A protein kinase bound to the projection portion of MAP 2 (microtubule-associated protein 2)

Abstract

The microtubule-associated protein 2 (MAP 2) molecule was previously demonstrated to consist of 2 structural parts. One part of the molecule, referred to as the assembly-promoting domain, binds to the microtubule surface and is responsible for promoting microtubule assembly; the other represents a filamentous projection observed on the microtubule surface that may be involved in the interaction of microtubules with other cellular structures. MAP 2 is specifically phosphorylated as the result of a protein kinase activity present in microtubule preparations. In the present study this enzymatic activity co-purifies with the projection portion of [calf brain] MAP 2 itself. Kinase activity co-eluted with MAP 2 when microtubule protein was subjected to either gel-filtration chromatography on Bio-Gel A-15m or ion-exchange chromatography on DEAE-Sephadex. The activity was released from microtubules by mild digestion with chymotrypsin in parallel with the removal by the protease of the MAP 2 projections from the microtubule surface. The association of the activity with the projection was demonstrated directly by gel filtration chromatography of the projections on Bio-Gel A-15m. Three protein species (MW = 39,000, 55,000 and 70,000) cofractionated with MAP 2, and 2 of these (MW = 39,000 and 55,000) may represent the subunits of an associated cAMP-dependent protein kinase. The projection-associated activity was stimulated 10-fold by cAMP and was inhibited > 95% by the cAMP-dependent protein kinase inhibitor from rabbit skeletal muscle. It appeared to represent the only significant activity associated with microtubules, almost no activity being found with tubulin, other MAP, or the assembly-promoting domain of MAP 2; it was estimated to account for 7-22% of the total brain cytosolic protein kinase activity. The location of the kinase on the projection is consistent with a role in regulating the function of the projection, although other roles for the enzyme are also possible.