Bw¢htmtca et Bwphyswa Acta, 755 (1983) 25-31 25 Elsevier Biomedmal Press BBA 21310 CHARACTERISTICS OF THE PROTEIN KINASE ACTIVITY ASSOCIATED WITH RAT NEUROFILAMENT PREPARATIONS JEAN-PIERREJULIEN, GERALDINE D SMOLUK and WALTER E. MUSHYNSKI* Department of Btochenustrv, McGtll Umverst O, 3655 Drummond St, Montreal. Quebec H3G 1 Y6 (Canada) (Received Jul) 7th, 1982) Ke~ uords Protein kmase, Neurofdament: (Rat) Some properties of the protein kinase activi~, associated with neurofilaments isolated from the brain stem and spinal cord of rats have been investigated. The activi~, had an apparent K m for ATP of 20 tx M, a pH optimum of 8.0 and phosphorylated both serine and threonine residues in neurofilament proteins. Cyclic AMP had no effect on the in vitro reaction and casein was a preferred exogenous substrate in comparison to histone. Phosphopeptide mapping of the 145 kDa subunit from neurofilaments phospho~,lated in the presence and absence of microtubule proteins indicated that the neurofilament-associated activity, was distinct from the microtubule-associated protein kinase. Limited proteolysis of neurofilaments with chymotrypsin indicated that the enzyme activity, was not associated with a domain of the 200 kDa subunit which may form the side-arm projections on neurofilaments. Introduction Phosphorylation appears to be a general prop- ertv of neurofilament proteins, hawng been ob- served under a variety of conditions in diverse >pecies such as the squid [1], the marine worm M1"xwola mfundibulum [2.3] and certain mammals [3-5]. The 68, 145 and 200 kDa subunits of mam- malian neurofilaments are phosphorylated in vitro by a protein kinase activity that co-purifies with neurofilament-enriched preparations isolated from central or peripheral nerve tissue by a varmty of methods [3-5]. A physmlogical basis for this as- sociation is indicated by the observed similarities between the phosphopeptide maps of the respec- tive in vivo and in vitro ~2p-labeled neurofilament subunits [5]. Available evidence suggests that microtubules may play a role in some aspect of neurofllament phosphorylation. Purified mtcrotubule-associated * To v, hom corre>pondence .',hould be addressed 0304-4165/83,0(}00-0000. $03 00 ' 1983 EIse',ter Btumedlt.al Pre,,,, protein 2 (microtubule-assoclated protein-2) is both an activator and a substrate of the bovine neuro- filament-associated protein kmase activity in vitro [4]. Since Microtubule-associated protein-2 con- tains a projection domain which extends from the microtubule wall [6] and may contact other organdies, including neurofilaments [7], this form of interaction may also take place in vivo. In addition, the microtubule-associated protein kinase can phosphorylate neurofilament proteins, and 145 kDa protein in particular, in vitro [8]. This micro- tubule-assocmted enzyme is a type II cyclic AMP- dependent protein kinase which is associated with the projection domain of microtubule-associated protein-2 [9]. Since the 200 kDa protein subunit of neurofilaments also contain.,, a projection domain which may correspond to the side-arms on neuro- filaments (unpublished data), it was of interest to determine whether the neurofilament-associated protein kinase is localized in a peripheral position analogous to that of the mlcrotubule-associated klnase.