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Tony Hunter - One of the best experts on this subject based on the ideXlab platform.

  • Phosphopeptide mapping and Phosphoamino Acid Analysis by electrophoresis and chromatography on thin‐layer cellulose plates
    Electrophoresis, 1994
    Co-Authors: Peter Van Der Geer, Tony Hunter
    Abstract:

    : Identification of protein phosphorylation sites is essential in order to evaluate the contribution of individual sites to the regulation of a particular protein by phosphorylation. Here we review a method we have developed for the identification of phosphorylation sites based on digestion of 32P-labeled proteins with site-specific proteases and separation of the digestion products in two dimensions on thin-layer cellulose plates using electrophoresis in the first dimension followed by chromatography. This method is very sensitive, requiring only a few hundred 32P-disintegrations per minute to obtain reproducible phosphopeptide maps. We also report methods for the Analysis of the Phosphoamino Acid content of both intact phosphoproteins and individual phosphopeptides recovered from two-dimensional separations, in which the material is subjected to partial Acid hydrolysis, and the hydrolysis products are separated on thin-layer cellulose plates by electrophoresis in one or two dimensions. Finally, we describe methods for analyzing the structure of isolated phosphopeptides by secondary digestion with site-specific proteases, by manual Edman degradation, and by immunoprecipitation, and indicate how this information can be used in conjunction with the two-dimensional mobility of the peptide to deduce the identity of a phosphopeptide from the known sequence of a protein.

  • phosphopeptide mapping and Phosphoamino Acid Analysis on cellulose thin layer plates
    Cell Biology#R##N#A Laboratory Handbook Volume 3, 1994
    Co-Authors: Peter Van Der Geer, Bartholomew M Sefton, Tony Hunter
    Abstract:

    Publisher Summary This chapter discusses the use of phosphopeptide mapping and Phosphoamino Acid Analysis on thin-layer cellulose. Phosphopeptide mapping is an important technique in the study of protein phosphorylation. It is used to determine the number and precise identity of sites of phosphorylation, to estimate the stoichiometry of phosphorylation at particular sites, and to deduce the identity of protein kinases responsible for their phosphorylation. Additionally, comparative phosphopeptide mapping is an invaluable tool for determining the identity, or lack of identity of phosphoproteins obtainable only in trace amounts. Phosphopeptide mapping has the advantage that it is extremely sensitive. In addition, individual phosphopeptides can be isolated from the inert cellulose coating of the plate, and used for further characterization including Phosphoamino Acid determination, N-terminal sequencing, and secondary digestion with additional proteases and chemicals.

  • phosphopeptide mapping and Phosphoamino Acid Analysis by electrophoresis and chromatography on thin layer cellulose plates
    Electrophoresis, 1994
    Co-Authors: Peter Van Der Geer, Tony Hunter
    Abstract:

    : Identification of protein phosphorylation sites is essential in order to evaluate the contribution of individual sites to the regulation of a particular protein by phosphorylation. Here we review a method we have developed for the identification of phosphorylation sites based on digestion of 32P-labeled proteins with site-specific proteases and separation of the digestion products in two dimensions on thin-layer cellulose plates using electrophoresis in the first dimension followed by chromatography. This method is very sensitive, requiring only a few hundred 32P-disintegrations per minute to obtain reproducible phosphopeptide maps. We also report methods for the Analysis of the Phosphoamino Acid content of both intact phosphoproteins and individual phosphopeptides recovered from two-dimensional separations, in which the material is subjected to partial Acid hydrolysis, and the hydrolysis products are separated on thin-layer cellulose plates by electrophoresis in one or two dimensions. Finally, we describe methods for analyzing the structure of isolated phosphopeptides by secondary digestion with site-specific proteases, by manual Edman degradation, and by immunoprecipitation, and indicate how this information can be used in conjunction with the two-dimensional mobility of the peptide to deduce the identity of a phosphopeptide from the known sequence of a protein.

  • phosphopeptide mapping and Phosphoamino Acid Analysis by two dimensional separation on thin layer cellulose plates
    Methods in Enzymology, 1991
    Co-Authors: William J Boyle, Peter Van Der Geer, Tony Hunter
    Abstract:

    Publisher Summary This chapter discusses the phosphopeptide mapping and Phosphoamino Acid Analysis by two-dimensional separation on thin-layer cellulose plates. Peptide mapping is a powerful technique used to help determine peptide structure and composition of proteins. Peptide maps or fingerprints of proteolyzed proteins are usually obtained by resolution on either one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), reversed-phase high-performance liquid chromatography (HPLC), or by two-dimensional separation on thin-layer cellulose (TLC) plates. The most common applications of peptide mapping are (1) to compare proteins encoded by the same or related genes, (2) to prepare individual peptides for determining amino Acid composition and sequence, and (3) to determine the precise location of amino Acid residues that are posttranslationally modified by fatty Acid acylation, glycosylation, methylation, acetylation, or phosphorylation.

Bartholomew M Sefton - One of the best experts on this subject based on the ideXlab platform.

  • unit 13 3 Phosphoamino Acid Analysis
    Current protocols in protein science, 2001
    Co-Authors: Bartholomew M Sefton
    Abstract:

    It is often valuable to identify the phosphorylated residue in a protein. This unit presents a protocol for partial Acid hydrolysis of proteins phosphorylated at serine, threonine, or tyrosine, followed by two-dimensional thin-layer electrophoresis of the labeled Phosphoamino Acid. Phosphothreonine and phosphotyrosine are more stable to hydrolysis in alkali than are RNA and phosphoserine. Therefore, an alternate procedure using mild alkaline hydrolysis of protein samples to enhance the detection of phosphothreonine and phosphotyrosine is also provided.

  • Phosphoamino Acid Analysis
    Current protocols in pharmacology, 1999
    Co-Authors: Bartholomew M Sefton
    Abstract:

    : Proteins involved in signal transduction are often phosphorylated. Determination of the specific amino Acid residue(s) involved is used in characterizing the particular pathway. Partial Acid hydrolysis of phosphorylated proteins followed by two-dimensional thin layer chromatography is used to identify the phosphorylated residues of the protein as phosphoserine, phosphothreonine, or phosphotyrosine. Mild alkaline hydrolysis is used to enhance detection of phosphothreonine and phosphotyrosine.

  • Phosphoamino Acid Analysis
    Current protocols in protein science, 1997
    Co-Authors: Bartholomew M Sefton
    Abstract:

    : It is often valuable to identify the phosphorylated residue in a protein. In the case of proteins phosphorylated at serine, threonine, or tyrosine, this is readily accomplished by partial Acid hydrolysis in HCl followed by two-dimensional thin-layer electrophoresis of the labeled Phosphoamino Acid, as described here. Phosphothreonine andphosphotyrosine are more stable to hydrolysis in alkali than are RNA andpho sphoserine. Therefore, a protocol for mild alkaline hydrolysis of protein samples is also provided to enhance the detection of phosphothreonine and phosphotyrosine. Although this procedure can be carried out with a protein eluted from a preparative gel and concentrated by trichloroacetic Acid or acetone precipitation, it is most easily accomplished by transfer of the protein of interest to a PVDF membrane.

  • Current Protocols in Molecular Biology - Phosphoamino Acid Analysis
    Current protocols in protein science, 1997
    Co-Authors: Bartholomew M Sefton
    Abstract:

    : It is often valuable to identify the phosphorylated residue in a protein. In the case of proteins phosphorylated at serine, threonine, or tyrosine, this is readily accomplished by partial Acid hydrolysis in HCl followed by two-dimensional thin-layer electrophoresis of the labeled Phosphoamino Acid, as described here. Phosphothreonine andphosphotyrosine are more stable to hydrolysis in alkali than are RNA andpho sphoserine. Therefore, a protocol for mild alkaline hydrolysis of protein samples is also provided to enhance the detection of phosphothreonine and phosphotyrosine. Although this procedure can be carried out with a protein eluted from a preparative gel and concentrated by trichloroacetic Acid or acetone precipitation, it is most easily accomplished by transfer of the protein of interest to a PVDF membrane.

  • phosphopeptide mapping and Phosphoamino Acid Analysis on cellulose thin layer plates
    Cell Biology#R##N#A Laboratory Handbook Volume 3, 1994
    Co-Authors: Peter Van Der Geer, Bartholomew M Sefton, Tony Hunter
    Abstract:

    Publisher Summary This chapter discusses the use of phosphopeptide mapping and Phosphoamino Acid Analysis on thin-layer cellulose. Phosphopeptide mapping is an important technique in the study of protein phosphorylation. It is used to determine the number and precise identity of sites of phosphorylation, to estimate the stoichiometry of phosphorylation at particular sites, and to deduce the identity of protein kinases responsible for their phosphorylation. Additionally, comparative phosphopeptide mapping is an invaluable tool for determining the identity, or lack of identity of phosphoproteins obtainable only in trace amounts. Phosphopeptide mapping has the advantage that it is extremely sensitive. In addition, individual phosphopeptides can be isolated from the inert cellulose coating of the plate, and used for further characterization including Phosphoamino Acid determination, N-terminal sequencing, and secondary digestion with additional proteases and chemicals.

Peter Van Der Geer - One of the best experts on this subject based on the ideXlab platform.

  • Phosphopeptide Mapping: A Basic Protocol
    Cell Biology, 2020
    Co-Authors: Jill Meisenhelder, Peter Van Der Geer
    Abstract:

    Publisher Summary Peptide mapping is a technique in which a radioactively labeled protein is digested with a sequence specific protease. The resulting peptides are separated in two dimensions on a thin-layer cellulose (TLC) plate by electrophoresis and chromatography. Proteins are usually separated from other contaminating proteins by SDS-PAGE and then subjected to phosphopeptide mapping or Phosphoamino Acid Analysis. Line the gel up with the film using the markings on the paper backing and autorad, localize the protein of interest, and cut the protein band out of the gel with a clean, single edge razor or a surgical blade. Remove the paper backing from the gel slices by scraping gently with a razor blade. Phosphopeptide mixtures are usually separated by electrophoresis in the horizontal dimension and chromatography in the vertical dimension. The mixture is spotted 3 cm from the bottom of the plate and 5 cm from the left side for electrophoresis at pH 1.9 or pH 4.72 or in the center of the plate for electrophoresis at pH 8.9.

  • Phosphopeptide mapping and Phosphoamino Acid Analysis by electrophoresis and chromatography on thin‐layer cellulose plates
    Electrophoresis, 1994
    Co-Authors: Peter Van Der Geer, Tony Hunter
    Abstract:

    : Identification of protein phosphorylation sites is essential in order to evaluate the contribution of individual sites to the regulation of a particular protein by phosphorylation. Here we review a method we have developed for the identification of phosphorylation sites based on digestion of 32P-labeled proteins with site-specific proteases and separation of the digestion products in two dimensions on thin-layer cellulose plates using electrophoresis in the first dimension followed by chromatography. This method is very sensitive, requiring only a few hundred 32P-disintegrations per minute to obtain reproducible phosphopeptide maps. We also report methods for the Analysis of the Phosphoamino Acid content of both intact phosphoproteins and individual phosphopeptides recovered from two-dimensional separations, in which the material is subjected to partial Acid hydrolysis, and the hydrolysis products are separated on thin-layer cellulose plates by electrophoresis in one or two dimensions. Finally, we describe methods for analyzing the structure of isolated phosphopeptides by secondary digestion with site-specific proteases, by manual Edman degradation, and by immunoprecipitation, and indicate how this information can be used in conjunction with the two-dimensional mobility of the peptide to deduce the identity of a phosphopeptide from the known sequence of a protein.

  • phosphopeptide mapping and Phosphoamino Acid Analysis on cellulose thin layer plates
    Cell Biology#R##N#A Laboratory Handbook Volume 3, 1994
    Co-Authors: Peter Van Der Geer, Bartholomew M Sefton, Tony Hunter
    Abstract:

    Publisher Summary This chapter discusses the use of phosphopeptide mapping and Phosphoamino Acid Analysis on thin-layer cellulose. Phosphopeptide mapping is an important technique in the study of protein phosphorylation. It is used to determine the number and precise identity of sites of phosphorylation, to estimate the stoichiometry of phosphorylation at particular sites, and to deduce the identity of protein kinases responsible for their phosphorylation. Additionally, comparative phosphopeptide mapping is an invaluable tool for determining the identity, or lack of identity of phosphoproteins obtainable only in trace amounts. Phosphopeptide mapping has the advantage that it is extremely sensitive. In addition, individual phosphopeptides can be isolated from the inert cellulose coating of the plate, and used for further characterization including Phosphoamino Acid determination, N-terminal sequencing, and secondary digestion with additional proteases and chemicals.

  • phosphopeptide mapping and Phosphoamino Acid Analysis by electrophoresis and chromatography on thin layer cellulose plates
    Electrophoresis, 1994
    Co-Authors: Peter Van Der Geer, Tony Hunter
    Abstract:

    : Identification of protein phosphorylation sites is essential in order to evaluate the contribution of individual sites to the regulation of a particular protein by phosphorylation. Here we review a method we have developed for the identification of phosphorylation sites based on digestion of 32P-labeled proteins with site-specific proteases and separation of the digestion products in two dimensions on thin-layer cellulose plates using electrophoresis in the first dimension followed by chromatography. This method is very sensitive, requiring only a few hundred 32P-disintegrations per minute to obtain reproducible phosphopeptide maps. We also report methods for the Analysis of the Phosphoamino Acid content of both intact phosphoproteins and individual phosphopeptides recovered from two-dimensional separations, in which the material is subjected to partial Acid hydrolysis, and the hydrolysis products are separated on thin-layer cellulose plates by electrophoresis in one or two dimensions. Finally, we describe methods for analyzing the structure of isolated phosphopeptides by secondary digestion with site-specific proteases, by manual Edman degradation, and by immunoprecipitation, and indicate how this information can be used in conjunction with the two-dimensional mobility of the peptide to deduce the identity of a phosphopeptide from the known sequence of a protein.

  • phosphopeptide mapping and Phosphoamino Acid Analysis by two dimensional separation on thin layer cellulose plates
    Methods in Enzymology, 1991
    Co-Authors: William J Boyle, Peter Van Der Geer, Tony Hunter
    Abstract:

    Publisher Summary This chapter discusses the phosphopeptide mapping and Phosphoamino Acid Analysis by two-dimensional separation on thin-layer cellulose plates. Peptide mapping is a powerful technique used to help determine peptide structure and composition of proteins. Peptide maps or fingerprints of proteolyzed proteins are usually obtained by resolution on either one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), reversed-phase high-performance liquid chromatography (HPLC), or by two-dimensional separation on thin-layer cellulose (TLC) plates. The most common applications of peptide mapping are (1) to compare proteins encoded by the same or related genes, (2) to prepare individual peptides for determining amino Acid composition and sequence, and (3) to determine the precise location of amino Acid residues that are posttranslationally modified by fatty Acid acylation, glycosylation, methylation, acetylation, or phosphorylation.

Kee Ryeon Kang - One of the best experts on this subject based on the ideXlab platform.

  • Association and Phosphorylation of Deoxyhypusine Synthase with CK2
    Key Engineering Materials, 2020
    Co-Authors: Kee Ryeon Kang
    Abstract:

    Deoxyhypusine synthase (DHS) catalyzes the first step in the posttranslational synthesis of hypusine in the eukaryotic initiation factor 5A (eIF5A) precursor protein. As such, the phosphorylation of DHS by the protein kinase CK2 was investigated to define the role of DHS in the regulation of eIF5A in cells. The results showed that DHS was phosphorylated by CK2 in vivo as well as in vitro. The endogenous CK2 in HeLa cells and cell lysates was able to phosphorylate DHS and this modification was enhanced or decreased by the addition of CK2 effectors, such as polylysine, heparin, or poly (Glu, Tyr). A Phosphoamino Acid Analysis of the enzyme revealed that the DHS was mainly phosphorylated into the Thr residue, with the remainder into the Ser residue. Therefore, it would appear that the phosphorylation of DHS was a CK2-dependent cellular event, thereby opening the path for possible regulation of the interaction with the eIF5A precursor for hypusine synthesis.

  • regulation of chicken protein tyrosine phosphatase 1 and human protein tyrosine phosphatase 1b activity by casein kinase ii and p56lck mediated phosphorylation
    Experimental and Molecular Medicine, 2000
    Co-Authors: Kee Ryeon Kang
    Abstract:

    Protein tyrosine phosphorylation and dephosphorylation are important in the regulation of cell proliferation and signaling cascade. In order to examine whether phosphatase activity of CPTP1 and HPTP1B, typical nontransmembrane protein tyrosine phosphatase, could be controlled by phosphorylation, affinity-purified PTPs were phosphorylated by CKII and p56 lck in vitro. Phosphoamino Acid Analysis revealed that CPTP1 was phosphorylated on both serine and threonine residues by CKII, and tyrosine residue by p56 lck . Phosphatase activity of CPTP1 was gradually increased by three-fold concomitant with phosporylation by CKII. Phosphorylation of HPTP1B by CKII resulted in quick two-fold enhancement of its phosphatase activity within 5 min of incubation and remained in that state. In the presence of CKII inhibitor, heparin or poly(Glu.Tyr), both phosphorylation and enhancement of phosphatase activity of CPTP1 and HPTP1B were mostly blocked. p56 lck catalyzed tyrosine phosphorylation of CPTP1 and HPTP1B was only observed by inhibiting the intrinsic tyrosine phosphatase activity. Taken together, these results indicate that CPTP1 or HPTP1B possesses a capability to regulate its phosphatase activity through phosphorylation processes and may participate in the cellular signal cascades.

Shain-dow Kung - One of the best experts on this subject based on the ideXlab platform.

  • Molecular cloning and biochemical characterization of a receptor-like serine/threonine kinase from rice
    Plant Molecular Biology, 1994
    Co-Authors: Yan Zhao, Xin-hua Feng, John C. Watson, Paul J. Bottino, Shain-dow Kung
    Abstract:

    A receptor-like protein kinase, OsPK10, has been cloned from rice ( Oryza sativa ). The 2.8 kb cDNA contains an open reading frame capable of encoding a peptide sequence of 824 amino Acids. The topological features of the predicted OsPK10 protein include an N-terminal signal peptide, a cysteine-rich extracellular ligand-binding domain, a membrane-spanning segment, and a cytoplasmic domain possessing all the hallmarks of catalytic domains of eukaryotic protein kinases. The cytoplasmic domain was selectively expressed in Escherichia coli and assayed for kinase activity. The results show the protein is capable of autophosphorylation using either ATP or GTP as the phosphate donor. Phosphoamino Acid Analysis reveals phosphorylation of threonines, consistent with the substrate specificity indicated by sequence motifs in the catalytic core. A single amino Acid substitution of Glu for Lys-528 completely abolishes autophosphorylation activity. DNA gel blot analyses suggest that the haploid rice genome contains a single copy of the OsPK10 gene. OsPK10 transcripts appear to be more abundant in shoots than in roots of rice seedlings.