Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 8 de 8
Filter
Add more filters










Database
Language
Publication year range
1.
Mol Cell ; 76(6): 953-964.e6, 2019 12 19.
Article in English | MEDLINE | ID: mdl-31585692

ABSTRACT

Dynamic protein phosphorylation constitutes a fundamental regulatory mechanism in all organisms. Phosphoprotein phosphatase 4 (PP4) is a conserved and essential nuclear serine and threonine phosphatase. Despite the importance of PP4, general principles of substrate selection are unknown, hampering the study of signal regulation by this phosphatase. Here, we identify and thoroughly characterize a general PP4 consensus-binding motif, the FxxP motif. X-ray crystallography studies reveal that FxxP motifs bind to a conserved pocket in the PP4 regulatory subunit PPP4R3. Systems-wide in silico searches integrated with proteomic analysis of PP4 interacting proteins allow us to identify numerous FxxP motifs in proteins controlling a range of fundamental cellular processes. We identify an FxxP motif in the cohesin release factor WAPL and show that this regulates WAPL phosphorylation status and is required for efficient cohesin release. Collectively our work uncovers basic principles of PP4 specificity with broad implications for understanding phosphorylation-mediated signaling in cells.


Subject(s)
Phosphoprotein Phosphatases/genetics , Phosphoprotein Phosphatases/metabolism , Phosphoprotein Phosphatases/ultrastructure , Amino Acid Sequence/genetics , Binding Sites , Conserved Sequence , Crystallography, X-Ray/methods , HEK293 Cells , HeLa Cells , Humans , Phosphorylation , Protein Binding/genetics , Substrate Specificity
2.
Nat Commun ; 10(1): 531, 2019 01 31.
Article in English | MEDLINE | ID: mdl-30705304

ABSTRACT

PGAM5 is a mitochondrial protein phosphatase whose genetic ablation in mice results in mitochondria-related disorders, including neurodegeneration. Functions of PGAM5 include regulation of mitophagy, cell death, metabolism and aging. However, mechanisms regulating PGAM5 activation and signaling are poorly understood. Using electron cryo-microscopy, we show that PGAM5 forms dodecamers in solution. We also present a crystal structure of PGAM5 that reveals the determinants of dodecamer formation. Furthermore, we observe PGAM5 dodecamer assembly into filaments both in vitro and in cells. We find that PGAM5 oligomerization into a dodecamer is not only essential for catalytic activation, but this form also plays a structural role on mitochondrial membranes, which is independent of phosphatase activity. Together, these findings suggest that modulation of the oligomerization of PGAM5 may be a regulatory switch of potential therapeutic interest.


Subject(s)
Cryoelectron Microscopy/methods , Phosphoprotein Phosphatases/metabolism , Phosphoprotein Phosphatases/ultrastructure , Animals , Cell Death/genetics , Cell Death/physiology , Mice , Mitochondrial Membranes/metabolism , Mitochondrial Membranes/ultrastructure , Mitophagy/genetics , Mitophagy/physiology , Polymerization
3.
Biochim Biophys Acta Mol Cell Res ; 1866(1): 153-166, 2019 01.
Article in English | MEDLINE | ID: mdl-30030002

ABSTRACT

Mammalian haloacid dehalogenase (HAD)-type phosphatases have evolved to dephosphorylate a wide range of small metabolites, but can also target macromolecules such as serine/threonine, tyrosine-, and histidine-phosphorylated proteins. To accomplish these tasks, HAD phosphatases are equipped with cap domains that control access to the active site and provide substrate specificity determinants. A number of capped HAD phosphatases impact protein phosphorylation, although structural data are consistent with small metabolite substrates rather than protein substrates. This review discusses the structures, functions and disease implications of the three closely related, capped HAD phosphatases pyridoxal phosphatase (PDXP or chronophin), phosphoglycolate phosphatase (PGP, also termed AUM or glycerol phosphatase) and phospholysine phosphohistidine inorganic pyrophosphate phosphatase (LHPP or HDHD2B). Evidence in support of small metabolite and protein phosphatase activity is discussed in the context of the diversity of their biological functions.


Subject(s)
Inorganic Pyrophosphatase/metabolism , Phosphoprotein Phosphatases/metabolism , Phosphoric Monoester Hydrolases/metabolism , Actin Cytoskeleton/physiology , Animals , Humans , Hydrolases , Inorganic Pyrophosphatase/chemistry , Inorganic Pyrophosphatase/physiology , Neoplasms/metabolism , Neoplasms/physiopathology , Phosphoprotein Phosphatases/chemistry , Phosphoprotein Phosphatases/physiology , Phosphoprotein Phosphatases/ultrastructure , Phosphoric Monoester Hydrolases/chemistry , Phosphoric Monoester Hydrolases/physiology , Phosphorylation , Protein Tyrosine Phosphatases/metabolism
4.
J Mol Biol ; 357(5): 1351-72, 2006 Apr 14.
Article in English | MEDLINE | ID: mdl-16487973

ABSTRACT

A eukaryotic-type signaling system in group A Streptococcus (GAS) was identified and characterized. This system comprises primarily the products of two co-transcribed genes, a eukaryotic-type Ser/Thr kinase (SP-STK) and phosphatase (SP-STP) and their endogenous substrate histone-like protein (SP-HLP). Enzyme activities of SP-STK and SP-STP primarily depended on Mn(2+). The site on the substrate for reversible phosphorylation by these enzymes was found to be only the threonine residue. Using specific antibodies generated against these proteins, SP-STK was found to be membrane-associated with its N-terminal kinase domain facing the cytoplasm and its C-terminal repeat domain outside the membrane and cell-wall associated. Further, SP-STP, primarily a cytoplasmic protein, was found to be a major secretory protein of GAS and essential for bacterial survival. Three isogenic mutants, lacking either the entire SP-STK, or one of its two domains, were found displaying distinct pleiotropic effects on growth, colony morphology, cell division/septation, surface protein/virulence factor expression, bacterial ability to adhere to and invade human pharyngeal cells, and resist phagocytosis by human neutrophils. In addition to these properties, the ability of these three proteins to modulate the expression of the major virulence factors, the M protein and the capsule, indicates that these proteins are structurally and functionally distinct from the kinases and phosphatases described in other microorganisms and play a key role in GAS pathogenesis.


Subject(s)
Bacterial Proteins/metabolism , Phosphoprotein Phosphatases/metabolism , Protein Serine-Threonine Kinases/metabolism , Signal Transduction/physiology , Streptococcus pyogenes/enzymology , Amino Acid Sequence , Bacterial Adhesion , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Cell Line , Cell Shape , Humans , Molecular Sequence Data , Phagocytosis , Pharynx/cytology , Pharynx/microbiology , Phosphoprotein Phosphatases/chemistry , Phosphoprotein Phosphatases/genetics , Phosphoprotein Phosphatases/ultrastructure , Protein Serine-Threonine Kinases/chemistry , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/ultrastructure , Protein Structure, Tertiary , Sequence Alignment , Streptococcus pyogenes/cytology , Streptococcus pyogenes/genetics , Substrate Specificity , Transcription, Genetic
5.
Trends Pharmacol Sci ; 18(4): 124-34, 1997 Apr.
Article in English | MEDLINE | ID: mdl-9149541

ABSTRACT

Calcineurin is a calcium (Ca2+)/calmodulin (CaM)-dependent protein phosphatase that has been shown to regulate the activity of ion channels, neurotransmitter and hormone release, synaptic plasticity and gene transcription. At glutamatergic synapses, the inhibition of calcineurin with immunosuppressant drugs has been reported to enhance both the presynaptic release of glutamate and postsynaptic responsiveness. Several other ligand- and voltage-gated ion channels are negatively regulated by calcineurin. Hormone release in insulin-secreting pancreatic beta cells and pituitary corticotrope tumour (AtT20) cells is also negatively regulated by calcineurin. In this article, Jerrel Yakel discusses the evidence that calcineurin plays a vital role in regulating neuronal excitability and hormone release.


Subject(s)
Calmodulin-Binding Proteins/physiology , Hormones/metabolism , Ion Channels/physiology , Neurotransmitter Agents/metabolism , Phosphoprotein Phosphatases/physiology , Synaptic Transmission/physiology , Transcription, Genetic/physiology , Animals , Calcineurin , Calmodulin-Binding Proteins/chemistry , Calmodulin-Binding Proteins/ultrastructure , Endocytosis/physiology , Exocytosis/physiology , Insulin/metabolism , Insulin Secretion , Ion Channel Gating/physiology , Islets of Langerhans/cytology , Islets of Langerhans/metabolism , Molecular Weight , Neuronal Plasticity/physiology , Patch-Clamp Techniques , Phosphoprotein Phosphatases/chemistry , Phosphoprotein Phosphatases/ultrastructure , Receptors, Glutamate/metabolism , Transcription, Genetic/genetics
6.
Microsc Res Tech ; 34(1): 77-86, 1996 May 01.
Article in English | MEDLINE | ID: mdl-8859891

ABSTRACT

Calcineurin is a heterodimeric phosphatase involved in the signal transduction of antigen-activated T cells. Coexpression of its two subunits, the regulatory subunit from human and the catalytic subunit from Neurospora crassa in cultured insect cells using the baculovirus expression system results in the formation of very large crystals in the cytoplasm. The crystals are formed initially in vesicles, but their subsequent growth appears to be uninhibited and continues without the need of an enclosing membrane until the host cell lyses. Although these in vivo crystals are low in population, ranging only 0-3 per cell, they are extremely large, over 10 mu m in some cases. Biochemical assays confirm their calcineurin origin, with the regulatory subunit incorporated being myristoylated, although both the myristoylated and unmyristoylated forms are expressed. The lattice structure of the in vivo crystals, with a spacing of 5.5 nm, is preserved with the regular electron microscopic (EM) specimen preparation procedure.


Subject(s)
Calmodulin-Binding Proteins/genetics , Calmodulin-Binding Proteins/metabolism , Gene Expression Regulation, Enzymologic , Phosphoprotein Phosphatases/genetics , Phosphoprotein Phosphatases/metabolism , Animals , Baculoviridae/genetics , Calcineurin , Calmodulin-Binding Proteins/ultrastructure , Cell Nucleus/ultrastructure , Cells, Cultured , Crystallization , Cytoplasm/metabolism , Electrophoresis, Polyacrylamide Gel , Genetic Vectors , Humans , Insecta , Microscopy, Electron , Neurospora crassa/genetics , Phosphoprotein Phosphatases/ultrastructure , Polymerase Chain Reaction , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Recombination, Genetic
7.
Nature ; 378(6557): 641-4, 1995 Dec 07.
Article in English | MEDLINE | ID: mdl-8524402

ABSTRACT

Calcineurin (CaN) is a calcium- and calmodulin-dependent protein serine/threonine phosphate which is critical for several important cellular processes, including T-cell activation. CaN is the target of the immunosuppressive drugs cyclosporin A and FK506, which inhibit CaN after forming complexes with cytoplasmic binding proteins (cyclophilin and FKBP12, respectively). We report here the crystal structures of full-length human CaN at 2.1 A resolution and of the complex of human CaN with FKBP12-FK506 at 3.5 A resolution. In the native CaN structure, an auto-inhibitory element binds at the Zn/Fe-containing active site. The metal-site geometry and active-site water structure suggest a catalytic mechanism involving nucleophilic attack on the substrate phosphate by a metal-activated water molecule. In the FKBP12-FK506-CaN complex, the auto-inhibitory element is displaced from the active site. The site of binding of FKBP12-FK506 appears to be shared by other non-competitive inhibitors of calcineurin, including a natural anchoring protein.


Subject(s)
Adaptor Proteins, Signal Transducing , Calmodulin-Binding Proteins/chemistry , Carrier Proteins/metabolism , DNA-Binding Proteins/metabolism , Heat-Shock Proteins/metabolism , Phosphoprotein Phosphatases/chemistry , Tacrolimus/metabolism , A Kinase Anchor Proteins , Amino Acid Sequence , Binding Sites , Calcineurin , Calcium/metabolism , Calmodulin-Binding Proteins/antagonists & inhibitors , Calmodulin-Binding Proteins/metabolism , Calmodulin-Binding Proteins/ultrastructure , Carrier Proteins/chemistry , Crystallization , Crystallography, X-Ray , DNA-Binding Proteins/chemistry , Enzyme Inhibitors/metabolism , Enzyme Inhibitors/pharmacology , Heat-Shock Proteins/chemistry , Humans , Hydrogen Bonding , Models, Molecular , Molecular Sequence Data , Phosphoprotein Phosphatases/antagonists & inhibitors , Phosphoprotein Phosphatases/metabolism , Phosphoprotein Phosphatases/ultrastructure , Protein Conformation , Protein Structure, Secondary , Proteins/metabolism , Proteins/pharmacology , Recombinant Proteins/chemistry , Tacrolimus/chemistry , Tacrolimus Binding Proteins , Water/metabolism
8.
J Mol Graph ; 11(1): 47-52, 45, 1993 Mar.
Article in English | MEDLINE | ID: mdl-8388712

ABSTRACT

The crystal structure of the calcium-binding protein calmodulin is used to model the immunologically important calcineurin subunit B. The rough structure is produced by computer-aided homology modeling. Refinement of this using molecular dynamics leads to a suggested structure which appears to satisfy reasonable hydrophilicity and hydrogen-bonding criteria. In the absence of a crystal structure, the model may prove useful in modeling of its interactions with the phosphatase catalytic subunit calcineurin A, and help to explain the calcium modulation of this protein.


Subject(s)
Calmodulin-Binding Proteins/chemistry , Phosphoprotein Phosphatases/chemistry , Amino Acid Sequence , Animals , Binding Sites , Calcineurin , Calmodulin/chemistry , Calmodulin/genetics , Calmodulin-Binding Proteins/genetics , Calmodulin-Binding Proteins/ultrastructure , Computer Graphics , Models, Molecular , Molecular Sequence Data , Molecular Structure , Phosphoprotein Phosphatases/genetics , Phosphoprotein Phosphatases/ultrastructure , Protein Conformation , Sequence Homology, Amino Acid
SELECTION OF CITATIONS
SEARCH DETAIL
...