Enrichment specificity of micro and nano-sized titanium and zirconium dioxides particles in phosphopeptide mapping

2013 ◽  
Vol 48 (11) ◽  
pp. 1188-1198 ◽  
Author(s):  
Annalisa Vilasi ◽  
Immacolata Fiume ◽  
Paolo Pace ◽  
Mosè Rossi ◽  
Gabriella Pocsfalvi
Keyword(s):  
Phosphopeptide Mapping

scholarly journals Cyclic AMP-dependent protein kinase phosphorylates rabbit reticulocyte elongation factor-2 kinase and induces calcium-independent activity

1993 ◽  
Vol 293 (1) ◽  
pp. 31-34 ◽  
Author(s):  
N T Redpath ◽  
C G Proud
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Elongation Factor ◽  
Total Activity ◽  
Dependent Protein Kinase ◽  
Calcium Calmodulin Dependent ◽  
Elongation Factor 2 ◽  
Eukaryotic Elongation Factor ◽  
Dependent Protein ◽  
Phosphopeptide Mapping

The catalytic subunit of cyclic AMP-dependent protein kinase (PKA) phosphorylated purified calcium/calmodulin-dependent eukaryotic elongation factor-2 (eEF-2) kinase, isolated from rabbit reticulocyte lysates. It maximally incorporated about 1 mol of phosphate/mol of eEF-2 kinase. The Km of eEF-2 kinase for PKA was calculated to be 7 microM. Phosphorylation of eEF-2 kinase by PKA induced calcium-independent activity which amounted to 40-50% of the total activity measured in the presence of calcium. Furthermore, the level of calcium-independent activity induced by phosphorylation by PKA was similar to that induced by the calcium-stimulated autophosphorylation of eEF-2 kinase. Phosphopeptide mapping of eEF-2 kinase labelled by autophosphorylation and by PKA revealed a number of common phosphopeptides. This suggests that PKA may phosphorylate the same site(s) which are phosphorylated autocatalytically and which are responsible for the induction of calcium-independent activity. The possible implications these findings have for the control of translation are discussed.

scholarly journals ErbB-1 and ErbB-2 Acquire Distinct Signaling Properties Dependent upon Their Dimerization Partner

1998 ◽  
Vol 18 (9) ◽  
pp. 5042-5051 ◽  
Author(s):  
Monilola A. Olayioye ◽  
Diana Graus-Porta ◽  
Roger R. Beerli ◽  
Jack Rohrer ◽  
Brigitte Gay ◽  
...  
Keyword(s):  
Intracellular Signaling ◽  
Adaptor Protein ◽  
Receptor Activation ◽  
Erbb Receptors ◽  
Antibody Treatment ◽  
Receptor Phosphorylation ◽  
Biological Responses ◽  
Monoclonal Antibody Treatment ◽  
Src Homology 2 Domain ◽  
Phosphopeptide Mapping

ABSTRACT The different epidermal growth factor (EGF)-related peptides elicit a diverse array of biological responses as the result of their ability to activate distinct subsets of ErbB receptor dimers, leading to the recruitment of different intracellular signaling networks. To specifically examine dimerization-dependent modulation of receptor signaling, we constructed NIH 3T3 cell lines expressing ErbB-1 and ErbB-2 singly and in pairwise combinations with each other ErbB family member. This model system allowed the comparison of EGF-activated ErbB-1 with ErbB-1 activated by Neu differentiation factor (NDF)-induced heterodimerization with ErbB-4. In both cases, ErbB-1 coupled to the adaptor protein Shc, but only when activated by EGF was it able to interact with Grb2. Compared to the rapid internalization of EGF-activated ErbB-1, NDF-activated ErbB-1 showed delayed internalization characteristics. Furthermore, the p85 subunit of phosphatidylinositol kinase (PI3-K) associated with EGF-activated ErbB-1 in a biphasic manner, whereas association with ErbB-1 transactivated by ErbB-4 was monophasic. The signaling properties of ErbB-2 following heterodimerization with the other ErbB receptors or homodimerization induced by point mutation or monoclonal antibody treatment were also analyzed. ErbB-2 binding to peptides containing the Src homology 2 domain of Grb2 or p85 and the phosphotyrosine binding domain of Shc varied according to the mode of receptor activation. Finally, tryptic phosphopeptide mapping of both ErbB-1 and ErbB-2 revealed that receptor phosphorylation is dependent on the dimerization partner. Differential receptor phosphorylation may, therefore, be the basis for the differences in the signaling properties observed.

2-D Phosphopeptide Mapping

2003 ◽  
pp. 673-680
Author(s):  
Hikaru Nagahara ◽  
Robert R. Latek ◽  
Sergei A. Ezhevsky ◽  
Steven F. Dowdy
Keyword(s):  
Phosphopeptide Mapping

Phosphopeptide Mapping and Phosphoamino Acid Analysis on Cellulose Thin-Layer Plates

Cell Biology ◽  
1994 ◽  
pp. 422-448 ◽  
Author(s):  
Peter van der Geer ◽  
Kunxin Luo ◽  
Bartholomew M. Sefton ◽  
Tony Hunter
Keyword(s):  
Thin Layer ◽  
Phosphoamino Acid ◽  
Phosphoamino Acid Analysis ◽  
Phosphopeptide Mapping

scholarly journals CaM kinase II regulation of CRHSP-28 phosphorylation in cultured mucosal T84 cells

2003 ◽  
Vol 285 (6) ◽  
pp. G1300-G1309 ◽  
Author(s):  
Kala M. Kaspar ◽  
Diana D. H. Thomas ◽  
William B. Taft ◽  
Eriko Takeshita ◽  
Ning Weng ◽  
...  
Keyword(s):  
Membrane Trafficking ◽  
Apical Membrane ◽  
Digestive Enzyme ◽  
Pancreatic Acinar Cells ◽  
Cellular Mechanisms ◽  
T84 Cells ◽  
Heat Stable ◽  
Heat Stable Protein ◽  
Phosphopeptide Mapping

Ca2+-regulated heat-stable protein of 28 kDa (CRHSP-28; a member of the tumor protein D52 family) is highly expressed in exocrine glands and was shown to regulate digestive enzyme secretion from pancreatic acinar cells. We found CRHSP-28 highly expressed in cultured mucosal secretory T84 cells, consistent with an important regulatory role in apical membrane trafficking. Stimulation of cells with carbachol (CCh) induced rapid, concentration-dependent phosphorylation of CRHSP-28 on at least two serine residues. Isoelectric focusing and immunoblotting were used to characterize cellular mechanisms governing CRHSP-28 phosphorylation. Phosphorylation depends on elevated cellular Ca2+, being maximally induced by ionomycin and thapsigargin and fully inhibited by BAPTAAM. In vitro phosphorylation of recombinant CRHSP-28 was 10-fold greater by casein kinase II (CKII) than Ca2+/calmodulin-dependent protein kinase II (CaMKII). However, phosphopeptide mapping studies demonstrated that CaMKII induced an identical phosphopeptide profile to endogenous CRHSP-28 immunoprecipitated from T84 cells. Although calmodulin antagonists had no effect on CCh-stimulated phosphorylation, disruption of actin filaments by cytochalasin D inhibited phosphorylation by 50%. Confocal microscopy indicated that CRHSP-28 is expressed in perinuclear regions of cells and accumulates immediately below the apical membrane of polarized monolayers following CCh stimulation. CaMKII was also localized to the subapical cytoplasm and was clearly displaced following actin filament disruption. These data suggest that CRHSP-28 phosphorylation is regulated by a CaMKII-like enzyme and likely involves a translocation of the protein within the apical cytoplasm of epithelial cells.

scholarly journals The major phosphorylation site of the NADPH oxidase component p67phox is Thr233

1999 ◽  
Vol 338 (1) ◽  
pp. 99-105 ◽  
Author(s):  
Louisa V. FORBES ◽  
Oanh TRUONG ◽  
Frans B. WIENTJES ◽  
Stephen J. MOSS ◽  
Anthony W. SEGAL
Keyword(s):  
Protein Kinase ◽  
Cyanogen Bromide ◽  
Tryptic Peptide ◽  
Phosphorylation Site ◽  
Mitogen Activated Protein Kinase ◽  
Rich Domain ◽  
Mitogen Activated Protein ◽  
Phosphopeptide Mapping ◽  
Major Phosphorylation Site

Phosphorylation of p67phox was shown to increase two- to three-fold upon stimulation by PMA, N-formylmethionyl-leucylphenylalanine or serum-opsonized zymosan. Phosphopeptide mapping showed one major tryptic peptide for p67phox immunoprecipitated from resting or stimulated cells. In vitro phosphorylation of p67phox by isolated cytosol or mitogen-activated protein kinase also generated the same phosphopeptide. Results of cyanogen bromide digestion and HPLC–MS suggested that Thr233 was the phosphorylated residue. Mutagenesis of Thr233 to alanine resulted in loss of phosphorylation in vitro. In the present work, Thr233 has been identified as the major phosphorylation site of p67phox, which is situated in a proline-rich domain.

scholarly journals PKCε Phosphorylates and Mediates the Cell Membrane Localization of RhoA

ISRN Oncology ◽  
2013 ◽  
Vol 2013 ◽  
pp. 1-9
Author(s):  
Tizhi Su ◽  
Samuel Straight ◽  
Liwei Bao ◽  
Xiujie Xie ◽  
Caryn L. Lehner ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Cell Membrane ◽  
Cell Invasion ◽  
Time Course ◽  
Time Lapse ◽  
Kinase C ◽  
Phosphorylation Event ◽  
Phosphopeptide Mapping ◽  
First Time ◽  
Initial Peak

Protein kinase Cε (PKCε) signals through RhoA to modulate cell invasion and motility. In this study, the multifaceted interaction between PKCε and RhoA was defined. Phosphopeptide mapping revealed that PKCε phosphorylates RhoA at T127 and S188. Recombinant PKCε bound to recombinant RhoA in the absence of ATP indicating that the association between PKCε and RhoA does not require an active ATP-docked PKCε conformation. Activation of PKCε resulted in a dramatic coordinated translocation of PKCε and RhoA from the cytoplasm to the cell membrane using time-lapse fluorescence microscopy. Stoichiometric FRET analysis revealed that the molecular interaction between PKCε and RhoA is a biphasic event, an initial peak at the cytoplasm and a gradual prolonged increase at the cell membrane for the entire time-course (12.5 minutes). These results suggest that the PKCε-RhoA complex is assembled in the cytoplasm and subsequently recruited to the cell membrane. Kinase inactive (K437R) PKCε is able to recruit RhoA to the cell membrane indicating that the association between PKCε and RhoA is proximal to the active catalytic site and perhaps independent of a PKCε-RhoA phosphorylation event. This work demonstrates, for the first time, that PKCε phosphorylates and modulates the cell membrane translocation of RhoA.

scholarly journals NF-κB/Rel family members are physically associated phosphoproteins

1994 ◽  
Vol 303 (2) ◽  
pp. 499-506 ◽  
Author(s):  
C C H Li ◽  
M Korner ◽  
D K Ferris ◽  
E Chen ◽  
R M Dai ◽  
...  
Keyword(s):  
Phorbol Ester ◽  
Family Members ◽  
Jurkat Cells ◽  
Nf Kappa B ◽  
I Kappa B Alpha ◽  
Jurkat T Cell ◽  
Phosphopeptide Mapping ◽  
First Time

We performed radioimmunoprecipitation followed by serial immunoblots to show that, in the unstimulated Jurkat T cell line, the NF-kappa B/Rel family proteins, p80-c-Rel, p105-NF-kappa B, p65-NF-kappa B, p50-NF-kappa B and p36-I kappa B alpha, can be detected as complexes using antisera against c-Rel, p105-NF-kappa B or p65-NF-kappa B. p36-I kappa B alpha and p105, both known inhibitors of NF-kappa B function, can physically associate with NF-kappa B/Rel family members, but not with each other. In vivo and in vitro phosphorylation experiments demonstrated that NF-kappa B/Rel family members, including p105, c-Rel, p50, p65 (for the first time for p50 and p65) and p36-I kappa B alpha are also phosphoproteins. Phosphoserine and phosphothreonine residues were identified in these proteins isolated from unstimulated Jurkat cells. Both unphosphorylated and hyperphosphorylated forms of p36-I kappa B alpha were found in the complexes, suggesting that hyperphosphorylated I kappa B alpha is still capable of associating with the NF-kappa B/Rel family members. After stimulation with phorbol 12-myristate 13-acetate and phytohaemagglutinin for 10 min, p105-NF-kappa B and p50-NF-kappa B, but not p36-I kappa B, were highly phosphorylated. Phosphopeptide mapping of p105 showed that phorbol ester/phytohaemagglutinin stimulation may change p105 phosphorylation qualitatively.

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