CHARACTERIZATION OF PI(CLN) PHOSPHORYLATION STATE AND A PI(CLN)-ASSOCIATED PROTEIN-KINASE

pI(Cln) is a ubiquitous cellular protein that has been proposed to be a volume-sensitive Cl- channel or a channel regulator. Detailed bioche mical, 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 protei ns that associate with it. Immunoprecipitation of pI(Cln) from (32) P- orthophosphoric acid-labeled C6 glioma cells revealed that the protein is phosphorylated constitutively, primarily on serine residues. Prote in kinase activity was detected in pI(Cln) immunoprecipitates, reveali ng 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) fusi on protein. The pI(Cln)-associated kinase displayed broad substrate sp ecificity and was inhibited in a concentration-dependent manner by hep arin, zinc and 5,6-dichloro-l-beta-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 disrupte d by increasing concentrations of NaCl in the washing buffer, suggesti ng that electrostatic interactions are involved in kinase binding. Mut agenesis experiments corroborated this observation. Truncation of pI(C ln) demonstrated that two highly charged clusters of acidic amino acid residues are both necessary and sufficient for kinase binding. Phosph opeptide mapping demonstrated that pI(Cln) contains at:least two phosp horylated serine residues that are located on trypsin cleavage fragmen ts rich in acidic amino acid residues;. We propose that the kinase or a kinase binding protein binds to acidic amino acids located between D 101 and Y156 and phosphorylates nearby serine residues. (C) 1998 Elsev ier Science B.V. All rights reserved.