Peter Lasch
Dec 1, 2020; 19:2125-2138
Technological Innovation and Resources

Sylwia Struk
Dec 28, 2020; 0:RA119.001766v1-mcp.RA119.001766
Research

Weixian Deng
Dec 22, 2020; 0:RA120.002411v1-mcp.RA120.002411
Research

Madison T Wright
Nov 18, 2020; 0:RA120.002168v1-mcp.RA120.002168
Research

Xinting Zhang
Dec 8, 2020; 0:RA120.002306v1-mcp.RA120.002306
Research

Ka-Won Kang
Nov 30, 2020; 0:RA120.002169v1-mcp.RA120.002169
Research

Kailun Fang
Nov 30, 2020; 0:RA120.002081v1-mcp.RA120.002081
Research

Ryan J Lumpkin
Dec 2, 2020; 0:RA120.002414v1-mcp.RA120.002414
Research

Colin T. McDowell
Nov 24, 2020; 0:RA120.002256v1-mcp.RA120.002256
Research

Matthias Schittmayer
Dec 1, 2020; 19:2104-2114
Research

Alison B. Ross
Nov 30, 2020; 0:R120.002190v1-mcp.R120.002190
Review

Diana Samodova
Dec 1, 2020; 19:2139-2156
Technological Innovation and Resources

The Wnt/β-catenin pathway is one of the major pathways that regulates embryonic development, adult homeostasis, and stem cell self-renewal. In this pathway, transcription factors T-cell factor and lymphoid enhancer factor (TCF/LEF) serve as a key switch to repress or activate Wnt target gene transcription by recruiting repressor molecules or interacting with the β-catenin effector, respectively. It has become evident that the protein stability of the TCF/LEF family members may play a critical role in controlling the activity of the Wnt/β-catenin signaling pathway. However, factors that regulate the stability of TCF/LEFs remain largely unknown. Here, we report that pVHL binding protein 1 (VBP1) regulates the Wnt/β-catenin signaling pathway by controlling the stability of TCF/LEFs. Surprisingly, we found that either overexpression or knockdown of VBP1 decreased Wnt/β-catenin signaling activity in both cultured cells and zebrafish embryos. Mechanistically, VBP1 directly binds to all four TCF/LEF family members and von Hippel-Lindau tumor-suppressor protein (pVHL). Either overexpression or knockdown of VBP1 increases the association between TCF/LEFs and pVHL and then decreases the protein levels of TCF/LEFs via proteasomal degradation. Together, our results provide mechanistic insights into the roles of VBP1 in controlling TCF/LEFs protein stability and regulating Wnt/β-catenin signaling pathway activity.

The extracellular matrix encompasses a reservoir of bioactive macromolecules that modulates a cornucopia of biological functions. A prominent body of work posits matrix constituents as master regulators of autophagy and angiogenesis and provides molecular insight into how these two processes are coordinated. Here, we review current understanding of the molecular mechanisms underlying hyaluronan and HAS2 regulation and the role of soluble proteoglycan in affecting autophagy and angiogenesis. Specifically, we assess the role of proteoglycan-evoked autophagy in regulating angiogenesis via the HAS2-hyaluronan axis and ATG9A, a novel HAS2 binding partner. We discuss extracellular hyaluronan biology and the post-transcriptional and post-translational modifications that regulate its main synthesizer, HAS2. We highlight the emerging group of proteoglycans that utilize outside-in signaling to modulate autophagy and angiogenesis in cancer microenvironments and thoroughly review the most up-to-date understanding of endorepellin signaling in vascular endothelia, providing insight into the temporal complexities involved.

The subcellular localization of Arf family proteins is generally thought to be determined by their corresponding guanine nucleotide exchange factors. By promoting GTP binding, guanine nucleotide exchange factors induce conformational changes of Arf proteins exposing their N-terminal amphipathic helices, which then insert into the membranes to stabilize the membrane association process. Here, we found that the N-terminal amphipathic motifs of the Golgi-localized Arf family protein, Arfrp1, and the endosome- and plasma membrane–localized Arf family protein, Arl14, play critical roles in spatial determination. Exchanging the amphipathic helix motifs between these two Arf proteins causes the switch of their localizations. Moreover, the amphipathic helices of Arfrp1 and Arl14 are sufficient for cytosolic proteins to be localized into a specific cellular compartment. The spatial determination mediated by the Arfrp1 helix requires its binding partner Sys1. In addition, the residues that are required for the acetylation of the Arfrp1 helix and the myristoylation of the Arl14 helix are important for the specific subcellular localization. Interestingly, Arfrp1 and Arl14 are recruited to their specific cellular compartments independent of GTP binding. Our results demonstrate that the amphipathic motifs of Arfrp1 and Arl14 are sufficient for determining specific subcellular localizations in a GTP-independent manner, suggesting that the membrane association and activation of some Arf proteins are uncoupled.

The glucagon receptor (GCGR) activated by the peptide hormone glucagon is a seven-transmembrane G protein–coupled receptor (GPCR) that regulates blood glucose levels. Ubiquitination influences trafficking and signaling of many GPCRs, but its characterization for the GCGR is lacking. Using endocytic colocalization and ubiquitination assays, we have identified a correlation between the ubiquitination profile and recycling of the GCGR. Our experiments revealed that GCGRs are constitutively ubiquitinated at the cell surface. Glucagon stimulation not only promoted GCGR endocytic trafficking through Rab5a early endosomes and Rab4a recycling endosomes, but also induced rapid deubiquitination of GCGRs. Inhibiting GCGR internalization or disrupting endocytic trafficking prevented agonist-induced deubiquitination of the GCGR. Furthermore, a Rab4a dominant negative (DN) that blocks trafficking at recycling endosomes enabled GCGR deubiquitination, whereas a Rab5a DN that blocks trafficking at early endosomes eliminated agonist-induced GCGR deubiquitination. By down-regulating candidate deubiquitinases that are either linked with GPCR trafficking or localized on endosomes, we identified signal-transducing adaptor molecule–binding protein (STAMBP) and ubiquitin-specific protease 33 (USP33) as cognate deubiquitinases for the GCGR. Our data suggest that USP33 constitutively deubiquitinates the GCGR, whereas both STAMBP and USP33 deubiquitinate agonist-activated GCGRs at early endosomes. A mutant GCGR with all five intracellular lysines altered to arginines remains deubiquitinated and shows augmented trafficking to Rab4a recycling endosomes compared with the WT, thus affirming the role of deubiquitination in GCGR recycling. We conclude that the GCGRs are rapidly deubiquitinated after agonist-activation to facilitate Rab4a-dependent recycling and that USP33 and STAMBP activities are critical for the endocytic recycling of the GCGR.

Elevated levels of fasting insulin release and insufficient glucose-stimulated insulin secretion (GSIS) are hallmarks of diabetes. Studies have established cross-talk between integrin signaling and insulin activity, but more details of how integrin-dependent signaling impacts the pathophysiology of diabetes are needed. Here, we dissected integrin-dependent signaling pathways involved in the regulation of insulin secretion in β-cells and studied their link to the still debated autocrine regulation of insulin secretion by insulin/insulin-like growth factor (IGF) 2–AKT signaling. We observed for the first time a cooperation between different AKT isoforms and focal adhesion kinase (FAK)–dependent adhesion signaling, which either controlled GSIS or prevented insulin secretion under fasting conditions. Indeed, β-cells form integrin-containing adhesions, which provide anchorage to the pancreatic extracellular matrix and are the origin of intracellular signaling via FAK and paxillin. Under low-glucose conditions, β-cells adopt a starved adhesion phenotype consisting of actin stress fibers and large peripheral focal adhesion. In contrast, glucose stimulation induces cell spreading, actin remodeling, and point-like adhesions that contain phospho-FAK and phosphopaxillin, located in small protrusions. Rat primary β-cells and mouse insulinomas showed an adhesion remodeling during GSIS resulting from autocrine insulin/IGF2 and AKT1 signaling. However, under starving conditions, the maintenance of stress fibers and the large adhesion phenotype required autocrine IGF2-IGF1 receptor signaling mediated by AKT2 and elevated FAK-kinase activity and ROCK-RhoA levels but low levels of paxillin phosphorylation. This starved adhesion phenotype prevented excessive insulin granule release to maintain low insulin secretion during fasting. Thus, deregulation of the IGF2 and adhesion-mediated signaling may explain dysfunctions observed in diabetes.

Gα proteins promote dynamic adjustments of cell shape directed by actin-cytoskeleton reorganization via their respective RhoGEF effectors. For example, Gα13 binding to the RGS-homology (RH) domains of several RH-RhoGEFs allosterically activates these proteins, causing them to expose their catalytic Dbl-homology (DH)/pleckstrin-homology (PH) regions, which triggers downstream signals. However, whether additional Gα proteins might directly regulate the RH-RhoGEFs was not known. To explore this question, we first examined the morphological effects of expressing shortened RH-RhoGEF DH/PH constructs of p115RhoGEF/ARHGEF1, PDZ-RhoGEF (PRG)/ARHGEF11, and LARG/ARHGEF12. As expected, the three constructs promoted cell contraction and activated RhoA, known to be downstream of Gα13. Intriguingly, PRG DH/PH also induced filopodia-like cell protrusions and activated Cdc42. This pathway was stimulated by constitutively active Gαs (GαsQ227L), which enabled endogenous PRG to gain affinity for Cdc42. A chemogenetic approach revealed that signaling by Gs-coupled receptors, but not by those coupled to Gi or Gq, enabled PRG to bind Cdc42. This receptor-dependent effect, as well as CREB phosphorylation, was blocked by a construct derived from the PRG:Gαs-binding region, PRG-linker. Active Gαs interacted with isolated PRG DH and PH domains and their linker. In addition, this construct interfered with GαsQ227L's ability to guide PRG's interaction with Cdc42. Endogenous Gs-coupled prostaglandin receptors stimulated PRG binding to membrane fractions and activated signaling to PKA, and this canonical endogenous pathway was attenuated by PRG-linker. Altogether, our results demonstrate that active Gαs can recognize PRG as a novel effector directing its DH/PH catalytic module to gain affinity for Cdc42.

AMP-activated protein kinase (AMPK) is a key regulator of energy metabolism that phosphorylates a wide range of proteins to maintain cellular homeostasis. AMPK consists of three subunits: α, β, and γ. AMPKα and β are encoded by two genes, the γ subunit by three genes, all of which are expressed in a tissue-specific manner. It is not fully understood, whether individual isoforms have different functions. Using RNA-Seq technology, we provide evidence that the loss of AMPKβ1 and AMPKβ2 lead to different gene expression profiles in human induced pluripotent stem cells (hiPSCs), indicating isoform-specific function. The knockout of AMPKβ2 was associated with a higher number of differentially regulated genes than the deletion of AMPKβ1, suggesting that AMPKβ2 has a more comprehensive impact on the transcriptome. Bioinformatics analysis identified cell differentiation as one biological function being specifically associated with AMPKβ2. Correspondingly, the two isoforms differentially affected lineage decision toward a cardiac cell fate. Although the lack of PRKAB1 impacted differentiation into cardiomyocytes only at late stages of cardiac maturation, the availability of PRKAB2 was indispensable for mesoderm specification as shown by gene expression analysis and histochemical staining for cardiac lineage markers such as cTnT, GATA4, and NKX2.5. Ultimately, the lack of AMPKβ1 impairs, whereas deficiency of AMPKβ2 abrogates differentiation into cardiomyocytes. Finally, we demonstrate that AMPK affects cellular physiology by engaging in the regulation of hiPSC transcription in an isoform-specific manner, providing the basis for further investigations elucidating the role of dedicated AMPK subunits in the modulation of gene expression.

Mitochondrial DNA (mtDNA) encodes proteins and RNAs that support the functions of mitochondria and thereby numerous physiological processes. Mutations of mtDNA can cause mitochondrial diseases and are implicated in aging. The mtDNA within cells is organized into nucleoids within the mitochondrial matrix, but how mtDNA nucleoids are formed and regulated within cells remains incompletely resolved. Visualization of mtDNA within cells is a powerful means by which mechanistic insight can be gained. Manipulation of the amount and sequence of mtDNA within cells is important experimentally and for developing therapeutic interventions to treat mitochondrial disease. This review details recent developments and opportunities for improvements in the experimental tools and techniques that can be used to visualize, quantify, and manipulate the properties of mtDNA within cells.

Genetic mutations related to ALS, a progressive neurological disease, have been discovered in the gene encoding σ-1 receptor (σ1R). We previously reported that σ1RE102Q elicits toxicity in cells. The σ1R forms oligomeric states that are regulated by ligands. Nevertheless, little is known about the effect of ALS-related mutations on oligomer formation. Here, we transfected NSC-34 cells, a motor neuronal cell line, and HEK293T cells with σ1R-mCherry (mCh), σ1RE102Q-mCh, or nontagged forms to investigate detergent solubility and subcellular distribution using immunocytochemistry and fluorescence recovery after photobleaching. The oligomeric state was determined using crosslinking procedure. σ1Rs were soluble to detergents, whereas the mutants accumulated in the insoluble fraction. Within the soluble fraction, peak distribution of mutants appeared in higher sucrose density fractions. Mutants formed intracellular aggregates that were co-stained with p62, ubiquitin, and phosphorylated pancreatic eukaryotic translation initiation factor-2-α kinase in NSC-34 cells but not in HEK293T cells. The aggregates had significantly lower recovery in fluorescence recovery after photobleaching. Acute treatment with σ1R agonist SA4503 failed to improve recovery, whereas prolonged treatment for 48 h significantly decreased σ1RE102Q-mCh insolubility and inhibited apoptosis. Whereas σ1R-mCh formed monomers and dimers, σ1RE102Q-mCh also formed trimers and tetramers. SA4503 reduced accumulation of the four types in the insoluble fraction and increased monomers in the soluble fraction. The σ1RE102Q insolubility was diminished by σ1R-mCh co-expression. These results suggest that the agonist and WT σ1R modify the detergent insolubility, toxicity, and oligomeric state of σ1RE102Q, which may lead to promising new treatments for σ1R-related ALS.

RAS genes are the most commonly mutated in human cancers and play critical roles in tumor initiation, progression, and drug resistance. Identification of targets that block RAS signaling is pivotal to develop therapies for RAS-related cancer. As RAS translocation to the plasma membrane (PM) is essential for its effective signal transduction, we devised a high-content screening assay to search for genes regulating KRAS membrane association. We found that the tyrosine phosphatase PTPN2 regulates the plasma membrane localization of KRAS. Knockdown of PTPN2 reduced the proliferation and promoted apoptosis in KRAS-dependent cancer cells, but not in KRAS-independent cells. Mechanistically, PTPN2 negatively regulates tyrosine phosphorylation of KRAS, which, in turn, affects the activation KRAS and its downstream signaling. Consistently, analysis of the TCGA database demonstrates that high expression of PTPN2 is significantly associated with poor prognosis of patients with KRAS-mutant pancreatic adenocarcinoma. These results indicate that PTPN2 is a key regulator of KRAS and may serve as a new target for therapy of KRAS-driven cancer.

Mutations in the GUCY2D gene coding for the dimeric human retinal membrane guanylyl cyclase (RetGC) isozyme RetGC1 cause various forms of blindness, ranging from rod dysfunction to rod and cone degeneration. We tested how the mutations causing recessive congenital stationary night blindness (CSNB), recessive Leber's congenital amaurosis (LCA1), and dominant cone–rod dystrophy-6 (CORD6) affected RetGC1 activity and regulation by RetGC-activating proteins (GCAPs) and retinal degeneration-3 protein (RD3). CSNB mutations R666W, R761W, and L911F, as well as LCA1 mutations R768W and G982VfsX39, disabled RetGC1 activation by human GCAP1, -2, and -3. The R666W and R761W substitutions compromised binding of GCAP1 with RetGC1 in HEK293 cells. In contrast, G982VfsX39 and L911F RetGC1 retained the ability to bind GCAP1 in cyto but failed to effectively bind RD3. R768W RetGC1 did not bind either GCAP1 or RD3. The co-expression of GUCY2D allelic combinations linked to CSNB did not restore RetGC1 activity in vitro. The CORD6 mutation R838S in the RetGC1 dimerization domain strongly dominated the Ca2+ sensitivity of cyclase regulation by GCAP1 in RetGC1 heterodimer produced by co-expression of WT and the R838S subunits. It required higher Ca2+ concentrations to decelerate GCAP-activated RetGC1 heterodimer—6-fold higher than WT and 2-fold higher than the Ser838-harboring homodimer. The heterodimer was also more resistant than homodimers to inhibition by RD3. The observed biochemical changes can explain the dominant CORD6 blindness and recessive LCA1 blindness, both of which affect rods and cones, but they cannot explain the selective loss of rod function in recessive CSNB.

Post-transcriptional modifications of pre-mRNAs expand the diversity of proteomes in higher eukaryotes. In the brain, these modifications diversify the functional output of many critical neuronal signal molecules. In this study, we identified a brain-specific A-to-I RNA editing that changed glutamine to arginine (Q/R) at exon 20 and an alternative splicing of exon 4 in Tmem63b, which encodes a ubiquitously expressed osmosensitive cation channel. The channel isoforms lacking exon 4 occurred in ∼80% of Tmem63b mRNAs in the brain but were not detected in other tissues, suggesting a brain-specific splicing. We found that the Q/R editing was catalyzed by Adar2 (Adarb1) and required an editing site complementary sequence located in the proximal 5' end of intron 20. Moreover, the Q/R editing was almost exclusively identified in the splicing isoform lacking exon 4, indicating a coupling between the editing and the splicing. Elimination of the Q/R editing in brain-specific Adar2 knockout mice did not affect the splicing efficiency of exon 4. Furthermore, transfection with the splicing isoform containing exon 4 suppressed the Q/R editing in primary cultured cerebellar granule neurons. Thus, our study revealed a coupling between an RNA editing and a distant alternative splicing in the Tmem63b pre-mRNA, in which the splicing plays a dominant role. Finally, physiological analysis showed that the splicing and the editing coordinately regulate Ca2+ permeability and osmosensitivity of channel proteins, which may contribute to their functions in the brain.

Bone morphogenetic protein-9 (BMP-9) is a circulating cytokine that is known to play an essential role in the endothelial homeostasis and the binding of BMP-9 to the receptor activin-like kinase 1 (ALK-1) promotes endothelial cell quiescence. Previously, using an unbiased screen, we identified ALK-1 as a high-capacity receptor for low-density lipoprotein (LDL) in endothelial cells that mediates its transcytosis in a nondegradative manner. Here we examine the crosstalk between BMP-9 and LDL and how it influences their interactions with ALK-1. Treatment of endothelial cells with BMP-9 triggers the extensive endocytosis of ALK-1, and it is mediated by caveolin-1 (CAV-1) and dynamin-2 (DNM2) but not clathrin heavy chain. Knockdown of CAV-1 reduces BMP-9–mediated internalization of ALK-1, BMP-9–dependent signaling and gene expression. Similarly, treatment of endothelial cells with LDL reduces BMP-9–induced SMAD1/5 phosphorylation and gene expression and silencing of CAV-1 and DNM2 diminishes LDL-mediated ALK-1 internalization. Interestingly, BMP-9–mediated ALK-1 internalization strongly re-duces LDL transcytosis to levels seen with ALK-1 deficiency. Thus, BMP-9 levels can control cell surface levels of ALK-1, via CAV-1, to regulate both BMP-9 signaling and LDL transcytosis.

When the drug Taxol® was approved by the United States Food and Drug Administration in 1993, it was a game changer for cancer patients. The compound, which arrests cell division by preventing the disassembly of tubulin microfibers, has been used over the past three decades to treat millions of cases of breast, lung, and ovarian cancer as well as Kaposi's sarcoma. In 1990, Bristol Myers Squibb applied to trademark the name Taxol, which was approved in 1992, changing the drug's generic name to paclitaxel.At the time that Taxol was entering clinical trials in the late 1970s, it also proved to be a valuable tool for cytoskeletal research. Tubulin had been discovered in the late 1960s, but it was still unclear how the soluble protein dimer polymerized (Fig. 1) to form the long, complex structures of the cytoskeleton.jbc;295/41/13994/F1F1F1Figure 1.Strands of tubulin, a protein in the cell's skeleton, photographed using a high-resolution microscopy technique. Image made by Pakorn Kanchanawong (National University of Singapore) and Clare Waterman (NHLBI, National Institutes of Health).“Back then, people were just discovering the most basic functions of tubulin and how it polymerized, and then they found a drug that affected this,” said Velia Fowler, a cell biologist at the University of Delaware and former Associate Editor at the Journal of Biological Chemistry.The drug and its cytoskeletal activity intersected in the 1981 JBC paper “Taxol-induced polymerization of purified tubulin” (1), the subject of this JBC Classic. In the single-author paper, Nirbhay Kumar, then a postdoctoral fellow at the National...

Priyanka Tripathi
Dec 30, 2020; 0:jlr.RA120001190v1-jlr.RA120001190
Research Articles

Alice Santonastaso
Dec 1, 2020; 61:1687-1696
Research Articles

Stephanie E. Hood
Dec 1, 2020; 61:1720-1732
Research Articles

Martin Prescher
Dec 1, 2020; 61:1605-1616
Research Articles

Frank C. Nichols
Dec 1, 2020; 61:1645-1657
Research Articles

Hideaki Kuge
Dec 1, 2020; 61:1747-1763
Research Articles

Chiara Pavanello
Dec 1, 2020; 61:1784-1788
Patient-Oriented and Epidemiological Research

Elisa Vidal
Dec 1, 2020; 61:1733-1746
Research Articles

Marco De Giorgi
Dec 1, 2020; 61:1675-1686
Research Articles

Montgomery Blencowe
Dec 23, 2020; 0:jlr.RA120000713v1-jlr.RA120000713
Research Articles

Douglas A. Andres
Dec 1, 2020; 61:1537-1537
Images in Lipid Research

Auguste Dargent
Dec 1, 2020; 61:1776-1783
Research Articles

Astrid M. Moerman
Dec 23, 2020; 0:jlr.RA120000974v1-jlr.RA120000974
Research Articles

Ryunosuke Ohkawa
Dec 1, 2020; 61:1577-1588
Research Articles

Genta Kakiyama
Dec 1, 2020; 61:1629-1644
Research Articles

Michael E. Abrams
Dec 1, 2020; 61:1538-1538
Images in Lipid Research

Junhwan Kim
Dec 1, 2020; 61:1707-1719
Research Articles

Thibaut Bourgeois
Dec 11, 2020; 0:jlr.RA120000737v1-jlr.RA120000737
Research Articles

Julia V Busik
Dec 23, 2020; 0:jlr.TR120000981v1-jlr.TR120000981
Thematic Reviews

Aloïs Dusuel
Dec 9, 2020; 0:jlr.RA120000704v1-jlr.RA120000704
Research Articles

Anja Jaeschke
Dec 9, 2020; 0:jlr.RA120001141v1-jlr.RA120001141
Research Articles

Abudukadier Abulizi
Dec 1, 2020; 61:1565-1576
Research Articles

Ying Zou
Dec 1, 2020; 61:1589-1604
Research Articles

Oktawia Nilsson
Nov 17, 2020; 0:jlr.RA120000920v1-jlr.RA120000920
Research Articles