title: Phosphorylation of the peripherin 58-kDa neuronal intermediate filament protein. Regulation by nerve growth factor and other agents.
abstract: Peripherin, a recently described member of the intermediate filament multigene family, is present in peripheral and certain central nervous system neurons as well as in cultured neuron-like cell lines, including PC12 pheochromocytoma cells. In PC12 cells, peripherin appears to be the major intermediate filament protein and its relative levels and synthesis are specifically increased during nerve growth factor (NGF)-promoted neuronal differentiation. The present study examines the phosphorylation of peripherin and the regulation thereof by nerve growth factor and other agents in cultured PC12 cells. Immunoblotting experiments using a peripherin-specific antiserum show five distinct isoforms of this protein in whole cell and cytoskeletal extracts resolved by two-dimensional isoelectric focusing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Three of these isoforms incorporate detectable quantities of [32P]phosphate during metabolic radiolabeling. The small proportion (approximately 6%) of total cellular peripherin that is extractable with 1% Triton X-100, does not appear to incorporate phosphate. NGF increases peripherin phosphorylation by 2-3-fold within 1-2 h of treatment. Epidermal growth factor and insulin have no effect. The relative levels of phosphorylated peripherin are markedly elevated (17-fold) by long term NGF exposure, and peripherin becomes a major cytoskeletal phosphoprotein. Activators of protein kinases A and C and treatment with depolarizing levels of K+ also enhance peripherin phosphorylation by 2-3-fold, in cultures both with and without prior long term NGF treatment. Evidence is presented that NGF regulates peripherin phosphorylation by a mechanism independent of protein kinases A and C and of depolarization. The large increase in phosphorylated peripherin brought about by NGF treatment suggests that this neuronal filament protein may play a role in the elaboration and maintenance of neurites. The presence of multiple independent pathways that acutely enhance peripherin phosphorylation indicates that this role is subject to modulation by extrinsic signals.
authors.affiliation: Department of Pathology, Columbia University College of Physicians and Surgeons, New York, New York 10032.
authors.forename: J M
authors.initials: JM
authors.lastname: Aletta
authors.forename: M L
authors.initials: ML
authors.lastname: Shelanski
authors.forename: L A
authors.initials: LA
authors.lastname: Greene
mesh_terms: D000310:Adrenal Gland Neoplasms; D000818:Animals; D002454:Cell Differentiation; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D015151:Immunoblotting; D007381:Intermediate Filament Proteins / Q000378:metabolism*; D007525:Isoelectric Focusing; D007526:Isoelectric Point; D008562:Membrane Glycoproteins; D008970:Molecular Weight; D009414:Nerve Growth Factors / Q000494:pharmacology*; D009419:Nerve Tissue Proteins; D009474:Neurons / Q000378:metabolism*; D064531:Peripherins; D010673:Pheochromocytoma; D010710:Phosphates / Q000378:metabolism; D010766:Phosphorylation; D011188:Potassium / Q000494:pharmacology; D051381:Rats; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013873:Thionucleotides / Q000494:pharmacology; D014407:Tumor Cells, Cultured
chemical_list: D007381:Intermediate Filament Proteins; D008562:Membrane Glycoproteins; D009414:Nerve Growth Factors; D009419:Nerve Tissue Proteins; D064531:Peripherins; D010710:Phosphates; D013873:Thionucleotides; C015929:8-((4-chlorophenyl)thio)cyclic-3',5'-AMP; D000242:Cyclic AMP; D013755:Tetradecanoylphorbol Acetate; D011188:Potassium