scholarly journals Revisiting the Role of Ser982 Phosphorylation in Stoichiometry Shift of the Electrogenic Na+/qHCO3− Cotransporter NBCe1

2021 ◽  
Vol 22 (23) ◽  
pp. 12817
Author(s):  
Thamer A. Alsufayan ◽  
Evan J. Myers ◽  
Bianca N. Quade ◽  
Clayton T. Brady ◽  
Aniko Marshall ◽  
...  
Keyword(s):  
Metabolic Acidosis ◽  
Xenopus Oocytes ◽  
Cell Types ◽  
Expression Systems ◽  
Phosphorylation State ◽  
Sodium Bicarbonate Cotransporter ◽  
Carboxy Terminal Domain ◽  
Heterologous Expression Systems ◽  
Carboxy Terminal

In most cell types and heterologous expression systems, the electrogenic sodium-bicarbonate cotransporter NBCe1 operates with a 1Na+–2HCO3− stoichiometry that, given typical transmembrane electrochemical gradients, promotes Na+ and HCO3− influx. However, NBCe1 in the kidney mediates HCO3− efflux (HCO3− reabsorption), a direction that has been predicted to be favored only if NBCe1 operates with a 1:3 stoichiometry. The phosphorylation state of Ser982 in the cytosolic carboxy-terminal domain of NBCe1 has been reported to be a key determinant of the transporter stoichiometry, with non-phosphorylated Ser982 favoring a 1:3 stoichiometry. Conversely, phosphoproteomic data from renal cortical preparations have revealed the presence of NBCe1 peptides including phosphoserine982 (pSer982) and/or pSer985 although it was not known what proportion of NBCe1 molecules were phosphorylated. In the present study, we report the generation, characterization, and application of a novel phosphospecific antibody raised against NBCe1/pSer982 and show that, contrary to expectations, Ser982 is more prevalently phosphorylated in murine kidneys (in which NBCe1 mediates HCO3− efflux) than in murine colons (in which NBCe1 mediates HCO3− influx). Using phosphomimetic mutants of murine NBCe1 expressed in Xenopus oocytes, we found no evidence that the phosphorylation state of Ser982 or Ser985 alone influences the transport stoichiometry or conductance. Furthermore, we found that the phosphorylation of NBCe1/Ser982 is enhanced in murine kidneys following a 24 h induction of metabolic acidosis. We conclude that the phosphorylation status of Ser982 is not a key determinant of NBCe1 stoichiometry but correlates with presumed NBCe1 activity.

scholarly journals What’s all the phos about? Insights into the phosphorylation state of the RNA polymerase II C-terminal domain via mass spectrometry

2021 ◽  
Author(s):  
Blase Matthew LeBlanc ◽  
Rosamaria Yvette Moreno ◽  
Edwin Escobar ◽  
Mukesh Kumar Venkat Ramani ◽  
Jennifer S Brodbelt ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Phosphorylation State ◽  
Terminal Domain ◽  
Carboxy Terminal Domain ◽  
Protein Encoding ◽  
Small Nuclear Rnas ◽  
Rnap Ii ◽  
Carboxy Terminal ◽  
Encoding Genes

RNA polymerase II (RNAP II) is one of the primary enzymes responsible for expressing protein-encoding genes and some small nuclear RNAs. The enigmatic carboxy-terminal domain (CTD) of RNAP II and...

scholarly journals The Carboxy-Terminal Tail of Human Cytomegalovirus (HCMV) US28 Regulates both Chemokine-Independent and Chemokine-Dependent Signaling in HCMV-Infected Cells

2009 ◽  
Vol 83 (19) ◽  
pp. 10016-10027 ◽  
Author(s):  
Melissa P. Stropes ◽  
Olivia D. Schneider ◽  
William A. Zagorski ◽  
Jeanette L. C. Miller ◽  
William E. Miller
Keyword(s):  
Human Cytomegalovirus ◽  
Calcium Release ◽  
Hcmv Infection ◽  
Calcium Signal ◽  
Terminal Domain ◽  
Carboxy Terminal Domain ◽  
Domain Truncation ◽  
Infected Cells ◽  
Heterologous Expression Systems ◽  
Carboxy Terminal

ABSTRACT The human cytomegalovirus (HCMV)-encoded G-protein-coupled receptor (GPCR) US28 is a potent activator of a number of signaling pathways in HCMV-infected cells. The intracellular carboxy-terminal domain of US28 contains residues critical for the regulation of US28 signaling in heterologous expression systems; however, the role that this domain plays during HCMV infection remains unknown. For this study, we constructed an HCMV recombinant virus encoding a carboxy-terminal domain truncation mutant of US28, FLAG-US28/1-314, to investigate the role that this domain plays in US28 signaling. We demonstrate that US28/1-314 exhibits a more potent phospholipase C-β (PLC-β) signal than does wild-type US28, indicating that the carboxy-terminal domain plays an important role in regulating agonist-independent signaling in infected cells. Moreover, HMCV-infected cells expressing the US28/1-314 mutant exhibit a prolonged calcium signal in response to CCL5, indicating that the US28 carboxy-terminal domain also regulates agonist-dependent signaling. Finally, while the chemokine CX3CL1 behaves as an inverse agonist or inhibitor of constitutive US28 signaling to PLC-β, we demonstrate that CX3CL1 functions as an agonist with regard to US28-stimulated calcium release. This study is the first to demonstrate that the carboxy terminus of US28 controls US28 signaling in the context of HCMV infection and indicates that chemokines such as CX3CL1 can decrease constitutive US28 signals and yet simultaneously promote nonconstitutive US28 signals.

scholarly journals Splicing Factors Associate with Hyperphosphorylated RNA Polymerase II in the Absence of Pre-mRNA

1997 ◽  
Vol 136 (1) ◽  
pp. 19-28 ◽  
Author(s):  
Euikyung Kim ◽  
Lei Du ◽  
David B. Bregman ◽  
Stephen L. Warren
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
Transcriptional Activity ◽  
Splicing Factors ◽  
Definitive Evidence ◽  
Carboxy Terminal Domain ◽  
Interphase Cells ◽  
Carboxy Terminal

The carboxy-terminal domain (CTD) of the largest subunit of RNA polymerase II (Pol II) contains multiple tandem copies of the consensus heptapeptide, TyrSerProThrSerProSer. Concomitant with transcription initiation the CTD is phosphorylated. Elongating polymerase has a hyperphosphorylated CTD, but the role of this modification is poorly understood. A recent study revealed that some hyperphosphorylated polymerase molecules (Pol IIo) are nonchromosomal, and hence transcriptionally unengaged (Bregman, D.B., L. Du, S. van der Zee, S.L. Warren. 1995. J. Cell Biol. 129: 287–298). Pol IIo was concentrated in discrete splicing factor domains, suggesting a possible relationship between CTD phosphorylation and splicing factors, but no evidence beyond immunolocalization data was provided to support this idea. Here, we show that Pol IIo co-immunoprecipitates with members of two classes of splicing factors, the Sm snRNPs and non-snRNP SerArg (SR) family proteins. Significantly, Pol IIo's association with splicing factors is maintained in the absence of pre-mRNA, and the polymerase need not be transcriptionally engaged. We also provide definitive evidence that hyperphosphorylation of Pol II's CTD is poorly correlated with its transcriptional activity. Using monoclonal antibodies (mAbs) H5 and H14, which are shown here to recognize phosphoepitopes on Pol II's CTD, we have quantitated the level of Pol IIo at different stages of the cell cycle. The level of Pol IIo is similar in interphase and mitotic cells, which are transcriptionally active and inactive, respectively. Finally, complexes containing Pol IIo and splicing factors can be prepared from mitotic as well as interphase cells. The experiments reported here establish that hyperphosphorylation of the CTD is a good indicator of polymerase's association with snRNP and SR splicing factors, but not of its transcriptional activity. Most importantly, the present study suggests that splicing factors may associate with the polymerase via the hyperphosphorylated CTD.

scholarly journals SAP97 interacts with Kv1.5 in heterologous expression systems

2001 ◽  
Vol 281 (6) ◽  
pp. H2575-H2584 ◽  
Author(s):  
Mitsunobu Murata ◽  
Peter D. Buckett ◽  
Jun Zhou ◽  
Michael Brunner ◽  
Eduardo Folco ◽  
...  
Keyword(s):  
Cardiac Myocytes ◽  
Xenopus Oocytes ◽  
Pdz Domain ◽  
Transient Transfection ◽  
Amino Acid Residues ◽  
Expression Systems ◽  
Terminal Amino Acid ◽  
Terminal Amino ◽  
Heterologous Expression Systems ◽  
Functional Analyses

PDZ domain-containing proteins such as SAP97 and ZO-1 have been implicated in the targeting and clustering of ion channels. We have explored the interactions of these polypeptides with a cardiac voltage-gated potassium channel. Immunocytochemistry in cardiac myocytes revealed colocalization of SAP97 and Kv1.5, both at the intercalated disks and the lateral membranes. Transient transfection experiments in COS-7 cells revealed that SAP97 and Kv1.5 polypeptides formed perinuclear clustered complexes that could be coimmunoprecipitated. Mutation of the three COOH-terminal amino acid residues of Kv1.5 (T-D-L to A-A-A) abolished these interactions. Whereas in most COS-7 cells the SAP97-Kv1.5 complexes were retained in the ER, functional analyses in Xenopus oocytes showed that Kv1.5-encoded outward potassium currents were augmented by coexpression with SAP97. By contrast, cotransfected ZO-1 and Kv1.5 polypeptides in COS-7 cells could not be coprecipitated nor did the coinjection of ZO-1 augment the Kv1.5-encoded currents in oocytes. Collectively, our results suggest that SAP97 may play an important role in the modulation of Kv1.5 channel function in cardiac myocytes.

Role of RNA Polymerase II Carboxy-terminal Domain in Coordinating Transcription with RNA Processing

1998 ◽  
Vol 63 (0) ◽  
pp. 301-310 ◽  
Author(s):  
S. MCCRACKEN ◽  
E. ROSONINA ◽  
N. FONG ◽  
M. SIKES ◽  
A. BEYER ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Rna Processing ◽  
Terminal Domain ◽  
Carboxy Terminal Domain ◽  
Carboxy Terminal

scholarly journals CITED2 modulation of trophoblast cell differentiation: insights from global transcriptome analysis

Reproduction ◽  
2016 ◽  
Vol 151 (5) ◽  
pp. 509-516 ◽  
Author(s):  
Kazuhiko Imakawa ◽  
Pramod Dhakal ◽  
Kaiyu Kubota ◽  
Kazuya Kusama ◽  
Damayanti Chakraborty ◽  
...  
Keyword(s):  
Cell Differentiation ◽  
Regulatory Network ◽  
Cell Types ◽  
Trophoblast Cell ◽  
Carboxy Terminal Domain ◽  
Trophoblast Stem ◽  
Global Transcriptome ◽  
Cell Transcriptome ◽  
Carboxy Terminal ◽  
The Impact

Trophoblast stem (TS) cells possess the capacity to differentiate along a multi-lineage pathway yielding several specialized cell types. The regulatory network controlling trophoblast cell differentiation is poorly understood. Cbp/p300-interacting transactivator with Glu/Asp-rich carboxy-terminal domain, 2 (CITED2) has been implicated in the regulation of placentation; however, we know little about how CITED2 acts to influence trophoblast cells. Rat Rcho-1 TS cells can be manipulated to proliferate or differentiate into specialized trophoblast lineages and are an excellent model for investigating trophoblast differentiation. CITED2 transcript and protein showed a robust induction during Rcho-1 TS cell differentiation. We used an shRNA knockdown approach to disrupt CITED2 expression in order to investigate its involvement in trophoblast cell differentiation. RNA-sequencing was used to examine the impact of CITED2 on trophoblast cell differentiation. CITED2 disruption affected the differentiating trophoblast cell transcriptome. CITED2 possessed a prominent role in the regulation of cell differentiation with links to several signal transduction pathways and to hypoxia-regulated and coagulation processes. In summary, our findings indicate that CITED2 contributes to the regulation of trophoblast cell differentiation.Reproduction (2016) 151 1–8

Role of the Cro repressor carboxy terminal domain and flexible dimer linkage in operator and nonspecific DNA binding

Biochemistry ◽  
1990 ◽  
Vol 29 (39) ◽  
pp. 9241-9249 ◽  
Author(s):  
Adrian J. Hubbard ◽  
Laurent P. Bracco ◽  
Scott J. Eisenbeis ◽  
Richard B. Gayle ◽  
Graham Beaton ◽  
...  
Keyword(s):  
Dna Binding ◽  
Terminal Domain ◽  
Carboxy Terminal Domain ◽  
Cro Repressor ◽  
Dimer Linkage ◽  
Carboxy Terminal
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