Dephosphorylation-dependent inhibitory activity of juxtanodin on filamentous actin disassembly.
In the vertebrate central nervous system, maturation of oligodendrocytes is accompanied by a dramatic transformation of cell morphology. Juxtanodin (JN) is an actin cytoskeleton-related oligodendroglial protein that promotes arborization of cultured oligodendrocytes. We performed in vitro and in culture experiments to further elucidate the biochemical effects, molecular interactions, and activity regulation of JN. Pulldown and co-sedimentation assays confirmed JN binding to filamentous but not globular beta-actin largely through a C-terminal domain of 14 amino acid residues. JN had much lower affinity to F-alpha-actin than to F-beta-actin. Bundling and actin polymerization assays revealed no JN influence on F-beta-actin cross-linking or G-beta-actin polymerization. Sedimentation assay, however, demonstrated that JN slowed the rate of F-beta-actin disassembly induced by dilution with F-actin depolymerization buffer. JN-S278E mutant, a mimic of phosphorylated JN at serine 278, exhibited a much diminished affinity/stabilizing effect on F-beta-actin. Immunoblotting revealed both phosphorylated and dephosphorylated native JN of the brain, with the former migrating slightly slower than the latter and becoming undetectable when brain lysate was subjected to in vitro dephosphorylation prior to being loaded for electrophoresis. In cultured OLN-93 cells, overexpression of JN promoted the formation of actin fibers and inhibited F-actin disassembly induced by latrunculin A. S278E phosphomimetic mutation resulted in loss of JN activity in cultured cells, whereas S278A, T258A, and T258E dephospho-/phosphomimetic mutations did not. These findings establish JN as an actin cytoskeleton-stabilizing protein that may play active roles in oligodendroglial differentiation and myelin formation. Specific phosphorylation of JN might serve as an important mechanism regulating JN functions.
Meng
Jun
J
Department of Anatomy, Yong Loo Lin School of Medicine, National University of Singapore, Singapore.
Xia
Wenhao
W
Tang
Junhong
J
Tang
Bor Luen
BL
Liang
Fengyi
F
D000199:Actins / Q000235:genetics / Q000378:metabolism*; D019943:Amino Acid Substitution; D000818:Animals; D001921:Brain / Q000166:cytology / Q000378:metabolism*; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000494:pharmacology; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology; D002460:Cell Line; D008840:Microfilament Proteins / Q000235:genetics / Q000378:metabolism*; D020125:Mutation, Missense; D009186:Myelin Sheath / Q000235:genetics / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D009836:Oligodendroglia / Q000166:cytology / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects / Q000502:physiology; D011485:Protein Binding / Q000187:drug effects / Q000502:physiology; D055550:Protein Stability; D017434:Protein Structure, Tertiary; D051381:Rats; D053778:Thiazolidines / Q000494:pharmacology
D000199:Actins; D019086:Bridged Bicyclo Compounds, Heterocyclic; C505830:Ermn protein, rat; D008840:Microfilament Proteins; D009419:Nerve Tissue Proteins; D053778:Thiazolidines; C037067:latrunculin A