Microtubule proteins in PC12 pheochromocytoma cells. Isolation, composition and in vitro phosphorylation

In the presence of nerve growth factor (NGF) PC12 pheochromocytoma cells develop properties of sympathetic neurons and extend long microtubule-containing neurites. PC12 cell microtubule protein was isolated using an assembly and disassembly procedure, either directly from an unlabeled cell extract or by copolymerization of a [35S]-methionine-labeled cell extract with rat brain microtubule proteins. Microtubule proteins of PC12 cells treated and untreated with NGF did not reveal any differences as analyzed by SDS-PAGE. The major constituents were the tubulin dimer and microtubule-associated proteins, mainly one high molecular weight component (HMW2) (Mr = 290,000), which resembles by several criteria the high molecular weight protein MAP2 of rat brain microtubule protein. PC12 cell microtubule protein contained intrinsic cAMP-dependent and cAMP-independent protein kinase activity. In vitro phosphorylation experiments indicated that, unlike in rat brain microtubule preparations, both subunits of the tubulin dimer were substrate for the endogenous kinase activity. The addition of cAMP to the incubation medium exerted a slight increase in phosphorylation of the HMW2 protein and an 80,000 polypeptide, and most effectively the phosphorylation of a Mr = 62,000 peptide was increased. This component could be identified as tyrosine hydroxylase by indirect immunoprecipitation procedures. Thus, the in vitro phosphorylation pattern of PC12 cell microtubule protein differed substantially from the one obtained from rat brain microtubule preparations.