scholarly journals The human Myt1 kinase preferentially phosphorylates Cdc2 on threonine 14 and localizes to the endoplasmic reticulum and Golgi complex.

1997 ◽  
Vol 17 (2) ◽  
pp. 571-583 ◽  
Author(s):  
F Liu ◽  
J J Stanton ◽  
Z Wu ◽  
H Piwnica-Worms
Keyword(s):  
Amino Acids ◽  
Endoplasmic Reticulum ◽  
Golgi Complex ◽  
Catalytic Domain ◽  
Cyclin B ◽  
Dependent Manner ◽  
Cdc2 Kinase ◽  
Dual Specificity ◽  
Catalytically Active ◽  
Specificity Protein

Entry into mitosis requires the activity of the Cdc2 kinase. Cdc2 associates with the B-type cyclins, and the Cdc2-cyclin B heterodimer is in turn regulated by phosphorylation. Phosphorylation of threonine 161 is required for the Cdc2-cyclin B complex to be catalytically active, whereas phosphorylation of threonine 14 and tyrosine 15 is inhibitory. Human kinases that catalyze the phosphorylation of threonine 161 and tyrosine 15 have been identified. Here we report the isolation of a novel human cDNA encoding a dual-specificity protein kinase (designated Myt1Hu) that preferentially phosphorylates Cdc2 on threonine 14 in a cyclin-dependent manner. Myt1Hu is 46% identical to Myt1Xe, a kinase recently characterized from Xenopus laevis. Myt1Hu localizes to the endoplasmic reticulum and Golgi complex in HeLa cells. A stretch of hydrophobic and uncharged amino acids located outside the catalytic domain of Myt1Hu is the likely membrane-targeting domain, as its deletion results in the localization of Myt1Hu primarily to the nucleus.

scholarly journals The Amino Terminus of Varicella-Zoster Virus (VZV) Glycoprotein E Is Required for Binding to Insulin-Degrading Enzyme, a VZV Receptor

2007 ◽  
Vol 81 (16) ◽  
pp. 8525-8532 ◽  
Author(s):  
Qingxue Li ◽  
Tammy Krogmann ◽  
Mir A. Ali ◽  
Wei-Jen Tang ◽  
Jeffrey I. Cohen
Keyword(s):  
Amino Acids ◽  
Varicella Zoster Virus ◽  
Catalytic Domain ◽  
Amino Terminus ◽  
Dependent Manner ◽  
Insulin Degrading Enzyme ◽  
Concentration Dependent Manner ◽  
Glycoprotein E ◽  
Degrading Enzyme ◽  
Varicella Zoster

ABSTRACT Varicella-zoster virus (VZV) glycoprotein E (gE) is required for VZV infection. Although gE is well conserved among alphaherpesviruses, the amino terminus of VZV gE is unique. Previously, we showed that gE interacts with insulin-degrading enzyme (IDE) and facilitates VZV infection and cell-to-cell spread of the virus. Here we define the region of VZV gE required to bind IDE. Deletion of amino acids 32 to 71 of gE, located immediately after the predicted signal peptide, resulted in loss of the ability of gE to bind IDE. A synthetic peptide corresponding to amino acids 24 to 50 of gE blocked its interaction with IDE in a concentration-dependent manner. However, a chimeric gE in which amino acids 1 to 71 of VZV gE were fused to amino acids 30 to 545 of herpes simplex virus type 2 gE did not show an increased level of binding to IDE compared with that of full-length HSV gE. Thus, amino acids 24 to 71 of gE are required for IDE binding, and the secondary structure of gE is critical for the interaction. VZV gE also forms a heterodimer with glycoprotein gI. Deletion of amino acids 163 to 208 of gE severely reduced its ability to form a complex with gI. The amino portion of IDE, as well an IDE mutant in the catalytic domain of the protein, bound to gE. Therefore, distinct motifs of VZV gE are important for binding to IDE or to gI.

scholarly journals The interaction of small domains between the subunits of phosphatidylinositol 3-kinase determines enzyme activity.

1994 ◽  
Vol 14 (4) ◽  
pp. 2675-2685 ◽  
Author(s):  
A Klippel ◽  
J A Escobedo ◽  
M Hirano ◽  
L T Williams
Keyword(s):  
Amino Acids ◽  
Catalytic Domain ◽  
Specific Interaction ◽  
Sh2 Domains ◽  
Catalytically Active ◽  
Functional Complex ◽  
Src Homology ◽  
Carboxy Terminal

Previous studies have suggested that the two subunits of phosphatidylinositol (PI) 3-kinase, p85 and p110, function as localizing and catalytic subunits, respectively. Using recombinant p85 and p110 molecules, we have reconstituted the specific interaction between the two subunits of mouse PI 3-kinase in cells and in vitro. We have previously shown that the region between the two Src homology 2 (SH2) domains of p85 is able to form a functional complex with the 110-kDa subunit in vivo. In this report, we identify the corresponding domain in p110 which directs the binding to p85. We demonstrate that the interactive domains in p85 and p110 are less than 103 and 124 amino acids, respectively, in size. We also show that the association of p85 and p110 mediated by these domains is critical for PI 3-kinase activity. Surprisingly, a complex between a 102-amino-acid segment of p85 and the full-length p110 molecule is catalytically active, whereas p110 alone has no activity. In addition to the catalytic domain in the carboxy-terminal region, 123 amino acids at the amino terminus of p110 were required for catalytic activity and were sufficient for the interaction with p85. These results indicate that the 85-kDa subunit, previously thought to have only a linking role in localizing the p110 catalytic subunit, is an important component of the catalytic complex.

BET1, BOS1, and SEC22 are members of a group of interacting yeast genes required for transport from the endoplasmic reticulum to the Golgi complex

1990 ◽  
Vol 10 (7) ◽  
pp. 3405-3414
Author(s):  
A P Newman ◽  
J Shim ◽  
S Ferro-Novick
Keyword(s):  
Endoplasmic Reticulum ◽  
Golgi Complex ◽  
Genomic Library ◽  
Genetic Interaction ◽  
Gene Dosage ◽  
Restrictive Temperature ◽  
Dependent Manner ◽  
Direct Role ◽  
Wide Range ◽  
Yeast Genes

A subset of the genes required for transport from the endoplasmic reticulum (ER) to the Golgi complex in Saccharomyces cerevisiae was found to interact genetically. While screening a yeast genomic library for genes complementing the ER-accumulating mutant bet1 (A. Newman and S. Ferro-Novick, J. Cell Biol. 105: 1587-1594, 1987), we isolated BET1 and BOS1 (bet one suppressor). BOS1 suppresses bet1-1 in a gene dosage-dependent manner, providing greater suppression when it is introduced on a multicopy vector than when one additional copy is present. The BET1 and BOS1 genes are not functionally equivalent; overproduction of BOS1 does not alleviate the lethality associated with disruption of BET1. We also identified a pattern of genetic interactions among these genes and another gene implicated in transport from the ER to the Golgi complex: SEC22. Overproduction of either BET1 or BOS1 suppresses the growth and secretory defects of the sec22-3 mutant over a wide range of temperatures. Further evidence for genetic interaction was provided by the finding that a bet1 sec22 double mutant is inviable. Another mutant which is blocked in transport from the ER to the Golgi complex, sec21-1, demonstrates a more limited ability to be suppressed by the BET1 gene. The interactions we observed are specific for genes required for transport from the ER to the Golgi complex. The products of the genes involved are likely to have a direct role in transport, as bet1-1 and sec22-3 begin to display their mutant phenotypes within 5 min of a shift to the restrictive temperature.

scholarly journals Cell Cycle-dependent Regulation of Structure of Endoplasmic Reticulum and Inositol 1,4,5-Trisphosphate-induced Ca2+ Release in Mouse Oocytes and Embryos

2003 ◽  
Vol 14 (1) ◽  
pp. 288-301 ◽  
Author(s):  
Greg FitzHarris ◽  
Petros Marangos ◽  
John Carroll
Keyword(s):  
Cell Cycle ◽  
Endoplasmic Reticulum ◽  
Mitotic Spindle ◽  
Mitotic Division ◽  
Cyclin B ◽  
Dependent Manner ◽  
Inositol 1,4,5 Trisphosphate ◽  
A Cell ◽  
Cycle Dependent ◽  
Mouse Eggs

The organization of endoplasmic reticulum (ER) was examined in mouse eggs undergoing fertilization and in embryos during the first cell cycle. The ER in meiosis II (MII)-arrested mouse eggs is characterized by accumulations (clusters) that are restricted to the cortex of the vegetal hemisphere of the egg. Monitoring ER structure with DiI18 after egg activation has demonstrated that ER clusters disappear at the completion of meiosis II. The ER clusters can be maintained by inhibiting the decrease in cdk1-cyclin B activity by using the proteasome inhibitor MG132, or by microinjecting excess cyclin B. A role for cdk1-cyclin B in ER organization is further suggested by the finding that the cdk inhibitor roscovitine causes the loss of ER clusters in MII eggs. Cortical clusters are specific to meiosis as they do not return in the first mitotic division; rather, the ER aggregates around the mitotic spindle. Inositol 1,4,5-trisphosphate-induced Ca2+ release is also regulated in a cell cycle-dependent manner where it is increased in MII and in the first mitosis. The cell cycle dependent effects on ER structure and inositol 1,4,5-trisphosphate-induced Ca2+ release have implications for understanding meiotic and mitotic control of ER structure and inheritance, and of the mechanisms regulating mitotic Ca2+signaling.

scholarly journals Inhibition of protein kinase C catalytic activity by additional regions within the human protein kinase Calpha-regulatory domain lying outside of the pseudosubstrate sequence

2003 ◽  
Vol 373 (2) ◽  
pp. 571-581 ◽  
Author(s):  
Angie F. KIRWAN ◽  
Ashley C. BIBBY ◽  
Thierry MVILONGO ◽  
Heimo RIEDEL ◽  
Thomas BURKE ◽  
...  
Keyword(s):  
Amino Acids ◽  
Protein Kinase ◽  
Catalytic Domain ◽  
Dependent Manner ◽  
Regulatory Domain ◽  
Direct Role ◽  
Independent Manner ◽  
Inhibitory Capacity ◽  
Pseudosubstrate Sequence

The N-terminal pseudosubstrate site within the protein kinase Cα (PKCα)-regulatory domain has long been regarded as the major determinant for autoinhibition of catalytic domain activity. Previously, we observed that the PKC-inhibitory capacity of the human PKCα-regulatory domain was only reduced partially on removal of the pseudosubstrate sequence [Parissenti, Kirwan, Kim, Colantonio and Schimmer (1998) J. Biol. Chem. 273, 8940–8945]. This finding suggested that one or more additional region(s) contributes to the inhibition of catalytic domain activity. To assess this hypothesis, we first examined the PKC-inhibitory capacity of a smaller fragment of the PKCα-regulatory domain consisting of the C1a, C1b and V2 regions [GST-Rα39–177: this protein contained the full regulatory domain of human PKCα fused to glutathione S-transferase (GST), but lacked amino acids 1–38 (including the pseudosubstrate sequence) and amino acids 178–270 (including the C2 region)]. GST-Rα39–177 significantly inhibited PKC in a phorbol-independent manner and could not bind the peptide substrate used in our assays. These results suggested that a region within C1/V2 directly inhibits catalytic domain activity. Providing further in vivo support for this hypothesis, we found that expression of N-terminally truncated pseudosubstrate-less bovine PKCα holoenzymes in yeast was capable of inhibiting cell growth in a phorbol-dependent manner. This suggested that additional autoinhibitory force(s) remained within the truncated holoenzymes that could be relieved by phorbol ester. Using tandem PCR-mediated mutagenesis, we observed that mutation of amino acids 33–86 within GST-Rα39–177 dramatically reduced its PKC-inhibitory capacity when protamine was used as substrate. Mutagenesis of a broad range of sequences within C2 (amino acids 159–242) also significantly reduced PKC-inhibitory capacity. Taken together, these observations support strongly the existence of multiple regions within the PKCα-regulatory domain that play a direct role in the inhibition of catalytic domain activity.

The family-wide structure and function of human dual-specificity protein phosphatases

2014 ◽  
Vol 70 (2) ◽  
pp. 421-435 ◽  
Author(s):  
Dae Gwin Jeong ◽  
Chun Hua Wei ◽  
Bonsu Ku ◽  
Tae Jin Jeon ◽  
Pham Ngoc Chien ◽  
...  
Keyword(s):  
Active Site ◽  
Catalytic Domain ◽  
Protein Phosphatases ◽  
Homology Modelling ◽  
Dual Specificity ◽  
Surface Charge Distribution ◽  
The Family ◽  
Characteristic Features ◽  
And Function ◽  
Specificity Protein

Dual-specificity protein phosphatases (DUSPs), which dephosphorylate both phosphoserine/threonine and phosphotyrosine, play vital roles in immune activation, brain function and cell-growth signalling. A family-wide structural library of human DUSPs was constructed based on experimental structure determination supplemented with homology modelling. The catalytic domain of each individual DUSP has characteristic features in the active site and in surface-charge distribution, indicating substrate-interaction specificity. The active-site loop-to-strand switch occurs in a subtype-specific manner, indicating that the switch process is necessary for characteristic substrate interactions in the corresponding DUSPs. A comprehensive analysis of the activity–inhibition profile and active-site geometry of DUSPs revealed a novel role of the active-pocket structure in the substrate specificity of DUSPs. A structure-based analysis of redox responses indicated that the additional cysteine residues are important for the protection of enzyme activity. The family-wide structures of DUSPs form a basis for the understanding of phosphorylation-mediated signal transduction and the development of therapeutics.

scholarly journals Identification of furin pro-region determinants involved in folding and activation

2004 ◽  
Vol 379 (3) ◽  
pp. 757-763 ◽  
Author(s):  
Lyne BISSONNETTE ◽  
Gabriel CHAREST ◽  
Jean-Michel LONGPRÉ ◽  
Pierre LAVIGNE ◽  
Richard LEDUC
Keyword(s):  
Amino Acids ◽  
Catalytic Domain ◽  
Homology Modelling ◽  
Site Directed Mutagenesis ◽  
Dependent Manner ◽  
Hydrophobic Core ◽  
Binding Interface ◽  
Golgi Network ◽  
Von Willebrand ◽  
Pro Region

The pro-region of the subtilisin-like convertase furin acts early in the biosynthetic pathway as an intramolecular chaperone to enable proper folding of the zymogen, and later on as an inhibitor to constrain the activity of the enzyme until it reaches the trans-Golgi network. To identify residues that are important for pro-region function, we initially identified amino acids that are conserved among the pro-regions of various mammalian convertases. Site-directed mutagenesis of 17 selected amino acids within the 89-residue pro-region and biosynthetic labelling revealed that I60A-furin and H66A-furin were rapidly degraded in a proteasome-dependent manner, while W34A-furin and F67A-furin did not show any autocatalytic activation. Intriguingly, the latter mutants proteolytically cleaved pro-von Willebrand factor precursor to the mature polypeptide, suggesting that the mutations permitted proper folding, but did not allow the pro-region to exercise its role in inhibiting the enzyme. Homology modelling of furin's pro-region revealed that residues Ile-60 and His-66 might be crucial in forming the binding interface with the catalytic domain, while residues Trp-34 and Phe-67 might be involved in maintaining a hydrophobic core within the pro-region itself. These results provide structural insights into the dual role of furin's pro-region.

Bnsro1: A new homologue of Arabidopsis thaliana rcd1 from Brassica napus

Biologia ◽  
2015 ◽  
Vol 70 (5) ◽  
Author(s):  
Sadia Anjum ◽  
Saboohi Raza ◽  
Abid Azhar ◽  
Syeda Qamarunnisa
Keyword(s):  
Amino Acids ◽  
Arabidopsis Thaliana ◽  
Brassica Napus ◽  
Catalytic Domain ◽  
Molecular Signaling ◽  
General Phenomenon ◽  
Signaling Mechanism ◽  
Different Types ◽  
Catalytically Active ◽  
Computational Basis

AbstractGeneration of reactive oxidation species in response to different types of stress is a general phenomenon observed in plants. It is considered to be a molecular signaling mechanism of plants to encounter adverse effects. Radical-induced cell death 1 (rcd1 or Atrcd1) gene of Arabidopsis thaliana is a stress responsive gene known to interact with several transcription factors during different types of stress. It is predicted to provide scaffold for mediating interactions between two proteins using its WWE and RST domains. It also has an inactive PARP catalytic domain forming the Similar like rcd1 (SRO) family of plant PARPs along with its homologs. In this study a new homolog from Brassica napus genome (Bnsro1) was identified. Analysis of Bnsro1 was done to predict function on computational basis by comparison with its homolog. Bnsro1 has similarities with Atrcd1 at sequence level and contains same globular domains. It is predicted to be catalytically active as it conserves the 16 amino acids required for NAD

scholarly journals Identification of residues essential for catalysis and binding of calmodulin in rat brain inositol 1,4,5-trisphosphate 3-kinase

1991 ◽  
Vol 280 (1) ◽  
pp. 125-129 ◽  
Author(s):  
K Takazawa ◽  
C Erneux
Keyword(s):  
Amino Acids ◽  
Rat Brain ◽  
Catalytic Domain ◽  
Protein A ◽  
Restriction Enzymes ◽  
Deletion Mutants ◽  
Amino Acid Residues ◽  
Inositol 1,4,5 Trisphosphate ◽  
Catalytically Active ◽  
Inactive Protein

In order to identify the amino acid residues involved in calmodulin (CaM) binding and catalytic activity, rat brain inositol 1,4,5-trisphosphate (InsP3) 3-kinase was expressed in Escherichia coli as a beta-galactosidase fusion protein [clone C5; Takazawa, Vandekerckhove, Dumont & Erneux (1990) Biochem. J. 272, 107-112]. Three deletion mutants in the plasmid of clone C5 were generated using convenient restriction enzymes. The results show that the removal of 34 amino acids from the C-terminal end of InsP3 3-kinase resulted in an inactive protein which still interacted with CaM-Sepharose in a Ca2(+)-dependent way. The catalytic domain is thus located at the C-terminal end of the protein. A series of 5′ deletion mutants was prepared and used to produce proteins with the same C-terminal end but shortened N-termini, varying in length by over 80 amino acids. Assay of InsP3 3-kinase activity in bacterial extracts indicated that a maximum of 275 amino acids in the C-terminal region may be sufficient for the construction of a catalytically active domain. Affinity chromatography on CaM-Sepharose of 5′ and 3′ deletion mutants revealed that the sequence stretching from Ser-156 to Leu-189 is involved in CaM binding and enzyme stimulation.

scholarly journals Orientia tsutsugamushi Modulates Endoplasmic Reticulum-Associated Degradation To Benefit Its Growth

2017 ◽  
Vol 86 (1) ◽  
Author(s):  
Kyle G. Rodino ◽  
Lauren VieBrock ◽  
Sean M. Evans ◽  
Hong Ge ◽  
Allen L. Richards ◽  
...  
Keyword(s):  
Amino Acids ◽  
Endoplasmic Reticulum ◽  
Amino Acid ◽  
Er Stress ◽  
Growth Defect ◽  
Dependent Manner ◽  
Amino Acid Supplementation ◽  
Orientia Tsutsugamushi ◽  
Content Type ◽  
Obligate Intracellular

ABSTRACT Orientia tsutsugamushi, an obligate intracellular bacterium that is auxotrophic for the aromatic amino acids and histidine, causes scrub typhus, a potentially deadly infection that threatens 1 billion people. O. tsutsugamushi growth is minimal during the first 24 to 48 h of infection but its growth becomes logarithmic thereafter. How the pathogen modulates cellular functions to support its growth is poorly understood. The unfolded protein response (UPR) is a cytoprotective pathway that relieves endoplasmic reticulum (ER) stress by promoting ER-associated degradation (ERAD) of misfolded proteins. Here, we show that O. tsutsugamushi invokes the UPR in the first 48 h and benefits from ER stress in an amino acid-dependent manner. O. tsutsugamushi also impedes ERAD during this time period. By 72 h, ER stress is alleviated and ERAD proceeds unhindered. Sustained inhibition of ERAD using RNA interference results in an O. tsutsugamushi growth defect at 72 h that can be rescued by amino acid supplementation. Thus, O. tsutsugamushi temporally stalls ERAD until ERAD-derived amino acids are needed to support its growth. The O. tsutsugamushi effector Ank4 is linked to this phenomenon. Ank4 interacts with Bat3, a eukaryotic chaperone that is essential for ERAD, and is transiently expressed by O. tsutsugamushi during the infection period when it inhibits ERAD. Ectopically expressed Ank4 blocks ERAD to phenocopy O. tsutsugamushi infection. Our data reveal a novel mechanism by which an obligate intracellular bacterial pathogen modulates ERAD to satisfy its nutritional virulence requirements.

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