CHARACTERIZATION OF PI(CLN) BINDING-PROTEINS - IDENTIFICATION OF P17 AND ASSESSMENT OF THE ROLE OF ACIDIC DOMAINS IN MEDIATING PROTEIN-PROTEIN INTERACTIONS

pI(Cln) is a ubiquitous and abundant 27 kDa soluble protein that is lo calized primarily to the cytoplasm. The protein has been proposed to b e a swelling-activated anion channel or a channel regulator. Recent st udies, however, have cast significant doubt on these hypotheses, and t he function of pI(Cln) therefore remains unknown. To further character ize the physiological role of pI(Cln), we have begun to identify the p roteins that bind to it and the amino acid domains that mediate pI(Cln ) protein-protein interactions. Using affinity assays and immunoprecip itation we have identified three proteins in C6 glioma cells with mole cular masses of 17 kDa, 29 kDa and 72 kDa that bind selectively to pI( Cln). Microsequencing revealed that p17 is the nonmuscle isoform of th e alkali myosin light chain. pI(Cln) contains three acidic amino acid domains termed AD1, AD2 and AD3. Mutation of AD1 and/or AD2 had no eff ect on p17, p29 and p72 binding. However, binding of p72 was lost when four acidic amino acid residues were mutated in AD3, which is located at the carboxy terminus. A truncation peptide containing the last 29 amino acids of pI(Cln) was able to bind p72 normally. These results in dicate that the carboxy terminus is necessary for p72-pI(Cln), interac tion. Based on these and other findings, we propose that pIcl, is a pr otein responsible for regulating the structure and function of the cyt oskeleton, and/or a protein involved in mediating interactions between components of intracellular signal transduction pathways. 0167-4889/9 8/$ - see front matter (C) 1998 Elsevier Science B.V. All rights reser ved.