scholarly journals Protein kinase B phosphorylates AHNAK and regulates its subcellular localization

2001 ◽  
Vol 154 (5) ◽  
pp. 1019-1030 ◽  
Author(s):  
Joshua Sussman ◽  
David Stokoe ◽  
Natalya Ossina ◽  
Emma Shtivelman
Keyword(s):  
Protein Kinase ◽  
Epithelial Cells ◽  
Transcriptional Repression ◽  
Nuclear Export ◽  
Protein Kinase B ◽  
Nuclear Export Signal ◽  
Nuclear Exclusion ◽  
Export Signal

AHNAK is a ubiquitously expressed giant phosphoprotein that was initially identified as a gene product subject to transcriptional repression in neuroblastoma. AHNAK is predominantly nuclear in cells of nonepithelial origin, but is cytoplasmic or associated with plasma membrane in epithelial cells. In this study we show that the extranuclear localization of AHNAK in epithelial cells depends on the formation of cell–cell contacts. We show that AHNAK is a phosphorylation substrate of protein kinase B (PKB) in vitro and in vivo. Nuclear exclusion of AHNAK is mediated through a nuclear export signal (NES) in a manner that depends on the phosphorylation of serine 5535 of AHNAK by PKB, a process that also plays a major role in determining extranuclear localization of AHNAK. AHNAK is a new PKB substrate whose function, though unknown, is likely to be regulated by its localization, which is in turn regulated by PKB.

scholarly journals The Phosphoinositol-3-Kinase–Protein Kinase B/Akt Pathway Is Critical forPseudomonas aeruginosaStrain PAK Internalization

2005 ◽  
Vol 16 (5) ◽  
pp. 2577-2585 ◽  
Author(s):  
A. Kierbel ◽  
A. Gassama-Diagne ◽  
K. Mostov ◽  
J. N. Engel
Keyword(s):  
Protein Kinase ◽  
Epithelial Cells ◽  
Mdck Cells ◽  
Protein Kinase B ◽  
Pi3k Signaling ◽  
Dependent Manner ◽  
Akt Phosphorylation ◽  
Pi3k Signaling Pathway

Several Pseudomonas aeruginosa strains are internalized by epithelial cells in vitro and in vivo, but the host pathways usurped by the bacteria to enter nonphagocytic cells are not clearly understood. Here, we report that internalization of strain PAK into epithelial cells triggers and requires activation of phosphatidylinositol 3-kinase (PI3K) and protein kinase B/Akt (Akt). Incubation of Madin-Darby canine kidney (MDCK) or HeLa cells with the PI3K inhibitors LY294002 (LY) or wortmannin abrogated PAK uptake. Addition of the PI3K product phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P3] to polarized MDCK cells was sufficient to increase PAK internalization. PtdIns(3,4,5)P3accumulated at the site of bacterial binding in an LY-dependent manner. Akt phosphorylation correlated with PAK invasion. The specific Akt phosphorylation inhibitor SH-5 inhibited PAK uptake; internalization also was inhibited by small interfering RNA-mediated depletion of Akt phosphorylation. Expression of constitutively active Akt was sufficient to restore invasion when PI3K signaling was inhibited. Together, these results demonstrate that the PI3K signaling pathway is necessary and sufficient for the P. aeruginosa entry and provide the first example of a bacterium that requires Akt for uptake into epithelial cells.

Fucoidans from Cucumaria frondosa ameliorates renal interstitial fibrosis via inhibition of PI3K/Akt/NF-κB signaling pathway

2022 ◽  
Author(s):  
Zhuo-yue Song ◽  
Mengru Zhu ◽  
Jun Wu ◽  
Tian Yu ◽  
Yao Chen ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Signaling Pathway ◽  
Interstitial Fibrosis ◽  
Protein Kinase B ◽  
Nuclear Factor Κb ◽  
Nuclear Factor ◽  
Renal Interstitial Fibrosis ◽  
Cucumaria Frondosa

The effects of Cucumaria frondosa polysaccharides (CFP) on renal interstitial fibrosis via regulating phosphatidylinositol-3-hydroxykinase/protein kinase-B/Nuclear factor-κB (PI3K/AKT/NF-κB) signaling pathway were investigated in vivo and in vitro in this research. A...

scholarly journals Reconstitution of nuclear protein export in isolated nuclear envelopes

2002 ◽  
Vol 158 (5) ◽  
pp. 849-854 ◽  
Author(s):  
Jan Peter Siebrasse ◽  
Elias Coutavas ◽  
Reiner Peters
Keyword(s):  
Protein Kinase ◽  
Nuclear Export ◽  
Xenopus Oocytes ◽  
Nuclear Protein ◽  
Nuclear Export Signal ◽  
Protein Export ◽  
Leptomycin B ◽  
Permeabilized Cells ◽  
Export Signal ◽  
Kinase A

Signal-dependent nuclear protein export was studied in perforated nuclei and isolated nuclear envelopes of Xenopus oocytes by optical single transporter recording. Manually isolated and purified oocyte nuclei were attached to isoporous filters and made permeable for macromolecules by perforation. Export of a recombinant protein (GG-NES) containing the nuclear export signal (NES) of the protein kinase A inhibitor through nuclear envelope patches spanning filter pores could be induced by the addition of GTP alone. Export continued against a concentration gradient, and was NES dependent and inhibited by leptomycin B and GTPγS, a nonhydrolyzable GTP analogue. Addition of recombinant RanBP3, a potential cofactor of CRM1-dependent export, did not promote GG-NES export at stoichiometric concentration but gradually inhibited export at higher concentrations. In isolated filter-attached nuclear envelopes, export of GG-NES was virtually abolished in the presence of GTP alone. However, a preformed export complex consisting of GG-NES, recombinant human CRM1, and RanGTP was rapidly exported. Unexpectedly, export was strongly reduced when the export complex contained RanGTPγS or RanG19V/Q69L-GTP, a GTPase-deficient Ran mutant. This paper shows that nuclear transport, previously studied in intact and permeabilized cells only, can be quantitatively analyzed in perforated nuclei and isolated nuclear envelopes.

scholarly journals Protein Kinase CK2 Phosphorylation of EB2 Regulates Its Function in the Production of Epstein-Barr Virus Infectious Viral Particles

2007 ◽  
Vol 81 (21) ◽  
pp. 11850-11860 ◽  
Author(s):  
Cahora Medina-Palazon ◽  
Henri Gruffat ◽  
Fabrice Mure ◽  
Odile Filhol ◽  
Valérie Vingtdeux-Didier ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Nuclear Export ◽  
Epstein Barr Virus ◽  
Nuclear Export Signal ◽  
Regulatory Subunit ◽  
Viral Mrnas ◽  
Barr Virus ◽  
Early Protein ◽  
Epstein Barr

ABSTRACT The Epstein-Barr Virus (EBV) early protein EB2 (also called BMLF1, Mta, or SM) promotes the nuclear export of a subset of early and late viral mRNAs and is essential for the production of infectious virions. We show here that in vitro, protein kinase CK2α and -β subunits bind both individually and, more efficiently, as a complex to the EB2 N terminus and that the CK2β regulatory subunit also interacts with the EB2 C terminus. Immunoprecipitated EB2 has CK2 activity that phosphorylates several sites within the 80 N-terminal amino acids of EB2, including Ser-55, -56, and -57, which are localized next to the nuclear export signal. EB2S3E, the phosphorylation-mimicking mutant of EB2 at these three serines, but not the phosphorylation ablation mutant EB2S3A, efficiently rescued the production of infectious EBV particles by HEK293BMLF1-KO cells harboring an EB2-defective EBV genome. The defect of EB2S3A in transcomplementing 293BMLF1-KO cells was not due to impaired nucleocytoplasmic shuttling of the mutated protein but was associated with a decrease in the cytoplasmic accumulation of several late viral mRNAs. Thus, EB2-mediated production of infectious EBV virions is regulated by CK2 phosphorylation at one or more of the serine residues Ser-55, -56, and -57.

scholarly journals A novel LIM protein Cal promotes cardiac differentiation by association with CSX/NKX2-5

2004 ◽  
Vol 164 (3) ◽  
pp. 395-405 ◽  
Author(s):  
Hiroshi Akazawa ◽  
Sumiyo Kudoh ◽  
Naoki Mochizuki ◽  
Noboru Takekoshi ◽  
Hiroyuki Takano ◽  
...  
Keyword(s):  
Heart Development ◽  
Nuclear Export ◽  
Target Genes ◽  
Nuclear Export Signal ◽  
Gene Promoter ◽  
Myocardial Cell ◽  
Cardiac Differentiation ◽  
Lim Protein

The cardiac homeobox transcription factor CSX/NKX2-5 plays an important role in vertebrate heart development. Using a yeast two-hybrid screening, we identified a novel LIM domain–containing protein, named CSX-associated LIM protein (Cal), that interacts with CSX/NKX2-5. CSX/NKX2-5 and Cal associate with each other both in vivo and in vitro, and the LIM domains of Cal and the homeodomain of CSX/NKX2-5 were necessary for mutual binding. Cal itself possessed the transcription-promoting activity, and cotransfection of Cal enhanced CSX/NKX2-5–induced activation of atrial natriuretic peptide gene promoter. Cal contained a functional nuclear export signal and shuttled from the cytoplasm into the nucleus in response to calcium. Accumulation of Cal in the nucleus of P19CL6 cells promoted myocardial cell differentiation accompanied by increased expression levels of the target genes of CSX/NKX2-5. These results suggest that a novel LIM protein Cal induces cardiomyocyte differentiation through its dynamic intracellular shuttling and association with CSX/NKX2-5.

scholarly journals RanGTP-Binding Protein NXT1 Facilitates Nuclear Export of Different Classes of RNA In Vitro

2000 ◽  
Vol 20 (13) ◽  
pp. 4562-4571 ◽  
Author(s):  
Batool Ossareh-Nazari ◽  
Christèle Maison ◽  
Ben E. Black ◽  
Lyne Lévesque ◽  
Bryce M. Paschal ◽  
...  
Keyword(s):  
Nuclear Export ◽  
Nuclear Export Signal ◽  
Small Gtpase ◽  
Dependent Manner ◽  
Vitro Assay ◽  
U1 Snrna ◽  
Rna Export ◽  
Gtpase Ran ◽  
Export Signal

ABSTRACT To better characterize the mechanisms responsible for RNA export from the nucleus, we developed an in vitro assay based on the use of permeabilized HeLa cells. This new assay supports nuclear export of U1 snRNA, tRNA, and mRNA in an energy- and Xenopusextract-dependent manner. U1 snRNA export requires a 5′ monomethylated cap structure, the nuclear export signal receptor CRM1, and the small GTPase Ran. In contrast, mRNA export does not require the participation of CRM1. We show here that NXT1, an NTF2-related protein that binds directly to RanGTP, strongly stimulates export of U1 snRNA, tRNA, and mRNA. The ability of NXT1 to promote export is dependent on its capacity to bind RanGTP. These results support the emerging view that NXT1 is a general export factor, functioning on both CRM1-dependent and CRM1-independent pathways of RNA export.

scholarly journals Targeting redox regulatory site of protein kinase B impedes neutrophilic inflammation in lung injury

2018 ◽  
Author(s):  
Po-Jen Chen ◽  
I-Ling Ko ◽  
Chia-Lin Lee ◽  
Hao-Chun Hu ◽  
Fang-Rong Chang ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Lung Injury ◽  
Inflammatory Responses ◽  
Protein Kinase B ◽  
Reduced Form ◽  
Human Neutrophils ◽  
Akt Inhibitor ◽  
Neutrophilic Inflammation

AbstractNeutrophil activation has a pathogenic effect in inflammatory diseases. Protein kinase B (PKB)/AKT regulates diverse cellular responses. However, the significance of AKT in neutrophilic inflammation is still not well understood. Here, we identified CLLV-1 as a novel AKT inhibitor. CLLV-1 inhibited respiratory burst, degranulation, chemotaxis, and AKT phosphorylation in activated human neutrophils and dHL-60 cells. Significantly, CLLV-1 blocked AKT activity and covalently reacted with AKT Cys310 in vitro. The AKT309-313 peptide-CLLV-1 adducts were determined by NMR or mass spectrometry assay. The alkylation agent-conjugated AKT (reduced form) level was also inhibited by CLLV-1. Additionally, CLLV-1 ameliorated lipopolysaccharide (LPS)-induced acute lung injury (ALI) in mice. CLLV-1 acts as a covalent allosteric AKT inhibitor by targeting AKT Cys310 to restrain inflammatory responses in human neutrophils and LPS-induced ALI in vivo. Our findings provide a mechanistic framework for redox modification of AKT that may serve as a novel pharmacological target to alleviate neutrophilic inflammation.

scholarly journals Protein Kinase B Inactivation Is Associated with Magnolol-Enhanced Therapeutic Efficacy of Sorafenib in Hepatocellular Carcinoma In Vitro and In Vivo

Cancers ◽  
2019 ◽  
Vol 12 (1) ◽  
pp. 87 ◽  
Author(s):  
Jiann-Hwa Chen ◽  
I-Tsang Chiang ◽  
Fei-Ting Hsu
Keyword(s):  
Hepatocellular Carcinoma ◽  
Protein Kinase ◽  
Protein Kinase B ◽  
Bioactive Compound ◽  
Critical Issue ◽  
Akt Inhibitor ◽  
Multikinase Inhibitor ◽  
Therapeutic Benefits

Although sorafenib, an oral multikinase inhibitor, was approved as a treatment drug of advance hepatocellular carcinoma (HCC), treatment efficacy still requires improvement. Searching for the adjuvant reagent for enhancing sorafenib efficacy remains as a critical issue. Sorafenib has been proved to suppress extracellular signal-regulated kinases (ERK) in HCC; however, protein kinase B (AKT) was not affected by it. Targeting AKT in combination with sorafenib could be an important breakthrough point of HCC treatment. Many herbal compounds and composite formulas have been shown to enhance anti-HCC activity of sorafenib. Magnolol is a bioactive compound extracted from the bark of the Magnolia officinalis and has been shown to induce apoptosis and inhibit cell invasion in HCC in vitro. However, whether magnolol sensitizes HCC to sorafenib is ambiguous. In this study, we indicated that magnolol significantly enhanced sorafenib-diminished tumor cell growth, expression of anti-apoptotic proteins, and migration/invasion ability compared to sorafenib alone. Magnolol significantly boosted sorafenib-induced extrinsic/intrinsic dependent apoptosis pathways in HCC. Notably sorafenib could not reduce protein level of AKT (Ser473), but expression of AKT (Ser473) was significantly decreased by magnolol or magnolol combined with sorafenib. LY294002 as specific AKT inhibitor was used to confirm that AKT inactivation may promote anticancer effect of sorafenib. Taken together, AKT inhibition is associated with magnolol-enhanced the therapeutic effect of sorafenib in HCC. We suggested magnolol as the potential adjuvant which may enhance therapeutic benefits of sorafenib in patients with HCC.

scholarly journals Identification of Three Functions of the Adenovirus E4orf6 Protein That Mediate p53 Degradation by the E4orf6-E1B55K Complex

2001 ◽  
Vol 75 (2) ◽  
pp. 699-709 ◽  
Author(s):  
Emmanuelle Querido ◽  
Megan R. Morisson ◽  
Huan Chu-Pham-Dang ◽  
Sarah W.-L. Thirlwell ◽  
Dominique Boivin ◽  
...  
Keyword(s):  
Nuclear Export ◽  
Mammalian Cells ◽  
Nuclear Export Signal ◽  
Direct Interaction ◽  
26S Proteasome ◽  
Productive Infection ◽  
Nuclear Retention ◽  
Hpv E6 ◽  
Export Signal

ABSTRACT Complexes containing adenovirus E4orf6 and E1B55K proteins play critical roles in productive infection. Both proteins interact directly with the cellular tumor suppressor p53, and in combination they promote its rapid degradation. To examine the mechanism of this process, degradation of exogenously expressed p53 was analyzed in p53-null human cells infected with adenovirus vectors encoding E4orf6 and/or E1B55K. Coexpression of E4orf6 and E1B55K greatly reduced both the level and the half-life of wild-type p53. No effect was observed with the p53-related p73 proteins, which did not appear to interact with E4orf6 or E1B55K. Mutant forms of p53 were not degraded if they could not efficiently bind E1B55K, suggesting that direct interaction between p53 and E1B55K may be required. Degradation of p53 was independent of both MDM2 and p19ARF, regulators of p53 stability in mammalian cells, but required an extended region of E4orf6 from residues 44 to 274, which appeared to possess three separate biological functions. First, residues 39 to 107 were necessary to interact with E1B55K. Second, an overlapping region from about residues 44 to 218 corresponded to the ability of E4orf6 to form complexes with cellular proteins of 19 and 14 kDa. Third, the nuclear retention signal/amphipathic arginine-rich α-helical region from residues 239 to 253 was required. Interestingly, neither the E4orf6 nuclear localization signal nor the nuclear export signal was essential. These results suggested that if nuclear-cytoplasmic shuttling is involved in this process, it must involve another export signal. Degradation was significantly blocked by the 26S proteasome inhibitor MG132, but unlike the HPV E6 protein, E4orf6 and E1B55K were unable to induce p53 degradation in vitro in reticulocyte lysates. Thus, this study implies that the E4orf6-E1B55K complex may direct p53 for degradation by a novel mechanism.

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