MAP-2, the most conspicuous protein (MW 270,000) in mammalian brain that coassembles with tubulin, has previously been found subject to multisite phosphorylation at 2 subsets of sites. The A subset, in the microtubule-projection domain, are occupied in MAP-2 isolated after assembly cycles; these sites have been labeled by intraventricular injection of 32-Pi, but the phosphatase and kinase involved had not been identified. The 10 B subset, primarilly in the binding domain, are unoccupied in MAP-2 thus isolated; they can then be phosphorylated by cAMP kinase. It seemed not unlikely that they might be phosphorylated also in vivo, since a third of this brain kinase is complexed with MAP-2. We now have 2 phosphatase which can release the A subset, an intestinal alkaline phosphatase and an enzyme purified from brain using MAP-2 as assay substrate. Using MAP-2 thus depleted in A subset, we have determined which kinases can add additional residues after such phosphatase treatment. In addition to cAMP kinase, Ca/calmodulin and Ca/Plipid kinases were chosen initially because they are abundant in brain. The first 2 added a maximum of 10 residues regardless of prior phosphatase treatment. With Ca/Plipid kinase the plateau of """"""""maximum"""""""" phosphorylation varied with enzyme concentration, but at both 20 and 30 residues, additional phosphates were added corresponding exactly to the amount removed by phosphatase. We find MAP-2 rapidly isolated from adult rat brain has 30 phosphates, i.e. the A subset and 20 more, presumably released by phosphatase during assembly cycles. Incubation with cAMP kinase will show whether the latter 20 include the B subset. The project concerning tubulin modification by tyrosinolation was resumed, using Crithidia in place of HeLa. We have also made progress toward purifying the brain detyrosinolating carboxypeptidase to homogeneity.
