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Divergent and convergent roles for kinases and phosphatases in neurofilament dynamics

ABSTRACT C-terminal neurofilament phosphorylation mediates cation-dependent self-association leading to neurofilament incorporation into the stationary axonal cytoskeleton. Multiple kinases phosphorylate the C-terminal domains of the heavy neurofilament subunit (NF-H), including cyclin-dependent protein kinase 5 (CDK5), mitogen-activated protein kinases (MAPKs), casein kinase 1 and 2 (CK1 and CK2) and glycogen synthase kinase 3&bgr; (GSK3&bgr;). The respective contributions of these kinases have been confounded because they phosphorylate multiple substrates in addition to neurofilaments and display extensive interaction. Herein, differentiated NB2a/d1 cells were transfected with constructs expressing GFP-tagged NF-H, isolated NF-H sidearms and NF-H lacking the distal-most 187 amino acids. Cultures were treated with roscovitine, PD98059, Li+, D4476, tetrabromobenzotriazole and calyculin, which are active against CDK5, MKK1 (also known as MAP2K1), GSK3&bgr;, CK1, CK2 and protein phosphatase 1 (PP1), respectively. Sequential phosphorylation by CDK5 and GSK3&bgr; mediated the neurofilament–neurofilament associations. The MAPK pathway (i.e. MKK1 to ERK1/2) was found to downregulate GSK3&bgr;, and CK1 activated PP1, both of which promoted axonal transport and restricted neurofilament–neurofilament associations to axonal neurites. The MAPK pathway and CDK5, but not CK1 and GSK3&bgr;, inhibited neurofilament proteolysis. These findings indicate that phosphorylation of neurofilaments by the proline-directed MAPK pathway and CDK5 counterbalance the impact of phosphorylation of neurofilaments by the non-proline-directed CK1 and GSK3&bgr;.