Identification and protein kinase C-dependent phosphorylation of alpha-adducin in human fibroblasts.
A protein of Mr approximately 120,000, related to the human erythrocyte membrane skeletal protein alpha-adducin, has been identified by immunological criteria in human fibroblasts. Using similar methods, beta-adducin (an Mr approximately 110,000 protein that forms a dimeric complex with alpha-adducin in the erythrocyte) is not present in fibroblasts. Subcellular distribution studies reveal that fibroblast alpha-adducin is largely associated with the particulate fraction and is most effectively solubilized from that fraction by a combination of nonionic detergent and high salt. Immunocytochemistry of quiescent fibroblasts shows that alpha-adducin is clustered in large perinuclear arrays that may correspond to vesicular structures; weak staining was also found in the sub-plasma membrane region. As in erythrocytes, the phosphorylation of fibroblast alpha-adducin is elevated on exposure of cells to phorbol esters that activate protein kinase C (PK-C). In addition, various mitogens such as serum, bradykinin and vasopressin also stimulate alpha-adducin phosphorylation by a PK-C-dependent pathway. The elevation in alpha-adducin phosphorylation is maintained for up to 30 min after mitogen addition. Peptide maps of phospho-alpha-adducin from both fibroblasts and erythrocytes after PK-C-mediated phosphorylation showed multiple phosphorylated peptides but with dissimilar migration patterns, suggesting divergence of structure around the phosphorylation sites. Adducin appears to play an important role in the regulation of spectrin-actin interactions in the red cell and may play a role in cytoskeletal function in the fibroblasts.
Waseem
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A
Department of Pharmacological and Physiological Sciences, University of Chicago, IL 60637.
Palfrey
H C
HC
D002148:Calmodulin-Binding Proteins / Q000378:metabolism*; D005347:Fibroblasts / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D006801:Humans; D066298:In Vitro Techniques; D008856:Microscopy, Fluorescence; D010766:Phosphorylation; D011493:Protein Kinase C / Q000502:physiology*; D000099291:Adducins
D002148:Calmodulin-Binding Proteins; D011493:Protein Kinase C; D000099291:Adducins