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Biomedicines
Special Issues
Signaling of Protein Kinases in Development and Disease
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Signaling of Protein Kinases in Development and Disease

A special issue of Biomedicines (ISSN 2227-9059). This special issue belongs to the section "Cell Biology and Pathology".

Deadline for manuscript submissions: 30 June 2025 | Viewed by 3012

Special Issue Editor

Dr. John Zheng Fu

E-Mail Website
Guest Editor
Department of Pharmacology, University of Virginia, Charlottesville, VA, USA
Interests: cell signaling of protein kinases; mechanism of ciliopathy

Special Issue Information

Dear Colleagues,

Protein kinases comprise one of the largest and most abundant gene families in humans and play a pivotal role in signal transduction during tissue development, patterning, and homeostasis through the phosphorylation and functional modulation of protein substrates. Both germ-line and somatic mutations in kinase genes have been associated with many human diseases. Protein kinases are the second most targeted group for drug development. Novel therapeutic strategies to target protein kinases and intervene in cell signaling are still limited due to our incomplete understanding of their signaling mechanisms.

This Special Issue welcomes both comprehensive reviews and original articles to highlight the recent progress in the discovery of new mechanisms by which protein kinases function and human mutations disrupt kinase signaling and impact signal transduction.

Dr. John Zheng Fu
Guest Editor

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Keywords

  • kinase
  • phosphorylation
  • signaling
  • mutation
  • mechanism
  • development
  • inhibitor
  • disease

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Published Papers (2 papers)

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Research

15 pages, 4218 KiB  
Article
A Protein Kinase Cε/Protein Kinase D3 Signalling Axis Modulates RhoA Activity During Cytokinesis
by Ursula Braun and Michael Leitges
Biomedicines 2025, 13(2), 345; https://doi.org/10.3390/biomedicines13020345 - 3 Feb 2025
Viewed by 499
Abstract
Background: Protein kinase D3 (PKD3) is a member of the PKD family that has been implicated in many intracellular signalling pathways. However, defined statements regarding PKD isoform specificity and in vivo functions are rare. Methods: Here, we use PKD3-depleted mouse embryonic fibroblast cells [...] Read more.
Background: Protein kinase D3 (PKD3) is a member of the PKD family that has been implicated in many intracellular signalling pathways. However, defined statements regarding PKD isoform specificity and in vivo functions are rare. Methods: Here, we use PKD3-depleted mouse embryonic fibroblast cells and employ various cell culture-based assays and fluorescence microscopy. Results: We show that PKD3 is involved in the regulation of cytokinesis after immortalisation by modulating RhoA activity through a PKCε/PKD3 signalling axis. Conclusions: PKD3 depletion leads to prolonged RhoA activity during cytokinesis, resulting in failed abscission and an increase in the number of multinucleated cells. This identifies a novel, previously unrecognised PKCε/PKD3 pathway involved in the modulation of cytokinesis. Full article
(This article belongs to the Special Issue Signaling of Protein Kinases in Development and Disease)
►▼ Show Figures

Figure 1

Figure 1
<p>Double/multi-nucleation in immortalized PKCε- and PKD3-deficient MEFs. (<b>A</b>) MEFs of the indicated genotypes were fixed with 4% PFA and stained with phalloidin and DAPI. The top row shows representative images of primary MEFs (passage number 2–4), and the second row shows representative images of MEFs after immortalisation (&gt;10 passages). The experimental details are described in <a href="#sec2-biomedicines-13-00345" class="html-sec">Section 2</a>. Scale bar, 24 μm. (<b>B</b>) The graph shows a calculation of single-nucleated versus double/multi-nucleated MEFs of various genotypes and compares primary with immortalized cells. The genotypes are indicated below, the primary values are shown in blue, the immortalized values are shown in red and the error bars represent the standard deviation; * indicates <span class="html-italic">p</span>-values 0.000072 for wt/PKCε<sup>-/-</sup> and 0.000028 for wt/PKD3<sup>-/-</sup> by <span class="html-italic">t</span>-test.</p> Full article ">Figure 2
<p>Localisation of protein kinase D during cytokinesis: The top row shows wild-type MEFs expressing PKD3/GFP, the 2nd row shows PKCε<sup>-/-</sup> MEFs expressing PKD3/GFP, the 3rd row shows PKD3<sup>-/-</sup> MEFs expressing PKD3/GFP, and the 4th and 5th rows show PKD3<sup>-/-</sup> MEFs expressing either PKD1/GFP or PKD2/GFP as indicated. All MEFs were fixed with 4% PFA and stained with phalloidin and DAPI. All experimental details are described in <a href="#sec2-biomedicines-13-00345" class="html-sec">Section 2</a>. Representative images of MEFs during cytokinesis are indicated by the presence of the cleavage furrow. The arrows indicate the position of the cleavage furrow, and the expression of the exogenous proteins is shown in <a href="#app1-biomedicines-13-00345" class="html-app">Supplementary Figure S2</a>; scale bar, 48 μm.</p> Full article ">Figure 3
<p>Characterisation of functional PKD3 mutations and their dynamic behavior during cytokinesis: (<b>A</b>) The top row depicts wild-type MEFs expressing a PKD3.DN/GFP construct, while the second row shows PKCε<sup>-/-</sup> MEFs expressing a PKD3.CA/GFP construct. MEFs of the indicated genotypes were fixed with 4% PFA and stained with phalloidin and DAPI. All experimental details are described in <a href="#sec2-biomedicines-13-00345" class="html-sec">Section 2</a>. Representative images of MEFs during cytokinesis are indicated by the presence of the cleavage furrow. (<b>B</b>) The graph shows a calculation of single-nucleated versus double/multi-nucleated MEFs of the wild-type and PKCε<sup>-/-</sup> genotype expressing either PKD3.DN or PKD3.CA constructs, as indicated. The error bars represent the standard deviation; * indicates <span class="html-italic">p</span>-values 0.002257 for wt/PKD3.DN and 0.004046 for PKCε<sup>-/-</sup>/PKD3.CA by <span class="html-italic">t</span>-test. (<b>C</b>) Representative images of wild-type MEFs expressing PKD3/GFP at various stages during cytokinesis are shown. The top row indicates an early phase of cytokinesis with a cleavage furrow staining of PKD3, the middle row demonstrates a midbody staining of PKD3 and the bottom row shows a late stage of cytokinesis with a split PKD3 domain at the midbody. The higher-magnification inset shows the cleavage furrow area in detail; scale bar, 48 μm.</p> Full article ">Figure 4
<p>Analysis of PKD3 activity in correlation to PKD3 and RhoA localisation during cytokinesis: (<b>A</b>) Cells expressing PKD3/GFP were fixed with 4% PFA and co-stained with an anti-phospho Ser 730/734 PKD antibody and DAPI at various stages of the cytokinesis. The top row represents an early stage of cytokinesis with PKD3 localized at the newly established cleavage furrow. The middle row indicates PKD3/GFP as a single spot at the midbody area overlapping with the phospho-PKD signal. The bottom row depicts late -stage cytokinesis with a split PKD3/GFP signal at the midbody area that overlaps with the phospho-PKD signal. All higher magnification inserts represent the furrow area of the corresponding merge, as indicated. (<b>B</b>) Cells expressing PKD3/GFP were fixed with 1% TCA and co-stained with an anti-RhoA antibody and DAPI. The first row represents a very early stage of cytokinesis, while the second row shows a more progressed stage when a cleavage furrow has already been established. Higher magnification of the first row shows the equatorial cortex, while the cleavage furrow is shown in the second blow up. (<b>C</b>) Wild-type MEFs were co-stained with anti-RhoA and anti-phospho-PKD antibodies. The top row indicates a stage when phospho-PKD is localized at the midbody, and the bottom row represents a stage when the phospho-PKD signal is split at the midbody. The high-magnification inserts represent the furrow area of the corresponding merge, as indicated. Scale bar, 48 μm.</p> Full article ">Figure 5
<p>RhoA activity is altered in PKCε- and PKD3-deficient MEFs: (<b>A</b>) RhoA<sup>+</sup> cells were analyzed during cytokinesis. The y-axis indicates percentage of cells, dark gray represents RhoA<sup>+</sup> cells, light gray represents RhoA<sup>−</sup> cells, the corresponding genotypes are indicated below and the error bars represent the standard deviation; * indicates <span class="html-italic">p</span>-values of 0.00383 for RhoA<sup>+</sup> cells in wt/PKCε<sup>-/-</sup> and 0.00471 in wt/PKD3<sup>-/-</sup> by <span class="html-italic">t</span>-test. (<b>B</b>) Wild-type and PKD3-deficient MEFs were assessed for RhoA activity using an activated RhoA pull down following nocodazole treatment (0, 1 and 3 h).</p> Full article ">
14 pages, 2738 KiB  
Article
Farnesol Inhibits PI3 Kinase Signaling and Inflammatory Gene Expression in Primary Human Renal Epithelial Cells
by Aline Müller, Maria Lozoya, Xiaoying Chen, Volkmar Weissig and Mahtab Nourbakhsh
Biomedicines 2023, 11(12), 3322; https://doi.org/10.3390/biomedicines11123322 - 15 Dec 2023
Cited by 3 | Viewed by 1716
Abstract
Chronic inflammation and elevated cytokine levels are closely associated with the progression of chronic kidney disease (CKD), which is responsible for the manifestation of numerous complications and mortality. In addition to conventional CKD therapies, the possibility of using natural compounds with anti-inflammatory potential [...] Read more.
Chronic inflammation and elevated cytokine levels are closely associated with the progression of chronic kidney disease (CKD), which is responsible for the manifestation of numerous complications and mortality. In addition to conventional CKD therapies, the possibility of using natural compounds with anti-inflammatory potential has attracted widespread attention in scientific research. This study aimed to study the potential anti-inflammatory effects of a natural oil compound, farnesol, in primary human renal proximal tubule epithelial cell (RPTEC) culture. Farnesol was encapsulated in lipid-based small unilamellar vesicles (SUVs) to overcome its insolubility in cell culture medium. The cell attachment of empty vesicles (SUVs) and farnesol-loaded vesicles (farnesol-SUVs) was examined using BODIPY, a fluorescent dye with hydrophobic properties. Next, we used multiple protein, RNA, and protein phosphorylation arrays to investigate the impact of farnesol on inflammatory signaling in RPTECs. The results indicated that farnesol inhibits TNF-α/IL-1β-induced phosphorylation of the PI3 kinase p85 subunit and subsequent transcriptional activation of the inflammatory genes TNFRSF9, CD27, TNFRSF8, DR6, FAS, IL-7, and CCL2. Therefore, farnesol may be a promising natural compound for treating CKD. Full article
(This article belongs to the Special Issue Signaling of Protein Kinases in Development and Disease)
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Figure 1

Figure 1
<p>Equal uptake of SUVs and farnesol-SUVs by RPTECs. RPTECs were either not treated (control) or were treated with farnesol, SUVs, or farnesol-SUVs as indicated on the top of each panel. RPTECs were stained using BODIPY. All transmitted light (TL, <b>upper panel</b>) or fluorescence (FL, <b>lower panel</b>) images were captured using the same magnification and exposure times. Scale bars indicate 50 μm.</p> Full article ">Figure 2
<p>Abundance and oxidative activity of mitochondria in RPTECs. RPTECs were left untreated (control) or treated with SUVs or farnesol-SUVs, as indicated at the top of each panel. Mitochondria were stained using MitoTracker Red (<b>upper panel</b>) or MitoTracker Orange (<b>lower panel</b>). The presented images were captured at the same settings and exposure times and are representative of three independent sets of experiments. Scale bars indicate 50 μm.</p> Full article ">Figure 3
<p>Farnesol-SUVs inhibit the TNF-α/IL-1β-induced expression of inflammatory proteins. RPTECs were left untreated or treated with SUV or farnesol-SUV for at least 48 h and then left unstimulated or stimulated with TNF-α /IL-1β for another 24 h. Cell extracts were analyzed for 55 chemokines/cytokines in triplicate. The relative protein expression was obtained through the normalization of the level of each protein to the total cell extract in each experiment (y-axis). The expression levels of seven proteins, TNFRSF9, CD27, TNFRSF8, DR6, FAS, IL-7, and CCL2, were inhibited by farnesol-SUV in RPTECS. The results are presented as the mean ± SD of the relative expression of each protein designated at the top of each diagram. Statistical significance was calculated using one-way ANOVA with the Tukey-Kramer multiple comparison test (TNFRSF9, CD27, FAS, IL-7, and CCL2) or Kruskal-Wallis test with Dunn’s multiple comparison test (TNFRSF8 and DR6) where appropriate. For the sake of simplicity, only the <span class="html-italic">p</span> values of significance for differences between TNF-α /IL-1β-stimulated cells, which were treated with farnesol-SUV or left untreated, are shown (asterisks). The differences between unstimulated (Ctrl, SUV, Farnesol-SUV) and respective TNF-α/IL-1β-stimulated cells (TNF-α/IL-1β, TNF-α/IL-1β+SUV, TNF-α/IL-1β+Farnesol-SUV) were &lt;0.05. The differences between unstimulated cells (Ctrl, SUV, Farnesol-SUV) and between untreated and SUV-treated TNF-α/IL-1β-stimulated cells (TNF-α/IL-1β, TNF-α/IL-1β+SUV) were insignificant. <span class="html-italic">p</span> ≤ 0.001 (***). <span class="html-italic">p</span> ≤ 0.0001 (****).</p> Full article ">Figure 4
<p>Farnesol inhibits TNF-α/IL-1β-mediated upregulation of inflammatory genes. RPTECs were left untreated or treated with SUV or farnesol-SUV for at least 48 h and then left unstimulated or stimulated with TNF-α/IL-1β for another 6 h. RPTECs were harvested, and total RNA was analyzed in triplicate using multiplex mRNA quantification to determine the mRNA expression levels of TNFRSF9, CD27, TNFRSF8, DR6, FAS, IL-7, and CCL2 that were normalized to the mRNA levels of GAPDH and HPRT1. The results are presented as the mean ± SD of the relative expression of mRNAs as indicated at the top of each diagram in three independent experiments. Statistical significance was calculated using one-way ANOVA with the Tukey-Kramer multiple comparison test. For the sake of simplicity, only the <span class="html-italic">p</span> value of significance for differences between TNF-α /IL-1β-stimulated cells which were treated with farnesol-SUV or left untreated are shown (asterisks). The differences between unstimulated (Ctrl, SUV, Farnesol-SUV) and respective TNF-α/IL-1β-stimulated cells (TNF-α/IL-1β, TNF-α/IL-1β+SUV, TNF-α/IL-1β+Farnesol-SUV) were &lt;0.001. The differences between unstimulated cells (Ctrl, SUV, Farnesol-SUV) and between untreated and SUV-treated TNF-α/IL-1β-stimulated cells (TNF-α/IL-1β, TNF-α/IL-1β+SUV) were insignificant. <span class="html-italic">p</span> ≤ 0.001 (***). <span class="html-italic">p</span> ≤ 0.0001 (****).</p> Full article ">Figure 5
<p>Farnesol inhibits the TNF-α/IL-1β-induced phosphorylation of signaling proteins. (<b>a</b>) RPTECs were left untreated or treated with farnesol-SUV and then stimulated with TNF-α/IL-1β for 0.5 h. The level of phosphorylated proteins was analyzed using preconfigured arrays of specific antibodies, each mounted as six replicates in equal amounts. The signals from SUV-farnesol-treated RPTECs were compared to the signals from control cells to obtain the relative inhibition of protein phosphorylation (y-axis) by farnesol-SUVs. Data are presented as the mean ± SD of six replicates. Statistical significance was calculated using one-sample <span class="html-italic">t</span> test (IκB-β (P-Ser23), PKR (P-Thr466), PI3K p85 (P-Tyr199/467), RAS-GRF1 (P-Ser916), PKCζ (P-Thr410), MKK6 (P-Serr207)) or Wilcoxon signed rank test (PAK2 (P-Ser192), PKCΔ (P-Ser645), HDAC5 (P-Ser207), NFκB-p65 (P-Ser311), IKK-a/b (P-Ser180/181)), as appropriate. (<b>b</b>) The diagram shows the mean integrated signal density of the PI3K p85 subunit or its phosphorylated isoforms, PI3K p85 (P-Tyr607) and PI3K p85 (P-Tyr199/467) (y-axis), in TNF-α/IL-1β-stimulated RPTECs that were left untreated (white bars) or treated with farnesol-SUVs (gray bars) before. Statistical significance was calculated using an unpaired <span class="html-italic">t</span> test or Mann-Whitney test, as appropriate. <span class="html-italic">p</span> ≤ 0.05 (**). <span class="html-italic">p</span> ≤ 0.0001 (****).</p> Full article ">Figure 6
<p>PI3K and its downstream signaling proteins. In unstimulated cells, the p110 catalytic subunit of PI3K is stabilized by dimerization with the regulatory p85 subunit. Upon activation, the p85 subunit becomes phosphorylated and releases p110, which leads to the production of PIP3 and the activation of the signaling kinases AKT and PDK1. AKT and PDK1 induce a cascade of downstream phosphorylation. Farnesol inhibits the phosphorylation of different members of the PI3K signaling pathway (gray ovals).</p> Full article ">
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