalinda A. Caday-Malcolm ◽  
Roshni R. Singaraja ◽  
Steven Zhou ◽  
Anthony Silverston ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Atp Binding ◽  
Site Specific ◽  
A Site ◽  
Kinase A ◽  
Atp Binding Cassette

scholarly journals Activity of Retinoic Acid Receptor-alpha Is Directly Regulated at Its Protein Kinase A Sites in Response to Follicle-Stimulating Hormone Signaling

Endocrinology ◽  
2010 ◽  
Vol 151 (5) ◽  
pp. 2361-2372 ◽  
Author(s):  
Nadine C. Santos ◽  
Kwan Hee Kim
Keyword(s):  
Cell Proliferation ◽  
Retinoic Acid ◽  
Protein Kinase ◽  
Protein Kinase A ◽  
Nuclear Localization ◽  
Transcriptional Activity ◽  
Retinoic Acid Receptor ◽  
Cell Proliferation And Differentiation ◽  
Proliferation And Differentiation ◽  
Kinase A

Retinoic acid receptor-α (RARA) is crucial for germ cell development in the testis, as shown by the degenerated testis in Rara gene knockout mice, which are sterile. Similarly, FSH is known to regulate Sertoli cell proliferation and differentiation, indirectly controlling the quantity of the spermatogenic output. Interestingly, FSH inhibited, via activation of FSH receptor, cAMP, and protein kinase A (PKA), the nuclear localization and transcriptional activity of RARA. Given that retinoic acid, the ligand for RARA, is known to regulate cell proliferation and differentiation, we investigated whether FSH regulates RARA by a direct posttranslational phosphorylation mechanism. Mutagenesis of serine 219 (S219) and S369 at the PKA sites on RARA to either double alanines or double glutamic acids showed that both PKA sites are important for RARA activity. The negative charges at the PKA sites, whether they are from glutamic acids or phosphorylation of serines, decreased the nuclear localization of RARA, heterodimerization with retinoid X receptor-α, and the transcriptional activity of the receptor. On the other hand, the double-alanine mutant that cannot be phosphorylated at the 219 and 369 amino acid positions did not respond to cAMP and PKA activation. Wild-type and double-mutant RARA interacted with PKA, but only in the presence of cAMP or FSH. These results together suggest that FSH may regulate cell proliferation and differentiation of Sertoli cells, at least partially, by directly affecting the PKA sites of RARA and controlling the transcriptional function of the receptor.

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.

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