Blog
About

4
views
0
recommends
+1 Recommend
0 collections
    0
    shares
      • Record: found
      • Abstract: found
      • Article: not found

      Characterization of pICln phosphorylation state and a pICln-associated protein kinase.

      Biochimica et Biophysica Acta

      Tumor Cells, Cultured, Amino Acid Sequence, Animals, Chloride Channels, metabolism, Molecular Sequence Data, Peptide Mapping, Phosphorylation, Protein Kinases, Rats

      Read this article at

      ScienceOpenPubMed
      Bookmark
          There is no author summary for this article yet. Authors can add summaries to their articles on ScienceOpen to make them more accessible to a non-specialist audience.

          Abstract

          pICln is a ubiquitous cellular protein that has been proposed to be a volume-sensitive Cl- channel or a channel regulator. Detailed biochemical, cellular and molecular characterization of pICln is required to understand its function. Our goal in the present investigation was to define further the biochemical properties of pICln and the proteins that associate with it. Immunoprecipitation of pICln from 32P-orthophosphoric acid-labeled C6 glioma cells revealed that the protein is phosphorylated constitutively, primarily on serine residues. Protein kinase activity was detected in pICln immunoprecipitates, revealing that a constitutively active protein kinase co-precipitates with pICln. A specific association between pICln and a protein kinase was also observed in affinity assays using a recombinant GST-pICln fusion protein. The pICln-associated kinase displayed broad substrate specificity and was inhibited in a concentration-dependent manner by heparin, zinc and 5,6-dichloro-1-beta-D-ribofuranosylbenose (DRB). These characteristics resembled those of casein kinase I and II. The pICln-associated kinase was not recognized, however, by antibodies against these two enzymes. Association of the kinase with pICln was disrupted by increasing concentrations of NaCl in the washing buffer, suggesting that electrostatic interactions are involved in kinase binding. Mutagenesis experiments corroborated this observation. Truncation of pICln demonstrated that two highly charged clusters of acidic amino acid residues are both necessary and sufficient for kinase binding. Phosphopeptide mapping demonstrated that pICln contains at least two phosphorylated serine residues that are located on trypsin cleavage fragments rich in acidic amino acid residues. We propose that the kinase or a kinase binding protein binds to acidic amino acids located between D101 and Y156 and phosphorylates nearby serine residues.

          Related collections

          Author and article information

          Journal
          9659371

          Comments

          Comment on this article