Phosphorylation of Neurofilament Proteins in Isolated Goldfish Mauthner Axoplasm

Abstract: The six neurofilament proteins (NFPs) in the goldfish Mauthner axon (M-axon) have molecular sizes of 235, 145, 123, 105, 80, and 60 kDa. To determine if NFPs in the M-axon are phosphorylated, isolated Mauthner axoplasm (M-axoplasm) and a neurofilament-enriched extract (NFE) prepared from M-axoplasm were incubated with ³²P, which resulted in the radiolabeling of NFPs as determined by their detection on autoradiograms. Kinase inhibitors directed against cyclic AMP-dependent kinases (PKAs) or cofactor-independent kinases significantly reduced the in vitro phosphorylation of NFPs in NFE, whereas inhibitors directed against protein kinase C did not significantly reduce the in vitro phosphorylation of NFPs in NFE. Experiments using two kinase inhibitors directed against different kinases significantly reduced the in vitro phosphorylation of NFPs in NFE to a greater extent than the reduction produced using any single kinase inhibitor. These data suggest that NFPs in the M-axon are phosphorylated and that the in vitro (and perhaps the in vivo) phosphorylation of NFPs is mediated by PKA and/or cofactor-independent kinases that copurify with NFPs.