scholarly journals Protein-N-myristoylation-dependent phosphorylation of serine 13 of tyrosine kinase Lyn by casein kinase 1γ at the Golgi during intracellular protein traffic

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Emiko Kinoshita-Kikuta ◽  
Toshihiko Utsumi ◽  
Aya Miyazaki ◽  
Chiharu Tokumoto ◽  
Kyosuke Doi ◽  
...  
Keyword(s):  
Casein Kinase ◽  
Src Family Kinases ◽  
The Other ◽  
Hek293 Cells ◽  
Intracellular Protein ◽  
Protein Traffic ◽  
Phosphorylation Level ◽  
Sds Page ◽  
Membrane Phosphorylation ◽  
Palmitoylation Site

Abstract Protein N-myristoylation of Src-family kinases (SFKs) is a critical co-translational modification to anchor the enzymes in the plasma membrane. Phosphorylation of SFKs is also an essential modification for regulating their enzymatic activities. In this study, we used Phos-tag SDS-PAGE to investigate N-myristoylation-dependent phosphorylation of SFKs and their non-N-myristoylated G2A mutants. The serine-13 residue of Lyn (Lyn-S13) was shown to be N-myristoylation-dependently phosphorylated. Although there have been more than 40 reports of mass spectrometric studies on phosphorylation at Lyn-S13, the kinase responsible remained unclear. We succeeded in identifying casein kinase 1γ (CK1γ) as the kinase responsible for phosphorylation of Lyn-S13. In HEK293 cells co-expressing Lyn and CK1γ, the phosphorylation level of Lyn-S13 increased significantly. CK1γ is unique among the CK1 family (α, γ, δ, and ε) in carrying an S-palmitoylation site for membrane binding. Co-expression with the non-S-palmitoylated CK1γ mutant, which localized in the cytosol, gave no increase in the phosphorylation level at Lyn-S13. In HEK293 cells expressing the non-S-palmitoylated Lyn-C3A mutant, on the other hand, the Lyn-C3A mutant was phosphorylated at Lyn-S13, and the mutant remained at the Golgi. These results showed that S-palmitoylated CK1γ can phosphorylate S13 of N-myristoylated Lyn at the Golgi during intracellular protein traffic.

O-linked but not N-linked glycosylation is necessary for the secretion of endoglucanases I and II by Trichoderma reesei

1987 ◽  
Vol 33 (8) ◽  
pp. 698-703 ◽  
Author(s):  
C. P. Kubicek ◽  
T. Panda ◽  
G. Schreferl-kunar ◽  
F. Gruber ◽  
R. Messner
Keyword(s):  
Trichoderma Reesei ◽  
Protein Glycosylation ◽  
The Other ◽  
Detectable Effect ◽  
Secreted Protein ◽  
Intracellular Protein ◽  
Molecular Weights ◽  
Sds Page ◽  
Wide Range ◽  
Molecular Masses

The effect of inhibiting protein glycosylation was studied in nongrowing mycelia and protoplasts of Trichoderma reesei which secreted two endoglucanases (I and II) upon addition of sophorose. Tunicamycin (40 μg∙mL−1) inhibited incorporation of N-acetylglucosamine into secreted protein, but had no effect on secretion of total protein or endoglucanases. The secreted endoglucanases I and II exhibited relative molecular masses of 58 and 45 kilodaltons, respectively, irrespective of the presence of tunicamycin. On the other hand 2-deoxy-D-glucose inhibited the biosynthesis of extracellular as well as intracellular protein over a wide range of concentrations; at 50 μg∙mL−1, however, it inhibited the synthesis of extracellular protein more strongly. The synthesis of endoglucanases I and II was decreased accordingly under these conditions. SDS–PAGE did not reveal the secretion of endoglucanases with smaller molecular weights. When the two endoglucanases were purified and subjected to Endo H treatment or β-elimination, the former had no detectable effect, whereas the latter released all carbohydrate from the protein. Nevertheless, endoglucanases I and II contained 1.3 and 0.5 mol of glucosamine per mol enzyme, respectively. It is concluded that endoglucanases I and II from T. reesei contain mainly O-linked neutral carbohydrate, which is required for their secretion.

scholarly journals A comprehensive analysis of novel disulfide bond introduction site into the constant domain of human Fab

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Hitomi Nakamura ◽  
Moeka Yoshikawa ◽  
Naoko Oda-Ueda ◽  
Tadashi Ueda ◽  
Takatoshi Ohkuri
Keyword(s):  
Thermal Stability ◽  
Pichia Pastoris ◽  
Protein Stability ◽  
Disulfide Bond ◽  
Binding Activity ◽  
Comprehensive Analysis ◽  
The Other ◽  
Constant Domain ◽  
Intermolecular Disulfide Bond ◽  
Sds Page

AbstractGenerally, intermolecular disulfide bond contribute to the conformational protein stability. To identify sites where intermolecular disulfide bond can be introduced into the Fab’s constant domain of the therapeutic IgG, Fab mutants were predicted using the MOE software, a molecular simulator, and expressed in Pichia pastoris. SDS-PAGE analysis of the prepared Fab mutants from P. pastoris indicated that among the nine analyzed Fab mutants, the F130C(H):Q124C(L), F174C(H):S176C(L), V177C(H):Q160C(L), F174C(H):S162C(L), F130C(H):S121C(L), and A145C(H):F116C(L) mutants mostly formed intermolecular disulfide bond. All these mutants showed increased thermal stability compared to that of Fab without intermolecular disulfide bond. In the other mutants, the intermolecular disulfide bond could not be completely formed, and the L132C(H):F118C(L) mutant showed only a slight decrease in binding activity and β-helix content, owing to the exertion of adverse intermolecular disulfide bond effects. Thus, our comprehensive analysis reveals that the introduction of intermolecular disulfide bond in the Fab’s constant domain is possible at various locations. These findings provide important insights for accomplishing human Fab stabilization.

Saccharomyces cerevisiae Apl2p, a homologue of the mammalian clathrin AP beta subunit, plays a role in clathrin-dependent Golgi functions

1995 ◽  
Vol 108 (4) ◽  
pp. 1605-1615 ◽  
Author(s):  
M.R. Rad ◽  
H.L. Phan ◽  
L. Kirchrath ◽  
P.K. Tan ◽  
T. Kirchhausen ◽  
...  
Keyword(s):  
Chain Gene ◽  
Beta Subunits ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Intracellular Protein ◽  
Protein Traffic ◽  
Specific Effects ◽  
Yeast Chromosome ◽  
Vacuolar Sorting ◽  
Golgi Network

Clathrin-coated vesicles mediate selective intracellular protein traffic from the plasma membrane and the trans-Golgi network. At these sites, clathrin-associated protein (AP) complexes have been implicated in both clathrin coat assembly and collection of cargo into nascent vesicles. We have found a gene on yeast chromosome XI that encodes a homologue of the mammalian AP beta subunits. Disruptions of this gene, APl2, and a previously identified beta homologue, APl1, have been engineered in cells expressing wild-type (CHC1) or temperature sensitive (chc1-ts) alleles of the clathrin heavy chain gene. APl1 or APl2 disruptions (apl1 delta or apl2 delta) yield no discernable phenotypes in CHC1 strains, indicating that the Apl proteins are not essential for clathrin function. However, the apl2 delta, but not the apl1 delta, allele enhances the growth and alpha-factor pheromone maturation defects of chc1-ts cells. Disruption of APl2 also partially suppresses the vacuolar sorting defect that occurs in chc1-ts cells immediately after imposition of the non-permissive temperature. These Golgi-specific effects of apl2 delta in chc1-ts cells provide evidence that Apl2p is a component of an AP complex that interacts with clathrin at the Golgi apparatus.

scholarly journals β-Arrestin 2 and ERK1/2 Are Important Mediators Engaged in Close Cooperation between TRPV1 and µ-Opioid Receptors in the Plasma Membrane

2020 ◽  
Vol 21 (13) ◽  
pp. 4626
Author(s):  
Barbora Melkes ◽  
Vendula Markova ◽  
Lucie Hejnova ◽  
Jiri Novotny
Keyword(s):  
Plasma Membrane ◽  
Signaling Pathways ◽  
Opioid Receptors ◽  
The Other ◽  
Hek293 Cells ◽  
Lateral Mobility ◽  
Pain Pathways ◽  
Close Cooperation ◽  
Signaling Interactions

The interactions between TRPV1 and µ-opioid receptors (MOR) have recently attracted much attention because these two receptors play important roles in pain pathways and can apparently modulate each other’s functioning. However, the knowledge about signaling interactions and crosstalk between these two receptors is still limited. In this study, we investigated the mutual interactions between MOR and TRPV1 shortly after their activation in HEK293 cells expressing these two receptors. After activation of one receptor we observed significant changes in the other receptor’s lateral mobility and vice versa. However, the changes in receptor movement within the plasma membrane were not connected with activation of the other receptor. We also observed that plasma membrane β-arrestin 2 levels were altered after treatment with agonists of both these receptors. Knockdown of β-arrestin 2 blocked all changes in the lateral mobility of both receptors. Furthermore, we found that β-arrestin 2 can play an important role in modulating the effectiveness of ERK1/2 phosphorylation after activation of MOR in the presence of TRPV1. These data suggest that β-arrestin 2 and ERK1/2 are important mediators between these two receptors and their signaling pathways. Collectively, MOR and TRPV1 can mutually affect each other’s behavior and β-arrestin 2 apparently plays a key role in the bidirectional crosstalk between these two receptors in the plasma membrane.

THE PHOSPHOLIPID-BINDING SITE OF FACTOR VIII IS LOCATED ON THE 80 kD LIGHT CHAIN

1987 ◽  
Author(s):  
G Kemball-Cook ◽  
S J A Edwards ◽  
K Sewerin ◽  
L-O Andersson ◽  
T W Barrowcliffe
Keyword(s):  
Binding Site ◽  
Light Chain ◽  
Binding Sites ◽  
Immunoaffinity Chromatography ◽  
The Other ◽  
Immunological Methods ◽  
Electrophoretic Separation ◽  
Antigenic Sites ◽  
Reducing Conditions ◽  
Sds Page

The binding of Factoi. VIII (F.VIII) peptides to phospholipid (PL) vesicles has been studied by two different methods involving the use of fractionated anti-F.VIII:C I-Fab123’pre viously reported, i-Fab123’ was fractionated by immunoadsorptionwith F.VIII-PL complexes into two pools:one binding only to PL-binding sites on F.VIIIsAg (PL-site antibody), the other directed against other antigenic sites (non-PL-site antibody).The first technique used was a modification of the method of Weinstein et al. (Proc.Natl.Acad.Sci.USA, 78, 5137-5141, 1981), and involved incubation of the two anti-F.VIII pool swith F.VIII-containing samples, followed by electrophoretic separation of the complexes on the basis of size in non-denaturing SDS gels: this technique allows qualitative analysis of antibody reactive peptides in highly impure samples. Non-PL-site pool reacted with a range of peptides with MrMapparent Mr 90 kD up to 280 kD, a similar pattern to that of ’heavy chain’(HC) peptides of F.VIII seen on SDS-PAGE under reducing conditions; the PL-site antibody, however, reacted only with peptides at apparent Mrs of 80 kD and sometimes150 kD, but not with bands of higher Mr a pattern more consistent with binding to light chain (LC) peptides. Thesame patterns with the two labels were seen in both plasma and F.VIII concentrateThe second approach employed the two labels described above in direct immunoradiometric assays (IFMA’s) on purified human F.VIII peptides prepared by immunoaffinity chromatography and ion exchange on Mono Q gel. Both PL-site and non-PL-site labels measured similar amounts of F.VIII m a sample containing both HC and LC peptides; however, on assaying a sample containing purified HC peptides alone, PL-site antibody measured only 2% of F.VIII:Ag found by non-PL-site label, indicating that PL-binding sites present in samples containing both HC and LC are absent in HC alone.Results from both these immunological methods indicate that the 80 kD LC peptide of F.VIII carries the PL-binding site.

Inhibition of glycogen synthase (casein) kinase-1 by divalent metal ions

1988 ◽  
Vol 66 (3) ◽  
pp. 238-243 ◽  
Author(s):  
Toolsee J. Singh
Keyword(s):  
Metal Ions ◽  
Kinase Activity ◽  
Glycogen Synthase ◽  
Divalent Metal ◽  
Casein Kinase ◽  
The Other ◽  
Divalent Metal Ions ◽  
Casein Kinase 1 ◽  
Competitive Inhibitors ◽  
Noncompetitive Inhibitors

The specificity of glycogen synthase (casein) kinase-1 (CK-1) for different divalent metal ions was explored in this study. Of nine metal ions (Mg2+, Mn2+, Zn2+, Cu2+, Ca2+, Ba2+, Ni2+, Co2+, Fe2+) tested, only Mg2+ supported significant kinase activity. Several of the other metals, however, inhibited the Mg2+-stimulated kinase activity. Half-maximal inhibitions by Mn2+, Zn2+, Co2+, Fe2+, and Ni2+ were observed at 55, 65, 110, 125, and 284 μM, respectively. Kinetic analyses indicate that the metal ions are acting as competitive inhibitors of CK-1 with respect to the protein substrate (casein) and as noncompetitive inhibitors with respect to the nucleotide substrate (ATP). The inhibition of CK-1 by the different metal ions can be reversed by EGTA.

Factor XI Homodimer Formation Is Mediated by Electrostatic (Lys331 with Glu287) and Hydrophobic (Ile290 with Leu284 and Tyr329 with Tyr329) Interactions in the Apple 4 Domain and Is Essential for Normal Proteolytic Activation of Factor XI by Factor XIIa, Thrombin and Factor XIa.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2706-2706
Author(s):  
Wenman Wu ◽  
Dipali Sinha ◽  
James D. Lear ◽  
Paul C. Billings ◽  
Peter N. Walsh
Keyword(s):  
Disulfide Bond ◽  
Analytical Ultracentrifugation ◽  
Hydrophobic Interactions ◽  
The Other ◽  
Size Exclusion ◽  
Hek293 Cells ◽  
Salt Bridges ◽  
Factor Xi ◽  
Proteolytic Activation ◽  
Interchain Disulfide Bond

Abstract Factor XI (FXI), a coagulation protein essential for normal hemostasis, is a homodimer consisting of two identical subunits of 80 KDa linked by a disulfide bond formed by Cys321 within the Apple 4 (A4) domain of each subunit. Prekallikrein (PK), in spite of its high homology with FXI both in amino acid sequence and domain structure, is a monomer. Cys321 in PK forms an intrachain disulfide bond with Cys326. However FXI/C321S (in which interchain disulfide bond formation is precluded) is a noncovalent dimer. Thus, there are interacting residues between the two subunits of FXI that are responsible for mediating its unique homodimeric structure. Examination of the crystal structure of FXI (Papagrigoriou E, McEwan P, Walsh PN, Emsley J. Nature Structural & Molecular Biology. 2006;13:557–8) shows salt bridges between Lys331 of one subunit with Glu287 of the other subunit as well as hydrophobic interactions at the interface of the A4 domains involving Ile290, Leu284 and Tyr329. FXI/C321S, FXI/C321S/K331A, FXI/C321S/E287A, FXI/C321S/I290A, FXI/C321S/Y329A, FXI/C321S/L284A and FXI/C321S/K331R were expressed in HEK293 cells and characterized using size exclusion chromatography (SEC), analytical ultracentrifugation (AUC), and functional assays. Whereas FXI/C321S existed in a monomer/dimer equilibrium (Kd ∼40 nM) all other mutants were predominantly monomers by SEC with impaired dimer formation by AUC (Kd 3.4–38 μM). All the monomeric mutants when converted to the active enzyme, FXIa, were able to hydrolyze the small chromogenic substrate S-2366 with normal values of Km and Vmax and cleaved the macromolecular substrate FIX at both its scissile bonds at rates similar to those observed with wtFXIa strongly suggesting that all mutant proteins were properly folded. However all the monomeric mutants displayed impaired clotting activity in an APTT assay and displayed markedly decreased rates of activation by FXIIa or thrombin and autoactivation in the presence or absence of dextran sulfate. We conclude that salt bridges formed between Lys331 of one subunit and Glu287 of the other together with hydrophobic interactions of residues Ile290 with Leu284 and Tyr329 with Tyr329 and are essential for normal homodimer formation, which is essential for normal proteolytic activation of FXI by FXIIa, thrombin and FXIa either in solution or on an anionic surface.

scholarly journals rlk/TXK Encodes Two Forms of a Novel Cysteine String Tyrosine Kinase Activated by Src Family Kinases

1999 ◽  
Vol 19 (2) ◽  
pp. 1498-1507 ◽  
Author(s):  
Jayantha Debnath ◽  
Mario Chamorro ◽  
Michael J. Czar ◽  
Edward M. Schaeffer ◽  
Michael J. Lenardo ◽  
...  
Keyword(s):  
Tyrosine Kinases ◽  
Protein Localization ◽  
Cell Receptor ◽  
Src Family Kinases ◽  
The Other ◽  
Ph Domain ◽  
Acid Modification ◽  
Critical Determinant ◽  
Initiation Of Translation ◽  
Tcr Activation

ABSTRACT Rlk/Txk is a member of the BTK/Tec family of tyrosine kinases and is primarily expressed in T lymphocytes. Unlike other members of this kinase family, Rlk lacks a pleckstrin homology (PH) domain near the amino terminus and instead contains a distinctive cysteine string motif. We demonstrate here that Rlk protein consists of two isoforms that arise by alternative initiation of translation from the same cDNA. The shorter, internally initiated protein species lacks the cysteine string motif and is located in the nucleus when expressed in the absence of the larger form. In contrast, the larger form is cytoplasmic. We show that the larger form is palmitoylated and that mutation of its cysteine string motif both abolishes palmitoylation and allows the protein to migrate to the nucleus. The cysteine string, therefore, is a critical determinant of both fatty acid modification and protein localization for the larger isoform of Rlk, suggesting that Rlk regulation is distinct from the other Btk family kinases. We further show that Rlk is phosphorylated and changes localization in response to T-cell-receptor (TCR) activation and, like the other Btk family kinases, can be phosphorylated and activated by Src family kinases. However, unlike the other Btk family members, Rlk is activated independently of the activity of phosphatidylinositol 3-kinase, consistent with its lack of a PH domain. Thus, Rlk has two distinct isoforms, each of which may have unique properties in signaling downstream from the TCR.

Sign in / Sign up

Export Citation Format

Share Document