Characterization of a cyclic nucleotide- and calcium-independent neurofilament protein kinase activity in axoplasm from the squid giant axon.
The phosphorylation activity associated with a neurofilament-enriched cytoskeletal preparation isolated from the squid giant axon has been studied and compared to the phosphorylation activities in intact squid axoplasm. The high molecular weight (greater than 300 kDa) and 220-kDa neurofilament proteins are the major endogenous substrates for the kinases in the axoplasm and the neurofilament preparation, whereas 95- and less than 60-kDa proteins are the major phosphoproteins in the ganglion cell preparation. The squid axon neurofilament (SANF) protein kinase activity appeared to be both cAMP and Ca2+ independent and could phosphorylate both casein (Km = 40 microM) and histone (Km = 180 microM). The SANF protein kinase could utilize either ATP or GTP in the phosphotransferase reaction, with a Km for ATP of 58 microM and 129.4 microM for GTP when casein was used as the exogenous substrate; and 25 and 98.1 microM for ATP and GTP, respectively, when the endogenous neurofilament proteins were used as substrates. The SANF protein kinase activity was only slightly inhibited by 2,3-diphosphoglycerate and various polyamines at high concentrations and was poorly inhibited by heparin (34% inhibition at 100 micrograms/ml). The failures of heparin to significantly inhibit and the polyamines to stimulate the SANF protein kinase indicate that it is not a casein type II kinase. The relative efficacy of GTP as a phosphate donor indicates that SANF protein kinase differs from known casein type I kinases. Phosphorylated (32P-labeled) neurofilament proteins were only slightly dephosphorylated in the presence of axoplasm or stellate ganglion cell supernatants, and the neurofilament-enriched preparation did not dephosphorylate 32P-labeled neurofilament proteins. The axoplasm and neurofilament preparations had no detectable protein kinase inhibitor activity, but a strong inhibitor activity, which was not dialyzable but was heat inactivatable, was found in ganglion cells. This inhibitor activity may account for the low phosphorylation activity found in the stellate ganglion cells and may indicate inhibitory regulation of SANF protein kinase activity in the ganglion cell bodies.
D000818:Animals; D001369:Axons / Q000166:cytology*; D002118:Calcium / Q000378:metabolism*; D000242:Cyclic AMP / Q000378:metabolism*; D006152:Cyclic GMP / Q000378:metabolism*; D003599:Cytoskeleton / Q000201:enzymology*; D049832:Decapodiformes; D006863:Hydrogen-Ion Concentration; D007382:Intermediate Filaments / Q000201:enzymology*; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D015636:Magnesium Chloride; D008854:Microscopy, Electron; D008970:Molecular Weight; D011494:Protein Kinases / Q000378:metabolism*; D012969:Sodium Fluoride / Q000494:pharmacology
D015636:Magnesium Chloride; D012969:Sodium Fluoride; D000242:Cyclic AMP; D011494:Protein Kinases; D006152:Cyclic GMP; D008274:Magnesium; D002118:Calcium