Divergent and convergent roles for kinases and phosphatases in neurofilament dynamics

Abstract

C-terminal neurofilament (NF) phosphorylation mediates cation-dependent associations leading to incorporation into the stationary axonal cytoskeleton. Multiple kinases phosphorylate NF-H C-terminal domains, including cyclin-dependent protein kinase 5 (cdk5), mitogen-activated protein kinase (MAPk), casein kinase 1 and 2 (CK1, CK2), glycogen synthase kinase 3 beta (GSK). The respective contributions of these kinases have been confounded since they phosphorylate multiple substrates in addition to NFs and display extensive interaction. Herein, differentiated NB2a/d1 cells were transfected with constructs expressing green fluorescent protein-tagged NF-H, isolated NF-H sidearms and NF-H lacking the distal-most 187 amino acids. Cultures were treated with roscovitine, PD98059, lithium, D4476, tetraBromoBenzoleTriazole, and calyculin, which are active against cdk5, MKK1, GSK, CK1, CK2, and protein phosphatase 1 (PP1), respectively. Sequential phosphorylation by cdk5 and GSK mediated NF-NF associations. MAPk downregulated GSK and CK1 activated PP1, both of which promoted axonal transport and restricted NF-NF associations to axonal neurites. MAPk and cdk5, but not CK1 and GSK, inhibited NF proteolysis. These findings indicate that phosphorylation of NFs by the proline-directed kinases MAPk and cdk5 counterbalance the impact of phosphorylation of NFs by the non-proline-directed CK1 and GSK.