Characterization of pI(Cln) binding proteins: identification of p17 and assessment of the role of acidic domains in mediating protein-protein interactions.
pICln is a ubiquitous and abundant 27 kDa soluble protein that is localized primarily to the cytoplasm. The protein has been proposed to be a swelling-activated anion channel or a channel regulator. Recent studies, however, have cast significant doubt on these hypotheses, and the function of pI(Cln) therefore remains unknown. To further characterize the physiological role of pI(Cln), we have begun to identify the proteins that bind to it and the amino acid domains that mediate pICln protein-protein interactions. Using affinity assays and immunoprecipitation we have identified three proteins in C6 glioma cells with molecular masses of 17 kDa, 29 kDa and 72 kDa that bind selectively to pI(Cln). Microsequencing revealed that p17 is the non-muscle isoform of the 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 effect 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 indicate that the carboxy terminus is necessary for p72-pI(Cln) interaction. Based on these and other findings, we propose that pI(Cln) is a protein responsible for regulating the structure and function of the cytoskeleton, and/or a protein involved in mediating interactions between components of intracellular signal transduction pathways.
Emma
F
F
Laboratory of Cellular and Molecular Physiology, Departments of Anesthesiology and Pharmacology, Vanderbilt University Medical Center, 504 Oxford House, 1313 21st Avenue South, Nashville, TN 37232, USA.
Sanchez-Olea
R
R
Strange
K
K
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry* / Q000302:isolation & purification; D002460:Cell Line; D003593:Cytoplasm / Q000737:chemistry; D003599:Cytoskeleton / Q000737:chemistry / Q000502:physiology*; D005910:Glioma; D007473:Ion Channels / Q000737:chemistry; D008969:Molecular Sequence Data; D018994:Myosin Light Chains / Q000737:chemistry / Q000302:isolation & purification; D011496:Protein Methyltransferases; D017433:Protein Structure, Secondary; D011506:Proteins / Q000302:isolation & purification; D051381:Rats; D015398:Signal Transduction
D000596:Amino Acids; D002352:Carrier Proteins; D007473:Ion Channels; D018994:Myosin Light Chains; D011506:Proteins; D011496:Protein Methyltransferases