Biology subjects

Cobb, M. H.

Publications and source records attributed to Cobb, M. H..

12 recordsLinked to original sources

bioRxiv2025

Coordination of nuclear RNA processing by speckle-localized kinase TAOK2

Nuclear speckles are membraneless organelles that act as active splicing hubs especially at sites of high transcription. Emerging views of this dynamic subnuclear structure place it as a hub of RNA processing, impacting steps from transcription to export. To manage this complex microcosm of RNA metabolism, nuclear speckles also require kinases and phosphorylation to execute their functions. The nuclear speckle-localized kinase, TAOK2, mediates the splicing and export of viral transcripts at the nuclear speckle, but its role in the processing of cellular transcripts was unknown. We used siRNA knockdown of TAOK2 and assessed RNA transcripts in both whole cell and nucleocytoplasmic fractions to characterize the complete endogenous effects of TAOK2. We found that TAOK2 knockdown impacts over 10% of the transcriptome, through changes in alternative splicing, export and transcript abundance. Cellular and biochemical phosphoproteomics further revealed nuclear speckle scaffolding proteins SRRM1 and SRRM2 as potential direct phosphorylation targets of TAOK2, mediating its large effects on speckle integrity and speckle-localized splicing. Indeed, knockdown of TAOK2 perturbs almost all speckle-resident serine/arginine (SR)-rich proteins while leaving heterogeneous ribonucleoproteins unperturbed. Altogether, we propose that phosphorylation of SRRM1/2 by TAOK2 plays a structural maintenance role that impacts SR protein-driven exon inclusion at the nuclear speckle.

molecular biology↗

bioRxiv2024

SMURF1/2 are novel regulators of WNK1 stability

Angiogenesis is essential for remodeling and repairing existing vessels, and this process requires signaling pathways including those controlled by transforming growth factor beta (TGF-{beta}). We have previously reported crosstalk between TGF-{beta} and the protein kinase With No lysine (K) 1 (WNK1). Homozygous disruption of the gene encoding WNK1 results in lethality in mice near embryonic day E12 due to impaired angiogenesis and this defect can be rescued by endothelial-specific expression of an activated form of the WNK1 substrate kinase Oxidative Stress-Responsive 1 (OSR1). However, molecular processes regulated via a collaboration between TGF-{beta} and WNK1/OSR1 are not well understood. Here we show that WNK1 interacts with the E3 ubiquitin ligases SMURF1/2. In addition, we discovered that WNK1 regulates SMURF1/2 protein stability and vice versa. We also demonstrate that WNK1 activity regulates TGF-{beta} receptor levels, in turn, controlling TGF-{beta} signaling.

biochemistry↗

bioRxiv2024

Predictive and Experimental Motif Interaction Analysis Identifies Functions of the WNK-OSR1/SPAK Pathway

The WNK-OSR1/SPAK protein kinase signaling pathway regulates ion homeostasis and cell volume, but its other functions are poorly understood. To uncover undefined signaling functions of the pathway we analyzed the binding specificity of the conserved C-terminal (CCT) domains of OSR1 and SPAK to find all possible interaction motifs in human proteins. These kinases bind the core consensus sequences R-F-x-V/I and R-x-F-x-V/I. Motifs were ranked based on sequence, conservation, cellular localization, and solvent accessibility. Out of nearly 3,700 motifs identified, 90% of previously published motifs were within the top 2% of those predicted. Selected candidates (TSC22D1, CAVIN1, ATG9A, NOS3, ARHGEF5) were tested. Upstream kinases WNKs 1-4 and their close relatives, the pseudokinases NRBP1/2, contain CCT-like domains as well. We identified additional distinct motif variants lacking the conserved arginine previously thought to be required, and found that the NRBP1 CCT-like domain binds TSC22D1 via the same motif as OSR1 and SPAK. Our results further highlight the rich and diverse functionality of CCT and CCT-like domains in connecting WNK signaling to cellular processes.

biochemistry↗

bioRxiv2024

WNKs regulate mouse behavior and alter central nervous system glucose uptake and insulin signaling

Certain areas of the brain involved in episodic memory and behavior, such as the hippocampus, express high levels of insulin receptors and glucose transporter-4 (GLUT4) and are responsive to insulin. Insulin and neuronal glucose metabolism improve cognitive functions and regulate mood in humans. Insulin-dependent GLUT4 trafficking has been extensively studied in muscle and adipose tissue, but little work has demonstrated either how it is controlled in insulin-responsive brain regions or its mechanistic connection to cognitive functions. In this study, we demonstrate that inhibition of WNK (With-No-lysine (K)) kinases improves learning and memory in mice. Neuronal inhibition of WNK enhances in vivo hippocampal glucose uptake. Inhibition of WNK enhances insulin signaling output and insulin-dependent GLUT4 trafficking to the plasma membrane in mice primary cortical neurons and hippocampal slices. Therefore, we propose that the extent of neuronal WNK kinase activity has an important influence on learning, memory and anxiety-related behaviors, in part, by modulation of neuronal insulin signaling.

animal behavior and cognition↗

bioRxiv2024

The Cancer Testis Antigen Testis Specific Serine Kinase 6 (TSSK6) is abnormally expressed in colorectal cancer and is essential for oncogenic behaviors

Cancer testis antigens (CTAs) are a collection of proteins whose expression is normally restricted to the gamete, but abnormally activated in a wide variety of tumors. The CTA, Testis specific serine kinase 6 (TSSK6), is essential for male fertility in mice. Functional relevance of TSSK6 to cancer, if any, has not previously been investigated. Here we find that TSSK6 is frequently anomalously expressed in colorectal cancer and patients with elevated TSSK6 expression have reduced relapse free survival. Depletion of TSSK6 from colorectal cancer cells attenuates anchorage independent growth, invasion and growth in vivo. Conversely, overexpression of TSSK6 enhances anchorage independence and invasion in vitro as well as in vivo tumor growth. Notably, ectopic expression of TSSK6 in semi-transformed human colonic epithelial cells is sufficient to confer anchorage independence and enhance invasion. In somatic cells, TSSK6 co-localizes with and enhances the formation of paxillin and tensin positive foci at the cell periphery, suggesting a function in focal adhesion formation. Importantly, TSSK6 kinase activity is essential to induce these tumorigenic behaviors. Our findings establish that TSSK6 exhibits oncogenic activity when abnormally expressed in colorectal cancer cells. Thus, TSSK6 is a previously unrecognized intervention target for therapy, which could exhibit an exceptionally broad therapeutic window.

cancer biology↗

bioRxiv2023

ASCL1-ERK1/2 Axis: ASCL1 restrains ERK1/2 via the dual specificity phosphatase DUSP6 to promote survival of a subset of neuroendocrine lung cancers

The transcription factor achaete-scute complex homolog 1 (ASCL1) is a lineage oncogene that is central for the growth and survival of small cell lung cancers (SCLC) and neuroendocrine non-small cell lung cancers (NSCLC-NE) that express it. Targeting ASCL1, or its downstream pathways, remains a challenge. However, a potential clue to overcoming this challenage has been information that SCLC and NSCLC-NE that express ASCL1 exhibit extremely low ERK1/2 activity, and efforts to increase ERK1/2 activity lead to inhibition of SCLC growth and surival. Of course, this is in dramatic contrast to the majority of NSCLCs where high activity of the ERK pathway plays a major role in cancer pathogenesis. A major knowledge gap is defining the mechanism(s) underlying the low ERK1/2 activity in SCLC, determining if ERK1/2 activity and ASCL1 function are inter-related, and if manipulating ERK1/2 activity provides a new therapeutic strategy for SCLC. We first found that expression of ERK signaling and ASCL1 have an inverse relationship in NE lung cancers: knocking down ASCL1 in SCLCs and NE-NSCLCs increased active ERK1/2, while inhibition of residual SCLC/NSCLC-NE ERK1/2 activity with a MEK inhibitor increased ASCL1 expression. To determine the effects of ERK activity on expression of other genes, we obtained RNA-seq from ASCL1-expressing lung tumor cells treated with an ERK pathway MEK inhibitor and identified down-regulated genes (such as SPRY4, ETV5, DUSP6, SPRED1) that potentially could influence SCLC/NSCLC-NE tumor cell survival. This led us to discover that genes regulated by MEK inhibition suppress ERK activation and CHIP-seq demonstrated these are bound by ASCL1. In addition, SPRY4, DUSP6, SPRED1 are known suppressors of the ERK1/2 pathway, while ETV5 regulates DUSP6. Survival of NE lung tumors was inhibited by activation of ERK1/2 and a subset of ASCL1-high NE lung tumors expressed DUSP6. Because the dual specificity phosphatase 6 (DUSP6) is an ERK1/2-selective phosphatase that inactivates these kinases and has a pharmacologic inhibitor, we focused mechanistic studies on DUSP6. These studies showed: Inhibition of DUSP6 increased active ERK1/2, which accumulated in the nucleus; pharmacologic and genetic inhibition of DUSP6 affected proliferation and survival of ASCL1-high NE lung cancers; and that knockout of DUSP6 "cured" some SCLCs while in others resistance rapidly developed indicating a bypass mechanism was activated. Thus, our findings fill this knowledge gap and indicate that combined expression of ASCL1, DUSP6 and low phospho-ERK1/2 identify some neuroendocrine lung cancers for which DUSP6 may be a therapeutic target.

cancer biology↗

bioRxiv2022

Balancing p38 MAPK Signaling with Proteostasis Mechanisms Supports Tissue Integrity during Aging in C. elegans

Like other p38 MAPKs, C. elegans PMK-1 is activated by phosphorylation during stress responses and inactivated by phosphatases. PMK-1 initiates immune response and blocks development when hyperactivated. Here we show that PMK-1 signaling is essential for tissue homeostasis during aging. Loss of PMK-1 accelerates progressive declines in neuronal integrity and lysosome function compromising longevity. Enhancing p38 signaling with caspase cleavage-resistant PMK-1 protects lysosomal and neuronal integrity extending a youthful phase. The cleavage-resistant PMK-1 mutant behaves oppositely to the pmk-1 null in regulating both transcriptional and protein degradation programs supporting tissue homeostasis. PMK-1 activates a complex transcriptional program and requires UNC-62 (MEIS), FOS/JUN, and DAF-16 (FOXO) transcription factors to regulate lysosome formation which is both cell autonomous and non-cell autonomous. We show that during early aging the absolute phospho-p38 amount is nearly constant but maintained at a small percentage of total p38. The reservoir of non-phospho-p38 diminishes during early aging to enhance signaling without hyperactivation. CED-3 caspase cleavage limits phosphorylated PMK-1 and truncated PMK-1 is rapidly degraded by the proteasome. Modulating phospho-p38 ratio confers dynamic control for tissue-homeostasis without activating stress response to support longevity.

molecular biology↗

bioRxiv2022

Small molecule-mediated insulin hypersecretion induces transient unfolded protein response and loss of function in pancreatic islet beta cells

Pancreatic islet beta cells require a fine-tuned ER stress response for normal function; abnormal ER stress contributes to diabetes pathogenesis. Here, we identified a small molecule, SW016789, with time-dependent effects on beta cell ER stress and function. Acute treatment with SW016789 potentiated nutrient-induced calcium influx and insulin secretion, while chronic exposure to SW016789 transiently induced ER stress and shut down secretory function in a reversible manner. Distinct from the effects of thapsigargin, SW016789 did not affect beta cell viability or apoptosis, potentially due to a rapid induction of adaptive genes, weak signaling through the eIF2 kinase PERK, and lack of oxidative stress gene Txnip induction. We determined that SW016789 acted upstream of voltage-dependent calcium channels (VDCCs) and potentiated nutrient- but not KCl-stimulated calcium influx. Measurements of metabolomics, oxygen consumption rate, and G protein-coupled receptor signaling did not explain the potentiating effects of SW016789. In chemical co-treatment experiments we discovered synergy between SW016789 and activators of protein kinase C (PKC) and VDCCs, suggesting involvement of these pathways in the mechanism of action. Finally, chronically elevated calcium influx was required for the inhibitory impact of SW016789, as blockade of VDCCs protected human islets and MIN6 beta cells from hypersecretion-induced dysfunction. We conclude that beta cells undergoing this type of pharmacological hypersecretion have the capacity to suppress their function to mitigate ER stress and avoid apoptosis. These results have the potential to uncover beta cell ER stress mitigation factors and add support to beta cell rest strategies to preserve function.

cell biology↗

bioRxiv2021

WNK1 Enhances Migration and Invasion in Breast Cancer Models

Metastasis is the major cause of mortality in breast cancer patients. Many signaling pathways have been linked to cancer invasiveness, but blockade of few protein components has succeeded in reducing metastasis. Thus, identification of proteins contributing to invasion that are manipulable by small molecules may be valuable in inhibiting spread of the disease. The protein kinase WNK1 (with no lysine (K) 1) has been suggested to induce migration of cells representing a range of cancer types. Analyses of mouse models and patient data have implicated WNK1 as one of a handful of genes uniquely linked to invasive breast cancer. Here we present evidence that inhibition of WNK1 slows breast cancer metastasis. We show that depletion or inhibition of WNK1 reduces migration of several breast cancer cell lines in wound healing assays and decreases invasion in collagen matrices. Furthermore, WNK1 depletion suppresses expression of AXL, a tyrosine kinase implicated in metastasis. Finally, we demonstrate that WNK inhibition in mice attenuates tumor progression and metastatic burden. These data showing reduced migration, invasion, and metastasis upon WNK1 depletion in multiple breast cancer models suggest that WNK1 contributes to the metastatic phenotype and that WNK1 inhibition may offer a therapeutic avenue for attenuating progression of invasive breast cancers.

cancer biology↗

bioRxiv2019

ERK2 phosphorylates the epigenetic regulator CXXC-finger protein 1 (Cfp1)

BackgroundThe Ras-Raf-MEK-ERK signaling pathway is essential for proper development and homeostatic regulation in eukaryotic cells and underlies progression of several types of cancer. Many pathway functions are performed by extracellular signal-regulated kinase (ERK)1 and 2 (ERK1/2), serine/threonine protein kinases of the mitogen-activated protein kinase (MAPK) family that interact with a large number of substrates and are highly active in the nucleus.\n\nResultsWe identified the epigenetic regulator CXXC-finger protein 1 (CFP1) as a protein that interacts with ERK2 on chromatin. CFP1 is involved in multiple aspects of chromatin regulation, including histone methylation and DNA methylation. Here, we demonstrate the overlapping roles for ERK1/2 and CFP1 in regulation of immediate early gene (IEG) induction. Our work suggests multiple modes of co-regulation and demonstrates that CFP1 is required for an optimal signal-dependent response. We also show that CFP1 is an ERK2 substrate in vitro and identify several phosphorylation sites. Furthermore, we provide evidence that Su(var)3-9, Enhancer-of-zeste and Trithorax (Set)1b, a CFP1-interacting histone methylase, is phosphorylated by ERK2 and is regulated by CFP1.\n\nConclusionOur work highlights ERK1/2 interactions with chromatin regulators that contribute to MAPK signaling diversity in the nucleus.

cell biology↗

bioRxiv2018

Anatomy of a pressure sensing protein kinase

Cells respond to hydrostatic pressure to maintain cellular, organ, and organism level functions, yet the direct pressure sensors are largely unknown. Here we show that hydrostatic pressure directly activates With No Lysine(K) kinase-3 (WNK3) 1, a soluble intracellular protein kinase. Using gel filtration we demonstrate that pressure induces a dimer to monomer transition in a construct of the unphosphorylated kinase domain of WNK3 (uWNK3-KDm or uWNK3). The uWNK3 has not been crystallized, but crosslinking data suggest that the uWNK3 dimer corresponds to crystallographically observed dimer of WNK1 (uWNK1-KDm, or uWNK1) 2,3. Sequence alignments with WNKs from species living in different pressure environments and mutational analysis lend further support for this idea. Unique features of the uWNK1 structure suggest a mechanism involving bound water. We further show that hydrostatic pressure activates full-length WNK3 in D. melanogaster tubules.

biophysics↗

bioRxiv2018

Chromomycin A2 potently inhibits glucose-stimulated insulin secretion from pancreatic beta cells.

Enhancers or inhibitors of insulin secretion could become therapeutics as well as lead to the identification of requisite {beta}-cell regulatory pathways and increase our understanding of pancreatic islet function. Toward this goal, we previously used an insulin-linked luciferase that is co-secreted with insulin in MIN6 {beta}-cells to perform a high-throughput natural product screen for chronic effects on glucose-stimulated insulin secretion. Using multiple phenotypic analyses, we identified that one of the top natural product hits, chromomycin A2 (CMA2), potently inhibited insulin secretion through at least three mechanisms: disruption of Wnt signaling, interfering with {beta}-cell gene expression, and suppression of triggering calcium (Ca2+) influx. Chronic treatment with CMA2 largely ablated glucose-stimulated insulin secretion even post-washout, but did not inhibit glucose-stimulated generation of ATP or Ca2+ influx. However, by using the KATP channel-opener diazoxide, we uncovered defects in depolarization-induced Ca2+ influx which may contribute to the suppressed secretory response. Glucose-responsive ERK1/2 and S6 phosphorylation were also disrupted by chronic CMA2 treatment. The FUSION bioinformatic database indicated that the phenotypic effects of CMA2 clustered with a number of Wnt/GSK3 pathway-related genes. Consistently, CMA2 decreased GSK3 phosphorylation and suppressed activation of a {beta}-catenin activity reporter. CMA2 and a related compound mithramycin are described to have DNA-interaction properties, possibly abrogating transcription factor binding to critical {beta}-cell gene promoters. We observed that CMA2, but not mithramycin, suppressed expression of PDX1 and UCN3. However, neither expression of INSI/II nor insulin content was affected by chronic CMA2. The mechanisms of CMA2-induced insulin secretion defects may involve components both proximal and distal to Ca2+ influx. Therefore, CMA2 is an example of a chemical that can simultaneously disrupt {beta}-cell function through both non-cytotoxic and cytotoxic mechanisms. Future applications of CMA2 and similar aureolic acid analogs for disease therapies should consider the potential impacts on pancreatic islet function.

cell biology↗