Neurofilament-associated protein phosphatase 2A: its possible role in preserving neurofilaments in filamentous states.
Neurofilament phosphatase (NF-phosphatase) activity, which dephosphorylates NF proteins phosphorylated by cyclic AMP-dependent protein kinase (A-kinase), was detected in NF fractions prepared from bovine spinal cords. This phosphatase was suggested to be associated with NFs by gel filtration and sedimentation analysis and was further demonstrated by dephosphorylation-dependent binding assay of NFs to microtubules. The NF-associated NF-phosphatase was identified as a type of protein phosphatase 2A (PP2A) by (i) its complete inhibition with 100 nM okadaic acid, at which concentration the purified type 1 protein phosphatase (PP1) was inhibited only 25%; (ii) the absence of effect of inhibitor-2, a specific inhibitor of PP1, on the NF-phosphatase activity; and (iii) the detection of 38-kDa catalytic and 65-kDa regulatory subunits of PP2A by immunoblotting. The NF-associated PP2A was partially solubilized from NFs by a high concentration of MgSO4, and the solubilized PP2A was suggested by gel filtration to be a dimeric holoenzyme consisting of a 38-kDa catalytic and a 65-kDa regulatory subunit. Phosphorylated NF-L, which is assembly incompetent, was induced to assemble into filaments by dephosphorylation with PP2A. These results suggest a role of NF-associated PP2A in preserving filamentous forms of NF in neurons.
Saito
T
T
Laboratory of Cell and Developmental Biology, Faculty of Biosciences, Tokyo Institute of Technology, Yokohama, Japan.
Shima
H
H
Osawa
Y
Y
Nagao
M
M
Hemmings
B A
BA
Kishimoto
T
T
Hisanaga
S
S
D008841:Actin Cytoskeleton / Q000648:ultrastructure; D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D002417:Cattle; D002458:Cell Fractionation; D002499:Centrifugation, Density Gradient; D002850:Chromatography, Gel; D007700:Kinetics; D008854:Microscopy, Electron; D008870:Microtubules / Q000648:ultrastructure; D018482:Muscle, Skeletal / Q000201:enzymology*; D016900:Neurofilament Proteins / Q000302:isolation & purification / Q000378:metabolism*; D010749:Phosphoprotein Phosphatases / Q000302:isolation & purification / Q000378:metabolism*; D011494:Protein Kinases / Q000378:metabolism; D054648:Protein Phosphatase 2; D011817:Rabbits; D013116:Spinal Cord / Q000737:chemistry / Q000648:ultrastructure; D013379:Substrate Specificity
D016900:Neurofilament Proteins; D011494:Protein Kinases; D000469:Alkaline Phosphatase; D010749:Phosphoprotein Phosphatases; D054648:Protein Phosphatase 2