PKD Phosphorylation as Novel Pathway of KV11.1 Regulation

Background/Aims: The voltage-gated potassium channel KV11.1 has been originally cloned from the brain and is expressed in a variety of tissues. The role of phosphorylation for channel function is a matter of debate. In this study, we aimed to elucidate the extent and role of protein kinase D mediated phosphorylation. Methods: We employed mass spectrometry, whole-cell patch clamp electrophysiology, confocal microscopy, site-directed mutagenesis, and western blotting. Results: Using brain tissue from rat and mouse, we mapped several phosphorylated KV11.1 residues by LC-MS mass spectrometry and identified protein kinase D (PKD1) as possible regulatory kinase. Co-expression of KV11.1 with PKD1 reduced current amplitudes without altering protein levels or surface expression of the channel. Based on LC-MS results from in vivo and HEK293 cell experiments we chose four KV11.1 mutant candidates for further functional analysis. Ablation of the putative PKD phosphorylation site in the mutant S284A increased the maximal current indicating S284 as a main PKD target in KV11.1. Conclusions: Our data might help mitigating a long-standing controversy in the field regarding PKC regulation of KV11.1. We propose that PKD1 mediates the PKC effects on KV11.1 and we found that PKD targets S284 in the N-terminus of the channel.

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The cAMP pathway regulates mRNA decay through phosphorylation of the RNA-binding protein TIS11b/BRF1

Molecular Biology of the Cell ◽

10.1091/mbc.e16-06-0379 ◽

2016 ◽

Vol 27 (24) ◽

pp. 3841-3854 ◽

Cited By ~ 13

Author(s):

Felicitas Rataj ◽

Séverine Planel ◽

Agnès Desroches-Castan ◽

Juliette Le Douce ◽

Khadija Lamribet ◽

...

Keyword(s):

Protein Kinase ◽

Protein Interactions ◽

Mrna Decay ◽

Rna Binding ◽

Phosphorylation Site ◽

P38 Map Kinase ◽

Site Directed Mutagenesis ◽

Protein Protein Interactions

TPA-inducible sequence 11b/butyrate response factor 1 (TIS11b/BRF1) belongs to the tristetraprolin (TTP) family of zinc-finger proteins, which bind to mRNAs containing AU-rich elements in their 3′-untranslated region and target them for degradation. Regulation of TTP family function through phosphorylation by p38 MAP kinase and Akt/protein kinase B signaling pathways has been extensively studied. In contrast, the role of cAMP-dependent protein kinase (PKA) in the control of TTP family activity in mRNA decay remains largely unknown. Here we show that PKA activation induces TIS11b gene expression and protein phosphorylation. Site-directed mutagenesis combined with kinase assays and specific phosphosite immunodetection identified Ser-54 (S54) and Ser-334 (S334) as PKA target amino acids in vitro and in vivo. Phosphomimetic mutation of the C-terminal S334 markedly increased TIS11b half-life and, unexpectedly, enhanced TIS11b activity on mRNA decay. Examination of protein–protein interactions between TIS11b and components of the mRNA decay machinery revealed that mimicking phosphorylation at S334 enhances TIS11b interaction with the decapping coactivator Dcp1a, while preventing phosphorylation at S334 potentiates its interaction with the Ccr4-Not deadenylase complex subunit Cnot1. Collectively our findings establish for the first time that cAMP-elicited phosphorylation of TIS11b plays a key regulatory role in its mRNA decay-promoting function.

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Structural characterization of the membrane-associated regulatory subunit of type I cAMP-dependent protein kinase by mass spectrometry: Identification of Ser81 as the in vivo phosphorylation site of RIα

Protein Science ◽

10.1110/ps.8.7.1515 ◽

1999 ◽

Vol 8 (7) ◽

pp. 1515-1522 ◽

Cited By ~ 10

Author(s):

Karen M. Boeshans ◽

Katheryn A. Resing ◽

John B. Hunt ◽

Natalie G. Ahn ◽

John B. Shabb

Keyword(s):

Mass Spectrometry ◽

Protein Kinase ◽

Phosphorylation Site ◽

Regulatory Subunit ◽

Type I ◽

Dependent Protein Kinase ◽

Mass Spectrometry Identification ◽

Dependent Protein

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A novel protein kinase D phosphorylation site in the tumor suppressor Rab interactor 1 is critical for coordination of cell migration

Molecular Biology of the Cell ◽

10.1091/mbc.e10-05-0427 ◽

2011 ◽

Vol 22 (5) ◽

pp. 570-580 ◽

Cited By ~ 29

Author(s):

Susanne Ziegler ◽

Tim Eiseler ◽

Rolf-Peter Scholz ◽

Alexander Beck ◽

Gisela Link ◽

...

Keyword(s):

Cell Migration ◽

Protein Kinase ◽

Phosphorylation Site ◽

Protein Kinase D ◽

Adapter Protein ◽

Actin Remodeling ◽

Abl Kinases ◽

Cell Processes

The multifunctional signal adapter protein Ras and Rab interactor 1 (RIN1) is a Ras effector protein involved in the regulation of epithelial cell processes such as cell migration and endocytosis. RIN1 signals via two downstream pathways, namely the activation of Rab5 and Abl family kinases. Protein kinase D (PKD) phosphorylates RIN1 at serine 351 in vitro, thereby regulating interaction with 14–3-3 proteins. Here, we report the identification of serine 292 in RIN1 as an in vivo PKD phosphorylation site. PKD-mediated phosphorylation at this site was confirmed with a phospho-specific antibody and by mass spectrometry. We demonstrate that phosphorylation at serine 292 controls RIN1-mediated inhibition of cell migration by modulating the activation of Abl kinases. We further provide evidence that RIN1 in vivo phosphorylation at serine 351 occurs independently of PKD. Collectively, our data identify a novel PKD signaling pathway through RIN1 and Abl kinases that is involved in the regulation of actin remodeling and cell migration.

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The Role of Protein Kinase D (PKD) Signaling in Breast Cancer Cell Migration and Invasion

10.21236/ada548836 ◽

2010 ◽

Author(s):

Claudine Christoforides

Keyword(s):

Breast Cancer ◽

Cell Migration ◽

Protein Kinase ◽

Cancer Cell ◽

Breast Cancer Cell ◽

Migration And Invasion ◽

Protein Kinase D ◽

Cancer Cell Migration ◽

Cell Migration And Invasion

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Topoisomerase II is regulated by translationally controlled tumor protein for cell survival during organ growth in Drosophila

Cell Death and Disease ◽

10.1038/s41419-021-04091-y ◽

2021 ◽

Vol 12 (9) ◽

Author(s):

Dae-Wook Yang ◽

Jung-Wan Mok ◽

Stephanie B. Telerman ◽

Robert Amson ◽

Adam Telerman ◽

...

Keyword(s):

Cell Survival ◽

Topoisomerase Ii ◽

Wing Disc ◽

Organ Development ◽

Translationally Controlled Tumor Protein ◽

Protein Levels ◽

Eye Disc ◽

Mutual Regulation

AbstractRegulation of cell survival is critical for organ development. Translationally controlled tumor protein (TCTP) is a conserved protein family implicated in the control of cell survival during normal development and tumorigenesis. Previously, we have identified a human Topoisomerase II (TOP2) as a TCTP partner, but its role in vivo has been unknown. To determine the significance of this interaction, we examined their roles in developing Drosophila organs. Top2 RNAi in the wing disc leads to tissue reduction and caspase activation, indicating the essential role of Top2 for cell survival. Top2 RNAi in the eye disc also causes loss of eye and head tissues. Tctp RNAi enhances the phenotypes of Top2 RNAi. The depletion of Tctp reduces Top2 levels in the wing disc and vice versa. Wing size is reduced by Top2 overexpression, implying that proper regulation of Top2 level is important for normal organ development. The wing phenotype of Tctp RNAi is partially suppressed by Top2 overexpression. This study suggests that mutual regulation of Tctp and Top2 protein levels is critical for cell survival during organ development.

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Protein kinase D promotes plasticity-induced F-actin stabilization in dendritic spines and regulates memory formation

The Journal of Cell Biology ◽

10.1083/jcb.201501114 ◽

2015 ◽

Vol 210 (5) ◽

pp. 771-783 ◽

Cited By ~ 8

Author(s):

Norbert Bencsik ◽

Zsófia Szíber ◽

Hanna Liliom ◽

Krisztián Tárnok ◽

Sándor Borbély ◽

...

Keyword(s):

Synaptic Plasticity ◽

Protein Kinase ◽

Dendritic Spines ◽

Morphological Changes ◽

Long Term Potentiation ◽

Memory Formation ◽

Protein Kinase D

Actin turnover in dendritic spines influences spine development, morphology, and plasticity, with functional consequences on learning and memory formation. In nonneuronal cells, protein kinase D (PKD) has an important role in stabilizing F-actin via multiple molecular pathways. Using in vitro models of neuronal plasticity, such as glycine-induced chemical long-term potentiation (LTP), known to evoke synaptic plasticity, or long-term depolarization block by KCl, leading to homeostatic morphological changes, we show that actin stabilization needed for the enlargement of dendritic spines is dependent on PKD activity. Consequently, impaired PKD functions attenuate activity-dependent changes in hippocampal dendritic spines, including LTP formation, cause morphological alterations in vivo, and have deleterious consequences on spatial memory formation. We thus provide compelling evidence that PKD controls synaptic plasticity and learning by regulating actin stability in dendritic spines.

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Role of Cathepsin S in Periodontal Inflammation and Infection

Mediators of Inflammation ◽

10.1155/2017/4786170 ◽

2017 ◽

Vol 2017 ◽

pp. 1-10 ◽

Cited By ~ 12

Author(s):

S. Memmert ◽

A. Damanaki ◽

A. V. B. Nogueira ◽

S. Eick ◽

M. Nokhbehsaim ◽

...

Keyword(s):

Periodontal Diseases ◽

Interleukin 1 ◽

Critical Role ◽

Gingival Tissue ◽

Cathepsin S ◽

Protein Levels ◽

Periodontal Inflammation

Cathepsin S is a cysteine protease and regulator of autophagy with possible involvement in periodontitis. The objective of this study was to investigate whether cathepsin S is involved in the pathogenesis of periodontal diseases. Human periodontal fibroblasts were cultured under inflammatory and infectious conditions elicited by interleukin-1β and Fusobacterium nucleatum, respectively. An array-based approach was used to analyze differential expression of autophagy-associated genes. Cathepsin S was upregulated most strongly and thus further studied in vitro at gene and protein levels. In vivo, gingival tissue biopsies from rats with ligature-induced periodontitis and from periodontitis patients were also analyzed at transcriptional and protein levels. Multiple gene expression changes due to interleukin-1β and F. nucleatum were observed in vitro. Both stimulants caused a significant cathepsin S upregulation. A significantly elevated cathepsin S expression in gingival biopsies from rats with experimental periodontitis was found in vivo, as compared to that from control. Gingival biopsies from periodontitis patients showed a significantly higher cathepsin S expression than those from healthy gingiva. Our findings provide original evidence that cathepsin S is increased in periodontal cells and tissues under inflammatory and infectious conditions, suggesting a critical role of this autophagy-associated molecule in the pathogenesis of periodontitis.

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Characterization of TNF receptor subtype expression and signaling on pulmonary endothelial cells in mice

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00326.2010 ◽

2011 ◽

Vol 300 (5) ◽

pp. L781-L789 ◽

Cited By ~ 17

Author(s):

Szabolcs Bertok ◽

Michael R. Wilson ◽

Anthony D. Dorr ◽

Justina O. Dokpesi ◽

Kieran P. O'Dea ◽

...

Keyword(s):

Flow Cytometry ◽

Adhesion Molecules ◽

Surface Expression ◽

Tnf Receptor ◽

Wild Type ◽

Tnf Receptors ◽

Pulmonary Endothelial Cells ◽

Microvascular Inflammation

TNF plays a crucial role in the pathogenesis of acute lung injury. However, the expression profile of its two receptors, p55 and p75, on pulmonary endothelium and their influence on TNF signaling during lung microvascular inflammation remain uncertain. Using flow cytometry, we characterized the expression profile of TNF receptors on the surface of freshly harvested pulmonary endothelial cells (PECs) from mice and found expression of both receptors with dominance of p55. To investigate the impact of stimulating individual TNF receptors, we treated wild-type and TNF receptor knockout mice with intravenous TNF and determined surface expression of adhesion molecules (E-selectin, VCAM-1, ICAM-1) on PECs by flow cytometry. TNF-induced upregulation of all adhesion molecules was substantially attenuated by absence of p55, whereas lack of p75 had a similar but smaller effect that varied between adhesion molecules. Selective blockade of individual TNF receptors by specific antibodies in wild-type primary PEC culture confirmed that the in vivo findings were due to direct effects of TNF receptor inhibition on endothelium and not other cells (e.g., circulating leukocytes). Finally, we found that PEC surface expression of p55 dramatically decreased in the early stages of endotoxemia following intravenous LPS, while no change in p75 expression was detected. These data demonstrate a crucial in vivo role of p55 and an auxiliary role of p75 in TNF-mediated adhesion molecule upregulation on PECs. It is possible that the importance of the individual receptors varies at different stages of pulmonary microvascular inflammation following changes in their relative expression.

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