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1.
Cell Mol Life Sci ; 75(22): 4235-4250, 2018 Nov.
Article in English | MEDLINE | ID: mdl-29987362

ABSTRACT

PTEN prevents tumor genesis by antagonizing the PI3 kinase/Akt pathway through D3 site phosphatase activity toward PI(3,4)P2 and PI(3,4,5)P3. The structural determinants of this important specificity remain unknown. Interestingly, PTEN shares remarkable homology to voltage-sensitive phosphatases (VSPs) that dephosphorylate D5 and D3 sites of PI(4,5)P2, PI(3,4)P2, and PI(3,4,5)P3. Since the catalytic center of PTEN and VSPs differ markedly only in TI/gating loop and active site motif, we wondered whether these differences explained the variation of their substrate specificity. Therefore, we introduced mutations into PTEN to mimic corresponding sequences of VSPs and studied phosphatase activity in living cells utilizing engineered, voltage switchable PTENCiV, a Ci-VSP/PTEN chimera that retains D3 site activity of the native enzyme. Substrate specificity of this enzyme was analyzed with whole-cell patch clamp in combination with total internal reflection fluorescence microscopy and genetically encoded phosphoinositide sensors. In PTENCiV, mutating TI167/168 in the TI loop into the corresponding ET pair of VSPs induced VSP-like D5 phosphatase activity toward PI(3,4,5)P3, but not toward PI(4,5)P2. Combining TI/ET mutations with an A126G exchange in the active site removed major sequence variations between PTEN and VSPs and resulted in D5 activity toward PI(4,5)P2 and PI(3,4,5)P3 of PTENCiV. This PTEN mutant thus fully reproduced the substrate specificity of native VSPs. Importantly, the same combination of mutations also induced D5 activity toward PI(3,4,5)P3 in native PTEN demonstrating that the same residues determine the substrate specificity of the tumor suppressor in living cells. Reciprocal mutations in VSPs did not alter their substrate specificity, but reduced phosphatase activity. In summary, A126 in the active site and TI167/168 in the TI loop are essential determinants of PTEN's substrate specificity, whereas additional features might contribute to the enzymatic activity of VSPs.


Subject(s)
PTEN Phosphohydrolase/chemistry , PTEN Phosphohydrolase/metabolism , Alanine/chemistry , Animals , CHO Cells , Catalytic Domain , Cell Line , Cricetulus , Mutation , PTEN Phosphohydrolase/genetics , Phosphatidylinositols/metabolism , Substrate Specificity , Threonine/chemistry
2.
Proc Natl Acad Sci U S A ; 110(37): 14972-7, 2013 Sep 10.
Article in English | MEDLINE | ID: mdl-23980138

ABSTRACT

Lumen formation is a critical event in biological tube formation, yet its molecular mechanisms remain poorly understood. Specifically, how lumen expansion is coordinated with other processes of tubulogenesis is not well known, and the role of membrane transporters in tubulogenesis during development has not been adequately addressed. Here we identify a solute carrier 26 (Slc26) family protein as an essential regulator of tubulogenesis using the notochord of the invertebrate chordate Ciona intestinalis as a model. Ci-Slc26aα is indispensable for lumen formation and expansion, but not for apical/luminal membrane formation and lumen connection. Ci-Slc26aα acts as an anion transporter, mediating the electrogenic exchange of sulfate or oxalate for chloride or bicarbonate and electroneutral chloride:bicarbonate exchange. Mutant rescue assays show that this transport activity is essential for Ci-Slc26aα's in vivo function. Our work reveals the consequences and relationships of several key processes in lumen formation, and establishes an in vivo assay for studying the molecular basis of the transport properties of SLC26 family transporters and their related diseases.


Subject(s)
Chloride-Bicarbonate Antiporters/metabolism , Ciona intestinalis/embryology , Ciona intestinalis/metabolism , Amino Acid Sequence , Animals , Chloride-Bicarbonate Antiporters/chemistry , Chloride-Bicarbonate Antiporters/genetics , Ciona intestinalis/genetics , Electrochemistry , Microscopy, Electron, Transmission , Models, Biological , Molecular Sequence Data , Mutant Proteins/genetics , Mutant Proteins/metabolism , Notochord/embryology , Notochord/metabolism , Notochord/ultrastructure , Phylogeny , Protein Structure, Tertiary , Sequence Homology, Amino Acid
3.
J Lipid Res ; 53(11): 2266-74, 2012 Nov.
Article in English | MEDLINE | ID: mdl-22896666

ABSTRACT

In voltage-sensitive phosphatases (VSPs), a transmembrane voltage sensor domain (VSD) controls an intracellular phosphoinositide phosphatase domain, thereby enabling immediate initiation of intracellular signals by membrane depolarization. The existence of such a mechanism in mammals has remained elusive, despite the presence of VSP-homologous proteins in mammalian cells, in particular in sperm precursor cells. Here we demonstrate activation of a human VSP (hVSP1/TPIP) by an intramolecular switch. By engineering a chimeric hVSP1 with enhanced plasma membrane targeting containing the VSD of a prototypic invertebrate VSP, we show that hVSP1 is a phosphoinositide-5-phosphatase whose predominant substrate is PI(4,5)P(2). In the chimera, enzymatic activity is controlled by membrane potential via hVSP1's endogenous phosphoinositide binding motif. These findings suggest that the endogenous VSD of hVSP1 is a control module that initiates signaling through the phosphatase domain and indicate a role for VSP-mediated phosphoinositide signaling in mammals.


Subject(s)
Phosphoric Monoester Hydrolases/metabolism , Animals , CHO Cells , Cricetinae , Electrophysiology , Humans , Microscopy, Fluorescence , Oocytes/metabolism , Phosphatidylinositols/metabolism , Phosphoric Monoester Hydrolases/chemistry , Phosphoric Monoester Hydrolases/genetics , Signal Transduction , Xenopus
4.
Br J Pharmacol ; 165(7): 2244-59, 2012 Apr.
Article in English | MEDLINE | ID: mdl-21951272

ABSTRACT

BACKGROUND AND PURPOSE: DFNA2 is a frequent hereditary hearing disorder caused by loss-of-function mutations in the voltage-gated potassium channel KCNQ4 (Kv7.4). KCNQ4 mediates the predominant K(+) conductance, I(K,n) , of auditory outer hair cells (OHCs), and loss of KCNQ4 function leads to degeneration of OHCs resulting in progressive hearing loss. Here we explore the possible recovery of channel activity of mutant KCNQ4 induced by synthetic KCNQ channel openers. EXPERIMENTAL APPROACH: Whole cell patch clamp recordings were performed on CHO cells transiently expressing KCNQ4 wild-type (wt) and DFNA2-relevant mutants, and from acutely isolated OHCs. KEY RESULTS: Various known KCNQ channel openers robustly enhanced KCNQ4 currents. The strongest potentiation was observed with a combination of zinc pyrithione plus retigabine. A similar albeit less pronounced current enhancement was observed with native I(K,n) currents in rat OHCs. DFNA2 mutations located in the channel's pore region abolished channel function and these mutant channels were completely unresponsive to channel openers. However, the function of a DFNA2 mutation located in the proximal C-terminus was restored by the combined application of both openers. Co-expression of wt and KCNQ4 pore mutants suppressed currents to barely detectable levels. In this dominant-negative situation, channel openers essentially restored currents back to wt levels, most probably through strong activation of only the small fraction of homomeric wt channels. CONCLUSIONS AND IMPLICATIONS: Our data suggest that by stabilizing the KCNQ4-mediated conductance in OHCs, chemical channel openers can protect against OHC degeneration and progression of hearing loss in DFNA2.


Subject(s)
Hearing Loss, Sensorineural/drug therapy , Hearing Loss, Sensorineural/genetics , KCNQ Potassium Channels/genetics , KCNQ Potassium Channels/metabolism , Mutant Proteins/genetics , Mutant Proteins/metabolism , Animals , CHO Cells , Carbamates/pharmacology , Cricetinae , Cricetulus , Hair Cells, Auditory, Outer/drug effects , Hair Cells, Auditory, Outer/metabolism , Hearing Loss, Sensorineural/metabolism , KCNQ Potassium Channels/agonists , KCNQ Potassium Channels/chemistry , Models, Molecular , Mutant Proteins/chemistry , Organometallic Compounds/pharmacology , Patch-Clamp Techniques , Phenylenediamines/pharmacology , Pyridines/pharmacology , Rats , Rats, Wistar , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism
5.
Hum Mol Genet ; 16(23): 2816-33, 2007 Dec 01.
Article in English | MEDLINE | ID: mdl-17761684

ABSTRACT

Emery-Dreifuss muscular dystrophy (EDMD) is a heterogeneous late-onset disease involving skeletal muscle wasting and heart defects caused, in a minority of cases, by mutations in either of two genes encoding the inner nuclear membrane (INM) proteins, emerin and lamins A/C. Nesprin-1 and -2 are multi-isomeric, spectrin-repeat proteins that bind both emerin and lamins A/C and form a network in muscle linking the nucleoskeleton to the INM, the outer nuclear membrane, membraneous organelles, the sarcomere and the actin cytoskeleton. Thus, disruptions in nesprin/lamin/emerin interactions might play a role in the muscle-specific pathogenesis of EDMD. Screening for DNA variations in the genes encoding nesprin-1 (SYNE1) and nesprin-2 (SYNE2) in 190 probands with EDMD or EDMD-like phenotypes identified four heterozygous missense mutations. Fibroblasts from these patients exhibited nuclear morphology defects and specific patterns of emerin and SUN2 mislocalization. In addition, diminished nuclear envelope localization of nesprins and impaired nesprin/emerin/lamin binding interactions were common features of all EDMD patient fibroblasts. siRNA knockdown of nesprin-1 or -2 in normal fibroblasts reproduced the nuclear morphological changes and mislocalization of emerin and SUN2 observed in patient fibroblasts. Taken together, these data suggest that EDMD may be caused, in part, by uncoupling of the nucleoskeleton and cytoskeleton because of perturbed nesprin/emerin/lamin interactions.


Subject(s)
Microfilament Proteins/genetics , Muscular Dystrophy, Emery-Dreifuss/genetics , Nerve Tissue Proteins/genetics , Nuclear Proteins/genetics , Amino Acid Sequence , Base Sequence , Cell Line , Cytoskeletal Proteins , DNA/genetics , DNA Mutational Analysis , Female , Fibroblasts/metabolism , Fibroblasts/ultrastructure , Heterozygote , Humans , Lamins/genetics , Lamins/metabolism , Male , Membrane Proteins/genetics , Membrane Proteins/metabolism , Microfilament Proteins/metabolism , Microscopy, Electron, Transmission , Molecular Sequence Data , Muscle, Skeletal/metabolism , Muscular Dystrophy, Emery-Dreifuss/etiology , Muscular Dystrophy, Emery-Dreifuss/metabolism , Mutation, Missense , Nerve Tissue Proteins/metabolism , Nuclear Envelope/metabolism , Nuclear Proteins/metabolism , Pedigree , RNA, Small Interfering/genetics , Sequence Homology, Amino Acid
6.
Ann Neurol ; 57(1): 148-51, 2005 Jan.
Article in English | MEDLINE | ID: mdl-15622532

ABSTRACT

We report a young girl with a phenotype combining early-onset myopathy and a progeria. She had myopathy and marked axial weakness during the first year of life; progeroid features, including growth failure, sclerodermatous skin changes, and osteolytic lesions, developed later. We identified the underlying cause to be a hitherto unreported de novo missense mutation in the LMNA gene (S143F) encoding the nuclear envelope proteins lamins A and C. Although LMNA mutations have been known to cause Hutchinson-Gilford progeria syndrome and Emery-Dreifuss muscular dystrophy, this is the first report of a patient combining features of these two phenotypes because of a single mutation in LMNA.


Subject(s)
Lamin Type A/genetics , Muscular Diseases/genetics , Mutation, Missense , Progeria/genetics , Blotting, Western/methods , Child , DNA Mutational Analysis/methods , Female , Humans , Lamin Type A/metabolism , Muscles/pathology , Muscles/physiopathology , Muscular Diseases/complications , Muscular Diseases/pathology , Phenylalanine/genetics , Progeria/complications , Progeria/pathology , Serine/genetics , Staining and Labeling/methods
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