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 thr ee putative neurofilament kinases, glycogen synthase kinase-3 (GSK-3)a lpha, GSK-3 beta, and cyclin-dependent kinase-5 (cdk-5), to phosphoryl ate NF-H in transfected cells, We analysed NF-H phosphorylation by usi ng a panel of phosphorylation-dependent antibodies and also by monitor ing the electrophoretic mobility of the transfected NF-H on sodium dod ecyl sulphate- polyacrylamide gel electrophoresis because this is know n 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 alp ha 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 mi gration to that of NF-H from brain. We conclude that cdk-5 and GSK-3 p hosphorylate different residues or sets of residues within NF-H sidear ms 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 a ctivator, p35. This suggests that there are other activators of cdk-5.