scholarly journals Phospho-Ser727 triggers a multistep inactivation of STAT3 by rapid dissociation of pY705–SH2 through C-terminal tail modulation

2019 ◽  
Author(s):  
Junhao Yang ◽  
Hiroyuki Kunimoto ◽  
Bumpei Katayama ◽  
Hong Zhao ◽  
Takashi Shiromizu ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Nuclear Export ◽  
Chromosome Region ◽  
Intramolecular Interactions ◽  
Nuclear Accumulation ◽  
Proper Function ◽  
Functional Studies ◽  
Reciprocal Interactions ◽  
The Stability ◽  
Transcription 3

Abstract Signal transducer and activator of transcription 3 (STAT3) is involved in many biological processes, including immunity and cancer. STAT3 becomes phosphorylated at Tyr705 and Ser727 on IL-6 stimulation. Phospho-Tyr705 (pY705) stabilizes the STAT3 dimer with reciprocal interactions between pY705 and the SH2 of the other molecule and phospho-Ser727 (pS727) accelerates pY705 dephosphorylation. We study how pS727 regulates STAT3 in both structural and biological perspectives. Using STAT3 reconstituted in HepG2-stat3-knockout cells, we show that pS727, together with a handshake N-terminal domain (NTD) interaction, causes rapid inactivation of STAT3 for pY705 dephosphorylation and a chromosome region maintenance 1 (CRM1)-independent nuclear export, which is critical for faithful STAT3 response to the cellular signals. The various N-terminal tags, GFP-related Ruby and FLAG, rendered the export CRM1-dependent and especially FLAG-tag caused nuclear accumulation of STAT3, indicating the presence of conformational changes in inactivation. Impaired reactivation of STAT3 by S727A or FLAG-tag delayed or inhibited the IL-6-induced saa1 mRNA expression, respectively. The detailed analysis of the pY705–SH2 structure identified the C-terminal tail (CTT) from L706 to P715 as a key regulator of the CTT–CTT intermolecular and the CTT–SH2 intramolecular interactions that support pY705–SH2 association. The functional studies using multiple STAT3 mutants indicated that the degree of the two interactions determines the stability of pY705–SH2 interaction. Importantly, Pro715 was critical for the pS727's destabilizing activity and the known phosphorylation and acetylation at the CTT structurally inhibited the pY705–SH2 interaction. Thus, pS727 triggers pY705–SH2 dissociation by weakening the supportive interactions likely through CTT modulation, inducing rapid cycles of STAT3 activation–inactivation for proper function of STAT3.

scholarly journals Kinetics of nuclear-cytoplasmic translocation of Foxo1 and Foxo3A in adult skeletal muscle fibers

2012 ◽  
Vol 303 (9) ◽  
pp. C977-C990 ◽  
Author(s):  
Tova Neustadt Schachter ◽  
Tiansheng Shen ◽  
Yewei Liu ◽  
Martin F. Schneider
Keyword(s):  
Skeletal Muscle ◽  
Nuclear Export ◽  
Time Course ◽  
Chromosome Region ◽  
Muscle Fibers ◽  
High Rate ◽  
Nuclear Accumulation ◽  
Experimental Conditions ◽  
Adult Skeletal Muscle ◽  
Skeletal Muscle Fibers

In skeletal muscle, the transcription factors Foxo1 and Foxo3A control expression of proteins that mediate muscle atrophy, making the nuclear concentration and nuclear-cytoplasmic movements of Foxo1 and Foxo3A of therapeutic interest in conditions of muscle wasting. Here, we use Foxo-GFP fusion proteins adenovirally expressed in cultured adult mouse skeletal muscle fibers to characterize the time course of nuclear efflux of Foxo1-GFP in response to activation of the insulin-like growth factor-1 (IGF-1)/phosphatidylinositol-3-kinase (PI3K)/Akt pathway to determine the time course of nuclear influx of Foxo1-GFP during inhibition of this pathway and to show that Akt mediates the efflux of nuclear Foxo1-GFP induced by IGF-1. Localization of endogenous Foxo1 in muscle fibers, as determined via immunocytochemistry, is consistent with that of Foxo1-GFP. Inhibition of the nuclear export carrier chromosome region maintenance 1 by leptomycin B (LMB) traps Foxo1 in the nucleus and results in a relatively rapid rate of Foxo1 nuclear accumulation, consistent with a high rate of nuclear-cytoplasmic shuttling of Foxo1 under control conditions before LMB application, with near balance of unidirectional influx and efflux. Expressed Foxo3A-GFP shuttles ∼20-fold more slowly than Foxo1-GFP. Our approach allows quantitative kinetic characterization of Foxo1 and Foxo3A nuclear-cytoplasmic movements in living muscle fibers under various experimental conditions.

scholarly journals Intersectin goes nuclear: secret life of an endocytic protein

2018 ◽  
Vol 475 (8) ◽  
pp. 1455-1472 ◽  
Author(s):  
Gualtiero Alvisi ◽  
Lucia Paolini ◽  
Andrea Contarini ◽  
Chiara Zambarda ◽  
Veronica Di Antonio ◽  
...  
Keyword(s):  
Nuclear Export ◽  
Chromosome Region ◽  
Coiled Coil ◽  
Nuclear Accumulation ◽  
Nucleocytoplasmic Trafficking ◽  
Multidomain Structure ◽  
Endocytic Protein ◽  
Localization Signal ◽  
Src Homology ◽  
Energy Dependent

Intersectin 1-short (ITSN1-s) is a 1220 amino acid ubiquitously expressed scaffold protein presenting a multidomain structure that allows to spatiotemporally regulate the functional interaction of a plethora of proteins. Besides its well-established role in endocytosis, ITSN1-s is involved in the regulation of cell signaling and is implicated in tumorigenesis processes, although the signaling pathways involved are still poorly understood. Here, we identify ITSN1-s as a nucleocytoplasmic trafficking protein. We show that, by binding to importin (IMP)α, a small fraction of ITSN1-s localizes in the cell nucleus at the steady state, where it preferentially associates with the nuclear envelope and interacts with lamin A/C. However, upon pharmacological ablation of chromosome region maintenance 1 (CRM-1)-dependent nuclear export pathway, the protein accumulates into the nucleus, thus revealing its moonlighting nature. Analysis of deletion mutants revealed that the coiled coil (CC) and Src homology (SH3) regions play the major role in its nucleocytoplasmic shuttling. While no evidence of nuclear localization signal (NLS) was detected in the CC region, a functional bipartite NLS was identified within the SH3D region of ITSN1-s (RKKNPGGWWEGELQARGKKRQIGW-1127), capable of conferring energy-dependent nuclear accumulation to reporter proteins and whose mutational ablation affects nuclear import of the whole SH3 region. Thus, ITSN1-s is an endocytic protein, which shuttles between the nucleus and the cytoplasm in a CRM-1- and IMPα-dependent fashion.

scholarly journals Regulation of the Src Family Kinase Lck by Hsp90 and Ubiquitination

2004 ◽  
Vol 24 (13) ◽  
pp. 5667-5676 ◽  
Author(s):  
Ana Giannini ◽  
Marie-José Bijlmakers
Keyword(s):  
Conformational Changes ◽  
Kinase Activity ◽  
Cell Receptor ◽  
Kinase Domain ◽  
Hsp90 Inhibitor ◽  
Intramolecular Interactions ◽  
Atp Binding Site ◽  
Regulatory Processes ◽  
The Stability ◽  
Lck Kinase

ABSTRACT Regulation of the Src-related tyrosine kinase Lck is crucial to the outcome of T-cell receptor (TCR) stimulation. It was previously shown that the stability of the constitutively active mutant LckY505F is controlled by Hsp90 (M. J. Bijlmakers and M. Marsh, Mol. Biol. Cell. 11:1585-1595, 2000). Here we establish that following TCR stimulation, endogenous activated Lck in T cells is also degraded in the presence of the Hsp90 inhibitor geldanamycin. Using Lck constructs expressed in COS-7 cells, we show that the presence of activating Lck mutations results not only in the enhanced dependence on Hsp90 but also in enhanced ubiquitination of Lck. Although both processes were induced by mutations Y505F and W97A that release the SH2 and SH3 inhibitory intramolecular interactions, respectively, neither process required Lck kinase activity or activation-dependent phosphorylation at serines 42 and 59 or tyrosine 394. By binding to the ATP-binding site, the Src family inhibitor PP2 reduced ubiquitination and overcame the need for Hsp90 monitoring of active Lck. We conclude that the levels of active Lck are influenced by two opposing processes, targeting for degradation by ubiquitination and rescue from degradation by Hsp90 monitoring. Based on the PP2 result, we propose that activation-induced conformational changes of the Lck kinase domain instigate both regulatory processes.

scholarly journals Role of Phosphatidylethanolamine in the Biogenesis of Mitochondrial Outer Membrane Proteins

2013 ◽  
Vol 288 (23) ◽  
pp. 16451-16459 ◽  
Author(s):  
Thomas Becker ◽  
Susanne E. Horvath ◽  
Lena Böttinger ◽  
Natalia Gebert ◽  
Günther Daum ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Outer Membrane ◽  
Outer Membrane Proteins ◽  
Proper Function ◽  
Mitochondrial Outer Membrane ◽  
Tom Complex ◽  
Precursor Proteins ◽  
Helical Proteins ◽  
The Stability

The mitochondrial outer membrane contains proteinaceous machineries for the import and assembly of proteins, including TOM (translocase of the outer membrane) and SAM (sorting and assembly machinery). It has been shown that the dimeric phospholipid cardiolipin is required for the stability of TOM and SAM complexes and thus for the efficient import and assembly of β-barrel proteins and some α-helical proteins of the outer membrane. Here, we report that mitochondria deficient in phosphatidylethanolamine (PE), the second non-bilayer-forming phospholipid, are impaired in the biogenesis of β-barrel proteins, but not of α-helical outer membrane proteins. The stability of TOM and SAM complexes is not disturbed by the lack of PE. By dissecting the import steps of β-barrel proteins, we show that an early import stage involving translocation through the TOM complex is affected. In PE-depleted mitochondria, the TOM complex binds precursor proteins with reduced efficiency. We conclude that PE is required for the proper function of the TOM complex.

scholarly journals p21Cip1Promotes Cyclin D1 Nuclear Accumulation via Direct Inhibition of Nuclear Export

2001 ◽  
Vol 277 (10) ◽  
pp. 8517-8523 ◽  
Author(s):  
Jodi R. Alt ◽  
Andrew B. Gladden ◽  
J. Alan Diehl
Keyword(s):  
Cyclin D1 ◽  
Nuclear Export ◽  
Nuclear Accumulation ◽  
Direct Inhibition

scholarly journals Structural and functional studies of pyruvate carboxylase regulation by cyclic di-AMP in lactic acid bacteria

2017 ◽  
Vol 114 (35) ◽  
pp. E7226-E7235 ◽  
Author(s):  
Philip H. Choi ◽  
Thu Minh Ngoc Vu ◽  
Huong Thi Pham ◽  
Joshua J. Woodward ◽  
Mark S. Turner ◽  
...  
Keyword(s):  
Binding Site ◽  
Conformational Changes ◽  
Pyruvate Carboxylase ◽  
Adenosine Monophosphate ◽  
Binding Mode ◽  
Functional Studies ◽  
Cellular Processes ◽  
Wide Range ◽  
A Site ◽  
Amp Inhibition

Cyclic di-3′,5′-adenosine monophosphate (c-di-AMP) is a broadly conserved bacterial second messenger that has been implicated in a wide range of cellular processes. Our earlier studies showed that c-di-AMP regulates central metabolism inListeria monocytogenesby inhibiting its pyruvate carboxylase (LmPC), a biotin-dependent enzyme with biotin carboxylase (BC) and carboxyltransferase (CT) activities. We report here structural, biochemical, and functional studies on the inhibition ofLactococcus lactisPC (LlPC) by c-di-AMP. The compound is bound at the dimer interface of the CT domain, at a site equivalent to that in LmPC, although it has a distinct binding mode in the LlPC complex. This binding site is not well conserved among PCs, and only a subset of these bacterial enzymes are sensitive to c-di-AMP. Conformational changes in the CT dimer induced by c-di-AMP binding may be the molecular mechanism for its inhibitory activity. Mutations of residues in the binding site can abolish c-di-AMP inhibition. InL. lactis, LlPC is required for efficient milk acidification through its essential role in aspartate biosynthesis. The aspartate pool inL. lactisis negatively regulated by c-di-AMP, and high aspartate levels can be restored by expression of a c-di-AMP–insensitive LlPC. LlPC has high intrinsic catalytic activity and is not sensitive to acetyl-CoA activation, in contrast to other PC enzymes.

scholarly journals Structural determinants of nuclear export signal orientation in binding to exportin CRM1

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Ho Yee Joyce Fung ◽  
Szu-Chin Fu ◽  
Chad A Brautigam ◽  
Yuh Min Chook
Keyword(s):  
Crystal Structures ◽  
Nuclear Export ◽  
Chromosome Region ◽  
Nuclear Export Signal ◽  
Opposite Orientation ◽  
Structural Determinants ◽  
Large Expansion ◽  
Export Signal

The Chromosome Region of Maintenance 1 (CRM1) protein mediates nuclear export of hundreds of proteins through recognition of their nuclear export signals (NESs), which are highly variable in sequence and structure. The plasticity of the CRM1-NES interaction is not well understood, as there are many NES sequences that seem incompatible with structures of the NES-bound CRM1 groove. Crystal structures of CRM1 bound to two different NESs with unusual sequences showed the NES peptides binding the CRM1 groove in the opposite orientation (minus) to that of previously studied NESs (plus). Comparison of minus and plus NESs identified structural and sequence determinants for NES orientation. The binding of NESs to CRM1 in both orientations results in a large expansion in NES consensus patterns and therefore a corresponding expansion of potential NESs in the proteome.

scholarly journals DNA methylation cues in nucleosome geometry, stability, and unwrapping

2021 ◽  
Author(s):  
Shuxiang Li ◽  
Yunhui Peng ◽  
David Landsman ◽  
Anna Panchenko
Keyword(s):  
Conformational Changes ◽  
Cytosine Methylation ◽  
Epigenetic Mark ◽  
Nucleosome Assembly ◽  
Functional Studies ◽  
Nucleosome Dynamics ◽  
Nucleosomal Dna ◽  
Dna Unwrapping ◽  
Dynamics Simulations ◽  
Eukaryotic Organisms

Cytosine methylation at the 5-carbon position is an essential DNA epigenetic mark in many eukaryotic organisms. Although countless structural and functional studies of cytosine methylation have been reported in both prokaryotes and eukaryotes, our understanding of how it influences the nucleosome assembly, structure, and dynamics remains obscure. Here we investigated the effects of cytosine methylation at CpG sites on nucleosome dynamics and stability. By applying long molecular dynamics simulations (five microsecond long trajectories, 60 microseconds in total), we generated extensive atomic level conformational full nucleosome ensembles. Our results revealed that methylation induces pronounced changes in geometry for both linker and nucleosomal DNA, leading to a more curved, under-twisted DNA, shifting the population equilibrium of sugar-phosphate backbone geometry. These conformational changes are associated with a considerable enhancement of interactions between methylated DNA and the histone octamer, doubling the number of contacts at some key arginines. H2A and H3 tails play important roles in these interactions, especially for DNA methylated nucleosomes. This, in turn, prevents a spontaneous DNA unwrapping of 3-4 helical turns for the methylated nucleosome with truncated histone tails, otherwise observed in the unmethylated system on several microsecond time scale.

Raman Spectroscopic Study of Ba2+-Surfactant Interactions in Aqueous Monodisperse Alkylpolyoxyethylene Surfactant Solutions

1993 ◽  
Vol 47 (11) ◽  
pp. 1784-1787
Author(s):  
Diana C. W. Siew ◽  
Ralph P. Cooney ◽  
Michael J. Taylor
Keyword(s):  
Conformational Changes ◽  
Influencing Factor ◽  
Difference Spectroscopy ◽  
Raman Spectroscopic ◽  
Chain Component ◽  
Raman Spectroscopic Study ◽  
Surfactant Solutions ◽  
Surfactant Complex ◽  
Surfactant Interactions ◽  
The Stability

Aqueous monodisperse alkylpolyoxyethylene surfactant-Ba2+ systems were investigated to clarify the coordination effects between the polyether chain and the cation which have been previously identified in polydisperse alkylphenoxy- and alkypolyoxyethylene surfactant systems. The coordination effects are simplified in the present study due to the presence of only a single polyether chain component. Formation of the surfactant complex resulted in conformational changes of the polyether chain which were monitored by Raman difference spectroscopy and curve fitting. The present studies confirm that surfactant-complex formation is dependent upon the polyether chain adopting the TGT-TĜT conformation and that the stability of the complex increases with the number of filled chain sites. The length of the polyether chain is also an influencing factor.

Requirements for the nuclear export of the small ribosomal subunit

2002 ◽  
Vol 115 (14) ◽  
pp. 2985-2995 ◽  
Author(s):  
Terence I. Moy ◽  
Pamela A. Silver
Keyword(s):  
Nuclear Export ◽  
Ribosome Biogenesis ◽  
Large Scale ◽  
Ribosomal Subunit ◽  
Small Subunit ◽  
Temperature Sensitive ◽  
Yeast Saccharomyces Cerevisiae ◽  
Small Ribosomal Subunit ◽  
Factors Affecting ◽  
The Stability

Eukaryotic ribosome biogenesis requires multiple steps of nuclear transport because ribosomes are assembled in the nucleus while protein synthesis occurs in the cytoplasm. Using an in situ RNA localization assay in the yeast Saccharomyces cerevisiae, we determined that efficient nuclear export of the small ribosomal subunit requires Yrb2, a factor involved in Crm1-mediated export. Furthermore, in cells lacking YRB2, the stability and abundance of the small ribosomal subunit is decreased in comparison with the large ribosomal subunit. To identify additional factors affecting small subunit export, we performed a large-scale screen of temperature-sensitive mutants. We isolated new alleles of several nucleoporins and Ran-GTPase regulators. Together with further analysis of existing mutants,we show that nucleoporins previously shown to be defective in ribosomal assembly are also defective in export of the small ribosomal subunit.

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