Differential cellular phosphorylation of neurofilament heavy side-arms by glycogen synthase kinase-3 and cyclin-dependent kinase-5.
To investigate the cellular mechanisms regulating neurofilament-heavy subunit (NF-H) side-arm phosphorylation, we studied the ability of three putative neurofilament kinases, glycogen synthase kinase-3 (GSK-3)alpha, GSK-3 beta, and cyclin-dependent kinase-5 (cdk-5), to phosphorylate NF-H in transfected cells. We analysed NF-H phosphorylation by using a panel of phosphorylation-dependent antibodies and also by monitoring the electrophoretic mobility of the transfected NF-H on sodium dodecyl sulphate-polyacrylamide gel electrophoresis because this is known to be affected by side-arm phosphorylation. Our results demonstrate that whereas GSK-3 alpha, GSK-3 beta, and cdk-5 will all phosphorylate NF-H, they generate different antibody reactivity profiles. GSK-3 alpha and GSK-3 beta induce a partial retardation of a proportion of the transfected NF-H, but only cdk-5 alters the rate of electrophoretic migration to that of NF-H from brain. We conclude that cdk-5 and GSK-3 phosphorylate different residues or sets of residues within NF-H sidearm in cells. We further show that cdk-5 is active in both the CNS and the PNS but that this activity is not dependent on expression of its activator, p35. This suggests that there are other activators of cdk-5.
Guidato
S
S
Department of Neuroscience, Institute of Psychiatry, London, England.
Tsai
L H
LH
Woodgett
J
J
Miller
C C
CC
D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D002460:Cell Line / Q000201:enzymology; D002490:Central Nervous System / Q000201:enzymology; D051360:Cyclin-Dependent Kinase 5; D018844:Cyclin-Dependent Kinases; D038362:Glycogen Synthase Kinase 3; D038341:Glycogen Synthase Kinases; D008869:Microtubule-Associated Proteins / Q000378:metabolism*; D016900:Neurofilament Proteins / Q000378:metabolism*; D017933:Peripheral Nervous System / Q000201:enzymology; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D051381:Rats
D008869:Microtubule-Associated Proteins; D016900:Neurofilament Proteins; D038341:Glycogen Synthase Kinases; D051360:Cyclin-Dependent Kinase 5; D017346:Protein Serine-Threonine Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; C495907:Cdk5 protein, rat; D018844:Cyclin-Dependent Kinases; D038362:Glycogen Synthase Kinase 3