Characterization of pI(Cln) phosphorylation state and a pI(Cln)-associated protein kinase
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abstract
pI(Cln) 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 pI(Cln) is required to understand its function. Our goal in the present investigation was to define further the biochemical properties of pI(Cln) and the proteins that associate with it. Immunoprecipitation of pI(Cln) 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 pI(Cln) immunoprecipitates, revealing that a constitutively active protein kinase co-precipitates with pI(Cln). A specific association between pI(Cln) and a protein kinase was also observed in affinity assays using a recombinant GST-pI(Cln) fusion protein. The pI(Cln)-associated kinase displayed broad substrate specificity and was inhibited in a concentration-dependent manner by heparin, zinc and 5,6-dichloro-1-β-d-ribofuranosylbenose (DRB). These characteristics resembled those of casein kinase I and II. The pI(Cln)-associated kinase was not recognized, however, by antibodies against these two enzymes. Association of the kinase with pI(Cln) 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 pI(Cln) demonstrated that two highly charged clusters of acidic amino acid residues are both necessary and sufficient for kinase binding. Phosphopeptide mapping demonstrated that pI(Cln) 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. Copyright (C) 1998 Elsevier Science B.V.
