Upstream Kinase Research Articles

The Hippo kinase cascade regulates a contractile cell behavior and cell density in a close unicellular relative of animals

The genomes of close unicellular relatives of animals encode orthologs of many genes that regulate animal development. However, little is known about the function of such genes in unicellular organisms or the evolutionary process by which these genes came to function in multicellular development. The Hippo pathway, which regulates cell proliferation and tissue size in animals, is present in some of the closest unicellular relatives of animals, including the amoeboid organism Capsaspora owczarzaki. We previously showed that the Capsaspora ortholog of the Hippo pathway nuclear effector Yorkie/YAP/TAZ (coYki) regulates actin dynamics and the three-dimensional morphology of Capsaspora cell aggregates, but is dispensable for cell proliferation control (Phillips et al., 2022). However, the function of upstream Hippo pathway components, and whether and how they regulate coYki in Capsaspora, remained unknown. Here, we analyze the function of the upstream Hippo pathway kinases coHpo and coWts in Capsaspora by generating mutant lines for each gene. Loss of either kinase results in increased nuclear localization of coYki, indicating an ancient, premetazoan origin of this Hippo pathway regulatory mechanism. Strikingly, we find that loss of either kinase causes a contractile cell behavior and increased density of cell packing within Capsaspora aggregates. We further show that this increased cell density is not due to differences in proliferation, but rather actomyosin-dependent changes in the multicellular architecture of aggregates. Given its well-established role in cell density-regulated proliferation in animals, the increased density of cell packing in coHpo and coWts mutants suggests a shared and possibly ancient and conserved function of the Hippo pathway in cell density control. Together, these results implicate cytoskeletal regulation but not proliferation as an ancestral function of the Hippo pathway kinase cascade and uncover a novel role for Hippo signaling in regulating cell density in a proliferation-independent manner.

Abstract 1216: Pan-RAS inhibition by a tumor-targeted biotherapeutic

Abstract Oncogenic RAS signaling drives tumor growth in about 30% of human malignancies. In recent years the first mutant-specific inhibitors of KRAS G12C (sotorasib and adagrasib) entered the clinic. While some durable responses were noted in KRAS-mutant non-small cell lung cancer, the clinical experience of KRAS G12C inhibitors is marked by rapid drug resistance, either intrinsic or acquired, causing re-activation of the MAPK pathway. Common resistance mechanisms include compensatory signaling through the related RAS isoforms NRAS and HRAS, as well as the selection of non-G12C KRAS mutations, among others. The bacterial peptidase RRSP potently cleaves all RAS mutants and isoforms in the conserved Switch I region. We have shown that receptor-mediated intracellular delivery of RRSP using an engineered protein delivery system (RASx) can inactivate all RAS signaling in a cell, causing apoptosis in RAS-addicted tumor cells in vitro and in vivo. Since systemic pan-RAS inhibition is not likely to be tolerated therapeutically, precise tumor targeting is a necessary feature of a true pan-RAS inhibitor. Here, we describe the unique ability of the RASx system to specify the cell surface receptor used by RRSP to enter cells, allowing exquisite targeting and killing of KRAS, NRAS and HRAS-driven tumor cells while sparing healthy tissues. We screened a panel of patient-derived tumor organoids (PDOs), to demonstrate broad tumor cell killing across pancreatic and colorectal cancer-derived PDOs. 97% of KRAS mutant organoids were sensitive to RASx, independent of the specific mutation. Intriguingly, 46% of wildtype RAS PDOs were sensitive to RASx, including organoids with activating mutations in EGFR. In contrast, PDOs with mutations downstream of RAS such as BRAF V600E were resistant to RASx. Indeed, RASx is effective not only against RAS-addicted tumors, but also wildtype RAS tumors driven by upstream receptor tyrosine kinases, potentially expanding the clinical utility of this platform. RASx is a first-in-class, tumor-targeted pan-RAS inhibitor that represents a unique entry into the growing armamentarium against oncogenic RAS. Citation Format: Greg L. Beilhartz, Huazhu Liang, Molly Udaskin, Christine Ng, Nikolina Radulovich, Roman A. Melnyk. Pan-RAS inhibition by a tumor-targeted biotherapeutic [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1216.

Abstract 7346: Optimizing pan-cancer driver pathway analysis from patient transcriptomics and phosphoproteomics

Abstract Alterations in cell signaling pathways drive cancer progression. Such changes can be detected by transcriptomics and proteomics profiling of tumor samples. Although proteomics profiling captures these alterations more directly, RNA-seq transcriptomics remains the most widely available and cost-effective source of data for elucidating tumor-specific cell signaling mechanisms. The eXpression2Kinases (X2K) pipeline is a computational workflow that starts with differentially expressed mRNAs, the identified differentially expressed genes are then used as input for transcription factor enrichment analysis. Then, the top-ranked transcription factors are connected via additional protein-protein interactions from several databases. Finally, the top-ranked transcription factors and the proteins that directly interact and connect these factors are subjected to kinase enrichment analysis. Such analysis identifies protein kinases most likely responsible for the observed changes in gene expression. The X2K pipeline can be applied to analyze tumor-specific transcriptomics to infer upstream regulatory transcription factors, protein intermediates, and kinases that are likely the drivers of cancer progression. While the X2K pipeline infers regulatory pathways from transcriptomics alone, phosphoproteomics can be used to independently validate the inferred kinases, and to calibrate the parameters of the X2K pipeline. Furthermore, this multiomics approach predicts cell signaling pathways with multiple layers of evidence by linking changes observed in the phosphoproteome to changes observed in the transcriptome. Here, we used the transcriptomics and phosphoproteomics profiles from the Clinical Proteomic Tumor Analysis Consortium (CPTAC) pan-cancer dataset, including 1,020 samples across 10 different cancer types, to optimize the parameters of the X2K pipeline and predict pan-cancer as well as tumor-specific signaling pathways. While inferred pathways were distinct between different tumor types, we observed global enrichment of receptor tyrosine kinases and cell cycle kinases. These pathways were subsequently tested for association with patient survival to identify pathways that may be effective therapeutic targets, for example, PTK2 signaling was identified for many lung cancer tumors (LUAD and LSCC) as well as some breast, head & neck squamous. Altogether, the X2K pipeline presents a rational approach to better identify driver regulatory mechanisms from patient-specific profiling of tumors with both transcriptomics and phosphoproteomics. Citation Format: Eden Z. Deng, Giacomo B. Marino, Daniel J. Clarke, Weiping Ma, Pei Wang, Avi Ma'ayan. Optimizing pan-cancer driver pathway analysis from patient transcriptomics and phosphoproteomics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7346.

Abstract 2994: MIR99AHG promotes ERα expression and ER-positive breast cancer cells proliferation via activating Hippo signaling

Abstract Background: Estrogen receptor (ER) positive breast cancer is the most common subtype of breast cancer. Elucidation of the regulatory network in estrogen receptor pathway is critical to develop and deliver potent targeted therapy for ER-positive breast cancer. The Mir-99a-let-7c cluster host gene MIR99AHG is a long non-coding RNA involving in a variety of tumor formation and disease progression processes. However, the biological functions of MIR99AHG in ER-positive breast cancer have not been reported. Methods: The Western-blot, qRT-PCR, Immunofluorescence and fluorescence in-situ hybridization were used to identify potential target genes and regulatory pathway of estrogen receptor α (ERa), which is a key proliferative driver of ER-positive breast cancer cells. The cell proliferation assay, clone formation assay, xenograft tumor model and rescue assay were used to investigate the effect of differential expression of MIR99AHG on the proliferative capacity of ER-positive breast cancer cells MCF7 and T47D. In addition, correlation analysis was performed to verify the relation of the expression of MIR99AHG, LATS1, LATS2, CYR61, CTGF and ESR1 from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) cohorts. And survival analysis was used to predict the prognostic impact of MIR99AHG in breast cancer patients. Results: MIR99AHG expression was significantly higher in ER-positive breast cancer cells MCF7 and T47D than in ER-negative breast cancer cells SKBR3, MDA-MB-231 and BT549. The high expression of MIR99AHG predicted poor prognosis in ER-positive breast cancer patients. In vitro and in vivo assays demonstrated that MIR99AHG positively regulated the proliferation of MCF7 and T47D cells. Importantly, the Hippo signaling was identified to be down-stream mediators of MIR99AHG in modulating ERα transcription. Furthermore, XMU-MP-1, which targets the Hippo signaling upstream kinase MST1/2, was able to reverse the effect of MIR99AHG on MCF7 cell proliferation. Conclusions: MIR99AHG up-regulates ERα expression and promotes proliferation in ER-positive breast cancer cells through activating Hippo signaling pathway. Targeting MIR99AHG/Hippo pathway is a potential strategy for the treatment of ER-positive breast cancer. Citation Format: Wende Wang, Danping Lin, Ziyang Lin, Yangyang Yan, Yusen Qin, Yuanke Liang, Haoyu Lin, De Zeng. MIR99AHG promotes ERα expression and ER-positive breast cancer cells proliferation via activating Hippo signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2994.

Phosphoproteomic Analysis Identified Mutual Phosphorylation of FAK and Src as a Mechanism of Osimertinib Resistance in EGFR-Mutant Lung Cancer

IntroductionOsimertinib is a standard treatment for patients with EGFR-mutant NSCLC. Although some osimertinib resistance mechanisms have been identified, nearly 50% of the mechanisms remain to be elucidated. This study was aimed at identifying non-genetic mechanisms underlying osimertinib resistance. MethodsWe established two osimertinib-resistant cell lines from EGFR mutation-positive PC-9 and HCC827 NSCLC cell lines (PC-9OR and HCC827OR, respectively) using a stepwise method. We compared the phosphoproteomic profiles of the osimertinib-resistant and parental cells using mass spectrometry. Upstream kinases were identified using the application Kinase Enrichment Analysis version 3. ResultsPhosphoproteomic analysis revealed 80 phosphorylation sites that were mutually up-regulated in PC-9OR and HCC827OR cells. The Kinase Enrichment Analysis version 3 analysis identified focal adhesion kinase (FAK) and proto-oncogene tyrosine-protein kinase Src (Src) as upstream kinases of these up-regulated phosphoproteins. The small-interfering RNA–mediated knockdown of FAK reduced Src phosphorylation and that of Src reduced FAK phosphorylation in both cell lines. Furthermore, FAK- or Src-specific small-interfering RNA treatments restored EGFR phosphorylation in PC-9OR and HCC827OR cells. The combination of FAK and Src inhibitors inhibited PC-9OR and HCC827OR cell proliferation in vitro and suppressed tumor growth in a xenograft mouse model. Immunohistochemistry of tumors from patients with EGFR-mutant NSCLC suggested that phosphorylated FAK and Src are involved in initial and acquired resistance to osimertinib. ConclusionsPhosphoproteomic analysis may help elucidate the mechanisms of resistance to molecular-targeted therapies in lung cancer. Mutual phosphorylation of FAK and Src is involved in osimertinib resistance. Thus, FAK and Src inhibition may be novel treatment strategies for osimertinib-resistant NSCLC.

Identifying Differences in Molecular Characteristics Relevant for Remodeling of Periodontal Ligament Stem Cells from the Upper and Lower Jaw.

Periodontal defects' localization affects wound healing and bone remodeling, with faster healing in the upper jaw compared to the lower jaw. While differences in blood supply, innervation, and odontogenesis contribute, cell-intrinsic variances may exist. Few studies explored cell signaling in periodontal ligament stem cells (PDLSC), overlooking mandible-maxilla disparitiesUsing kinomics technology, we investigated molecular variances in PDLSC. Characterization involved stem cell surface markers, proliferation, and differentiation capacities. Kinase activity was analyzed via multiplex kinase profiling, mapping differential activity in known gene regulatory networks. Upstream kinase analysis identified stronger EphA receptor expression in the mandible, potentially inhibiting osteogenic differentiation. The PI3K-Akt pathway showed higher activity in lower-jaw PDLSC. PDLSC from the upper jaw exhibit superior proliferation and differentiation capabilities. Differential activation of gene regulatory pathways in upper vs. lower-jaw PDLSC suggests implications for regenerative therapies.

Precise Readout of MEK1 Proteoforms upon MAPK Pathway Modulation by Individual Ion Mass Spectrometry.

The functions of proteins bearing multiple post-translational modifications (PTMs) are modulated by their modification patterns, yet precise characterization of them is difficult. MEK1 (also known as MAP2K1) is one such example that acts as a gatekeeper of the mitogen-activating protein kinase (MAPK) pathway and propagates signals via phosphorylation by upstream kinases. In principle, top-down mass spectrometry can precisely characterize whole MEK1 proteoforms, but fragmentation methods that would enable the site-specific characterization of labile modifications on 43 kDa protein ions result in overly dense tandem mass spectra. By using the charge-detection method called individual ion mass spectrometry, we demonstrate how complex mixtures of phosphoproteoforms and their fragment ions can be reproducibly handled to provide a "bird's eye" view of signaling activity through mapping proteoform landscapes in a pathway. Using this approach, the overall stoichiometry and distribution of 0-4 phosphorylations on MEK1 was determined in a cellular model of drug-resistant metastatic melanoma. This approach can be generalized to other multiply modified proteoforms, for which PTM combinations are key to their function and drug action.

LKB1 regulates autophagy through AMPK/TOR signaling pathway to alleviate the damage caused by Vibrio alginolyticus infection

LKB1 (liver kinase B1) is a key upstream kinase of AMPK and plays an important role in various cellular activities. While the function and mechanism of LKB1 have been widely reported in the study of tumor, there are few reports on its role in bacterial infectious diseases, especially in shrimp. In the present study, molecular characterization revealed that LvLKB1 has an open reading frame (ORF) of 1266 bp encoding 421 amino acids with a molecular weight of about 48 KDa, including the kinase region, N-terminal regulatory domain and C-terminal regulatory domain. LvLKB1 in hepatopancreas and hemocytes was significantly upregulated after infection with Vibrio alginolyticus (V. alginolyticus). After silencing LvLKB1 gene in Litopenaeus vannamei (L. vannamei) and artificially infecting V. alginolyticus, the survival rate of L. vannamei was significantly decreased. Subsequently, it was found that the expression of inflammatory factors in hepatopancreas and hemocytes of shrimp was up-regulated, and the expression of lipid oxidation factors was decreased after silencing LKB1, leading to the phenomenon of lipid accumulation in hepatopancreas. In order to explore the mechanism, autophagy levels of shrimp were detected after silencing LKB1, which showed that autophagy levels in hepatopancreas and hemocytes were significantly reduced. Further studies conclusively showed that silencing LvLKB1 inhibited AMPK phosphorylation induced by V. alginolyticus infection, thereby activating TOR pathway and inhibiting autophagy in shrimp. These results indicate that LvLKB1 regulates autophagy through AMPK/TOR signaling pathway to alleviate the damage caused by V. alginolyticus infection.

AMPK stimulation inhibits YAP/TAZ signaling to ameliorate hepatic fibrosis

Hepatic fibrosis is driven by the activation of hepatic stellate cells (HSCs). The Hippo pathway and its effectors, YAP and TAZ, are key regulators of HSC activation and fibrosis. However, there is a lack of mechanistic understanding of YAP/TAZ regulation in HSCs. Here we show that AMPK activation leads to YAP/TAZ inhibition and HSC inactivation in vitro, while the expression of a kinase-inactive mutant reversed these effects compared to wild type AMPKɑ1. Notably, the depletion of LATS1/2, an upstream kinase of YAP/TAZ signaling, rescues YAP/TAZ activation, suggesting that AMPK may be mediating YAP/TAZ inhibition via LATS1/2. In the carbon tetrachloride mouse model of fibrosis, pharmacologic activation of AMPK in HSCs inhibits YAP/TAZ signaling and reduces fibrosis. The findings implicate AMPK as a critical regulator of YAP/TAZ signaling and HSC inactivation and highlight AMPK activation as a therapeutic target for the treatment of hepatic fibrosis.

TRIM28 facilitates type I interferon activation by targeting TBK1.

Type I interferons play a fundamental role in innate host defense against viral infections by eliciting the induction of an antiviral gene program that serves to inhibit viral replication. Activation of type I interferon is regulated by the IRF3 transcription factor, which undergoes phosphorylation-dependent activation by the upstream kinase, TBK1, during viral infection. However, the mechanisms by which TBK1 achieves activation to support signaling to IRF3 remain incompletely understood. Here we identified the E3 ubiquitin ligase, tripartite motif containing 28 (TRIM28), as a positive regulator of type I interferon activation by facilitating TBK1 signaling. Genetic deletion of TRIM28 via CRISPR-Cas9 editing resulted in impaired type I interferon activation upon both RNA and DNA virus challenge, corresponding with increased susceptibility to virus infections in TRIM28 knockout cells. Mechanistically, TRIM28 interacted with TBK1 and mediated the assembly of K63-linked ubiquitin chains onto TBK1, a post-translational modification shown to augment TBK1 signal transmission events. TRIM28 knockout cells further displayed defective TBK1 phosphorylation and complex assembly with IRF3, resulting in impaired IRF3 phosphorylation. Altogether, our data demonstrate TBK1 to be a novel substrate for TRIM28 and identify TRIM28 as an essential regulatory factor in controlling innate antiviral immune responses.

Nuclear transport of phosphorylated LanCL2 promotes invadopodia formation and tumor progression of glioblastoma by activating STAT3/Cortactin signaling

IntroductionOur previous study showed that the abscisic acid receptor lanthionine synthetase C-like 2 (LanCL2) is a significant prognostic factor for overall survival in young glioblastoma patients. However, the role of LanCL2 in glioblastoma remains unclear yet. ObjectivesThis study aims to investigate the role of LanCL2 in regulating in-vitro cell invasion and in-vivo tumor progression of glioblastoma and its underlying mechanism. MethodsTyrosine 198 or 295 residue of LanCL2 was mutated using site-directed mutagenesis to block its phosphorylation. The role of LanCL2 in glioblastoma was investigated using transwell or 3D invasion assay, matrix degradation assay and intracranial xenograft model. ResultsThis study showed that nuclear transport of LanCL2 was enhanced by overexpression of LanCL2 or its ligand abscisic acid in glioblastoma cells. Knockdown of LanCL2 suppressed migration, invasion and invadopodia formation of glioblastoma cells, whereas overexpression of wild-type LanCL2 enhanced them. Blocking of Tyr295 residue phosphorylation of LanCL2 impeded its nuclear transport, retarded glioblastoma cell motility and invadopodia formation, and deceased the phosphorylation of Cortactin and STAT3. c-Met was identified as the upstream tyrosine kinase of Tyr295 residue of LanCL2, and inhibition of c-Met markedly suppressed the nuclear transport of LanCL2. Moreover, overexpression of wild-type LanCL2 significantly promoted orthotopic tumor growth of glioblastoma in vivo and led to poor survival of mice with median survival time of 33.5 days, whereas Tyr295 mutation rescued it with median survival time of 49 days. ConclusionOur findings suggested that Tyr295 phosphorylation is crucial to the activation and nuclear transport of LanCL2, as well as invadopodia formation and tumor progression of glioblastoma, providing the evidence of a novel signaling axis c-Met/LanCL2/STAT3/Cortactin and the first observation of the importance of Tyr295 phosphorylation to LanCL2.

SPAK inhibitor ZT-1a attenuates reactive astrogliosis and oligodendrocyte degeneration in a mouse model of vascular dementia.

Astrogliosis and white matter lesions (WML) are key characteristics of vascular contributions to cognitive impairment and dementia (VCID). However, the molecular mechanisms underlying VCID remain poorly understood. Stimulation of Na-K-Cl cotransport 1 (NKCC1) and its upstream kinases WNK (with no lysine) and SPAK (the STE20/SPS1-related proline/alanine-rich kinase) play a role in astrocytic intracellular Na+ overload, hypertrophy, and swelling. Therefore, in this study, we assessed the effect of SPAK inhibitor ZT-1a on pathogenesis and cognitive function in a mouse model of VCID induced by bilateral carotid artery stenosis (BCAS). Following sham or BCAS surgery, mice were randomly assigned to receive either vehicle (DMSO) or SPAK inhibitor ZT-1a treatment regimen (days 14-35 post-surgery). Mice were then evaluated for cognitive functions by Morris water maze, WML by exvivo MRI-DTI analysis, and astrogliosis/demyelination by immunofluorescence and immunoblotting. Compared to sham control mice, BCAS-Veh mice exhibited chronic cerebral hypoperfusion and memory impairments, accompanied by significant MRI DTI-detected WML and oligodendrocyte (OL) death. Increased activation of WNK-SPAK-NKCC1-signaling proteins was detected in white matter tissues and in C3d+ GFAP+ cytotoxic astrocytes but not in S100A10+ GFAP+ homeostatic astrocytes in BCAS-Veh mice. In contrast, ZT-1a-treated BCAS mice displayed reduced expression and phosphorylation of NKCC1, decreased astrogliosis, OL death, and WML, along with improved memory functions. BCAS-induced upregulation of WNK-SPAK-NKCC1 signaling contributes to white matter-reactive astrogliosis, OL death, and memory impairment. Pharmacological inhibition of the SPAK activity has therapeutic potential for alleviating pathogenesis and memory impairment in VCID.

Development of a Small Molecule Downmodulator for the Transcription Factor Brachyury

AbstractBrachyury is an oncogenic transcription factor whose overexpression drives chordoma growth. The downmodulation of brachyury in chordoma cells has demonstrated therapeutic potential, however, as a transcription factor it is classically deemed “undruggable”. Given that direct pharmacological intervention against brachyury has proven difficult, attempts at intervention have instead targeted upstream kinases. Recently, afatinib, an FDA‐approved kinase inhibitor, has been shown to modulate brachyury levels in multiple chordoma cell lines. Herein, we use afatinib as a lead to undertake a structure‐based drug design approach, aided by mass‐spectrometry and X‐ray crystallography, to develop DHC‐156, a small molecule that more selectively binds brachyury and downmodulates it as potently as afatinib. We eliminated kinase‐inhibition from this novel scaffold while demonstrating that DHC‐156 induces the post‐translational downmodulation of brachyury that results in an irreversible impairment of chordoma tumor cell growth. In doing so, we demonstrate the feasibility of direct brachyury modulation, which may further be developed into more potent tool compounds and therapies.

Development of a Small Molecule Downmodulator for the Transcription Factor Brachyury.

Brachyury is an oncogenic transcription factor whose overexpression drives chordoma growth. The downmodulation of brachyury in chordoma cells has demonstrated therapeutic potential, however, as a transcription factor it is classically deemed "undruggable". Given that direct pharmacological intervention against brachyury has proven difficult, attempts at intervention have instead targeted upstream kinases. Recently, afatinib, an FDA-approved kinase inhibitor, has been shown to modulate brachyury levels in multiple chordoma cell lines. Herein, we use afatinib as a lead to undertake a structure-based drug design approach, aided by mass-spectrometry and X-ray crystallography, to develop DHC-156, a small molecule that more selectively binds brachyury and downmodulates it as potently as afatinib. We eliminated kinase-inhibition from this novel scaffold while demonstrating that DHC-156 induces the post-translational downmodulation of brachyury that results in an irreversible impairment of chordoma tumor cell growth. In doing so, we demonstrate the feasibility of direct brachyury modulation, which may further be developed into more potent tool compounds and therapies.

Activation of the YAP/KLF5 transcriptional cascade in renal tubular cells aggravates kidney injury

The Hippo/YAP pathway plays a critical role in tissue homeostasis. Our previous work demonstrated that renal tubular YAP activation induced by double knockout of the upstream Hippo kinases Mst1 and Mst2 (Mst1/2 dKO) promotes tubular injury and renal inflammation under basal conditions. However, the importance of tubular YAP activation remains to be established in injured kidneys in which many other injurious pathways are simultaneously activated. Here, we show that tubular YAP was already activated 6 h after unilateral ureteral obstruction (UUO). Tubular YAP deficiency greatly attenuated tubular cell over-proliferation, tubular injury and renal inflammation induced by UUO or cisplatin. YAP promoted the transcription of the transcription factor KLF5. Consistently, the elevated expression of KLF5 and its target genes in Mst1/2 dKO or UUO kidneys was blocked by ablation of Yap in tubular cells. Inhibition of KLF5 prevented tubular cell over-proliferation, tubular injury and renal inflammation in Mst1/2 dKO kidneys. Therefore, our results demonstrate that tubular YAP is a key player in kidney injury. YAP and KLF5 form a transcriptional cascade, in which tubular YAP activation induced by kidney injury promotes KLF5 transcription. Activation of this cascade induces tubular cell over-proliferation, tubular injury and renal inflammation.

Distinct functional constraints driving conservation of the cofilin N-terminal regulatory tail

Cofilin family proteins have essential roles in remodeling the cytoskeleton through filamentous actin depolymerization and severing. The short, unstructured N-terminal region of cofilin is critical for actin binding and harbors the major site of inhibitory phosphorylation. Atypically for a disordered sequence, the N-terminal region is highly conserved, but specific aspects driving this conservation are unclear. Here, we screen a library of 16,000 human cofilin N-terminal sequence variants for their capacity to support growth in S. cerevisiae in the presence or absence of the upstream regulator LIM kinase. Results from the screen and biochemical analysis of individual variants reveal distinct sequence requirements for actin binding and regulation by LIM kinase. LIM kinase recognition only partly explains sequence constraints on phosphoregulation, which are instead driven to a large extent by the capacity for phosphorylation to inactivate cofilin. We find loose sequence requirements for actin binding and phosphoinhibition, but collectively they restrict the N-terminus to sequences found in natural cofilins. Our results illustrate how a phosphorylation site can balance potentially competing sequence requirements for function and regulation.

CURTAIN—A unique web-based tool for exploration and sharing of MS-based proteomics data

To facilitate analysis and sharing of mass spectrometry (MS)-based proteomics data, we created online tools called CURTAIN (https://curtain.proteo.info) and CURTAIN-PTM (https://curtainptm.proteo.info) with an accompanying series of video tutorials (https://www.youtube.com/@CURTAIN-me6hl). These are designed to enable non-MS experts to interactively peruse volcano plots and deconvolute primary experimental data so that replicates can be visualized in bar charts or violin plots and exported in publication-ready format. They also allow assessment of overall experimental quality by correlation matrix and profile plot analysis. After making a selection of protein "hits", the user can analyze known domain structure, AlphaFold predicted structure, reported interactors, relative expression as well as disease links. CURTAIN-PTM permits analysis of all identified PTM sites on protein(s) of interest with selected databases. CURTAIN-PTM also links with the Kinase Library to predict upstream kinases that may phosphorylate sites of interest. We provide examples of the utility of CURTAIN and CURTAIN-PTM in analyzing how targeted degradation of the PPM1H Rab phosphatase that counteracts the Parkinson's LRRK2 kinase impacts cellular protein levels and phosphorylation sites. We also reanalyzed a ubiquitylation dataset, characterizing the PINK1-Parkin pathway activation in primary neurons, revealing data of interest not highlighted previously. CURTAIN and CURTAIN-PTM are free to use and open source, enabling researchers to share and maximize the impact of their proteomics data. We advocate that MS data published in volcano plot format be reported containing a shareable CURTAIN weblink, thereby allowing readers to better analyze and exploit the data.

Dietary sodium alters aldosterone's effect on renal sodium transporter expression and distal convoluted tubule remodelling.

Aldosterone is responsible for maintaining volume and potassium homeostasis. Although high salt consumption should suppress aldosterone production, individuals with hyperaldosteronism lose this regulation, leading to a state of high aldosterone despite dietary sodium consumption. The present study examines the effects of elevated aldosterone, with or without high salt consumption, on the expression of key Na+ transporters and remodelling in the distal nephron. Epithelial sodium channel (ENaC) α-subunit expression was increased with aldosterone regardless of Na+ intake. However, ENaC β- and γ-subunits unexpectedly increased at both a transcript and protein level with aldosterone when high salt was present. Expression of total and phosphorylated Na+ Cl- cotransporter (NCC) significantly increased with aldosterone, in association with decreased blood [K+ ], but the addition of high salt markedly attenuated the aldosterone-dependent NCC increase, despite equally severe hypokalaemia. We hypothesized this was a result of differences in distal convoluted tubule length when salt was given with aldosterone. Imaging and measurement of the entire pNCC-positive tubule revealed that aldosterone alone caused a shortening of this segment, although the tubule had a larger cross-sectional diameter. This was not true when salt was given with aldosterone because the combination was associated with a lengthening of the tubule in addition to increased diameter, suggesting that differences in the pNCC-positive area are not responsible for differences in NCC expression. Together, our results suggest the actions of aldosterone, and the subsequent changes related to hypokalaemia, are altered in the presence of high dietary Na+ . KEY POINTS: Aldosterone regulates volume and potassium homeostasis through effects on transporters in the kidney; its production can be dysregulated, preventing its suppression by high dietary sodium intake. Here, we examined how chronic high sodium consumption affects aldosterone's regulation of sodium transporters in the distal nephron. Our results suggest that high sodium consumption with aldosterone is associated with increased expression of all three epithelial sodium channel subunits, rather than just the alpha subunit. Aldosterone and its associated decrease in blood [K+ ] lead to an increased expression of Na-Cl cotransporter (NCC); the addition of high sodium consumption with aldosterone partially attenuates this NCC expression, despite similarly low blood [K+ ]. Upstream kinase regulators and tubule remodelling do not explain these results.

Hydrocortisone cardioprotection in ischaemia/reperfusion injury involves antioxidant mechanisms.

Glucocorticoid (GR) and mineralocorticoid (MR) receptors are highly expressed in cardiac tissue, and both can be activated by corticosteroids. MR activation, in acute myocardial infarction (AMI), worsens cardiac function, and increase NHE activity contributing to the deleterious process. In contrast, effects of GR activation are not fully understood, probably because of the controversial scenario generated by using different doses or potencies of corticosteroids. We tested the hypothesis that an acute dose of hydrocortisone (HC), a low-potency glucocorticoid, in a murine model of AMI could be cardioprotective by regulating NHE1 activity, leading to a decrease in oxidative stress. Isolated hearts from Wistar rats were subjected to regional ischemic protocol. HC (10 nmol/L) was added to the perfusate during early reperfusion. Infarct size and oxidative stress were determined. Isolated papillary muscles from non-infarcted hearts were used to evaluate HC effect on sodium-proton exchanger 1 (NHE1) by analysing intracellular pH recovery from acute transient acidosis. HC treatment decreased infarct size, improved cardiac mechanics, reduced oxidative stress after AMI, while restoring the decreased level of the pro-fusion mitochondrial protein MFN-2. Co-treatment with the GR-blocker Mifepristone avoided these effects. HC reduced NHE1 activity by increasing the NHE1 pro-inhibiting Ser648 phosphorylation site and its upstream kinase AKT. HC restored the decreased AKT phosphorylation and anti-apoptotic BCL-2 protein expression detected after AMI. Our results provide the first evidence that acute HC treatment during early reperfusion induces cardioprotection against AMI, associated with a non-genomic HC-triggered NHE1 inhibition by AKT and antioxidant action that might involves mitochondrial dynamics improvement.

Proline-directed yeast and human MAP kinases phosphorylate the Dot1p/DOT1L histone H3K79 methyltransferase.

Disruptor of telomeric silencing 1 (Dot1p) is an exquisitely conserved histone methyltransferase and is the sole enzyme responsible for H3K79 methylation in the budding yeast Saccharomyces cerevisiae. It has been shown to be highly phosphorylated in vivo; however, the upstream kinases that act on Dot1p are almost entirely unknown in yeast and all other eukaryotes. Here, we used in vitro and in vivo kinase discovery approaches to show that mitogen-activated protein kinase HOG1 (Hog1p) is a bona fide kinase of the Dot1p methyltransferase. In vitro kinase assays showed that Hog1p phosphorylates Dot1p at multiple sites, including at several proline-adjacent sites that are consistent with known Hog1p substrate preferences. The activity of Hog1p was specifically enhanced at these proline-adjacent sites on Dot1p upon Hog1p activation by the osmostress-responsive MAP kinase kinase PBS2 (Pbs2p). Genomic deletion of HOG1 reduced phosphorylation at specific sites on Dot1p in vivo, providing further evidence for Hog1p kinase activity on Dot1p in budding yeast cells. Phenotypic analysis of knockout and phosphosite mutant yeast strains revealed the importance of Hog1p-catalysed phosphorylation of Dot1p for cellular responses to ultraviolet-induced DNA damage. In mammalian systems, this kinase-substrate relationship was found to be conserved: human DOT1L (the ortholog of yeast Dot1p) can be phosphorylated by the proline-directed kinase p38β (also known as MAPK11; the ortholog of yeast Hog1p) at multiple sites in vitro. Taken together, our findings establish Hog1p and p38β as newly identified upstream kinases of the Dot1p/DOT1L H3K79 methyltransferase enzymes in eukaryotes.