A MAP-2-Stimulated Protein Kinase Activity Associated with Neurofilaments

Preparations of mammalian neurofilaments purified from brain consist primarily of four proteins: a triplet of molecular weights 210000,160000, and 69000, and tubulin. Incubation of bovine brain neurofilaments in the presence of adenosine 5'-[γ-32P] triphosphate ([γ-32P]ATP) brought about phosphorylation of each of these four proteins, indicating that a protein kinase was associated with the neurofilaments. The activity was not removed by centrifugal washing of the neurofilaments in a number of different buffers. The pattern, rate, and extent of phosphorylation were found to be unaffected by adenosine cyclic 3’,5'-monophosphate (cAMP), by preincubation with adenosine 5'-diphosphate, or by addition of a protein kinase inhibitor. The protein kinase is thus cAMP independent. When the high molecular weight microtubuleassociated protein MAP-2 was added to neurofilaments and the mixture was incubated with [γ-32P]ATP, the MAP-2 became extensively phosphorylated. In addition, the inclusion of MAP-2 brought about an increase in the extent of phosphorylation of the neurofilament proteins. These results show that neurofilaments possess a firmly associated protein kinase activity and that this activity is stimulated by the presence of MAP-2. Microtubule preparations made by cycles of assembly and disassembly can contain both protein kinase activity and small amounts of contaminating neurofilaments. Two distinct protein kinases were resolved from such microtubule protein by gel filtration on Bio-Gel A-150m. The cAMP-independent protein kinase activity was found in the void volume fractions, together with the contaminating neurofilaments. A cAMPdependent protein kinase activity was found in the included peak, together with the major proteins of microtubules. The neurofilament-associated protein kinase is thus distinct from, and separable from, the microtubule-associated protein kinase.