Purification of a Heat-Stable Activator for Phospholipase C-γ1 from Bovine Brain Cytosol and its Identification as Microtubule-Associated Protein Tau

Abstract

During the purification of phospholipase C (PLC)-¡l which was over-expressed in HeLa cells transfected with a recombinant vaccinia virus carrying a complete cDNA sequence for PLC-¡l´, we noticed the presence of a heat-stable activator in crude cytosolic extract of HeLa cells which markedly stimulated phosphatidylinositol (PI) hydrolysis, when reconstituted with purified PLC-gl. Moreover, this putative factor was also found to be present in bovine brain cytosol. Subsequently, based on its ability to stimulate PI hydrolysis of PLC-gl as an assay, this activation factor was purified to homogeneity from bovine brain cytosol. It was purified by heat treatment, trichloroacetic acid precipitation, and successive chromatographic steps on DEAE-5PW, phenyl-SPW, and heparin-5PW HPLC columns. The purified protein, as seen on SDS-PAGE, consisted of 4 or 5 closely spaced bands of apparent molecular weight between 48 and 62 kDa. However, on gel filtration chromatography on TSK-G3000SW HPLC, the estimated molecular weight was revealed to be approximately 350 kDa. The purified activator was identified as a microtubule-associated protein tau by electroelution from an SDS-polyacrylamide gel, partial peptide mapping by Staphylococcal V8 protease, and amino acid sequencing of cyanogen bromide cleaved peptides. The identity of the activator was re-confirmed by an immunoblotting using a specific anti-tau monoclonal antibody. In addition, reconstituting PLC-gl with tau protein purified by an alternative method described elsewhere2 resulted in the same magnitude of activation. Tau protein mediated activation of PLC isozymes was calcium dependent. Isozyme specific activation of PLCs in 0.1% deoxycholate substrate appeared to be preferential toward PI hydrolysis. The magnitude of activation of PLC isozymes in PI substrate were PLC-gl > PLC-g2 > PLC-d1 > PLC-b in decreasing order. Approximately 200 nM concentration of tau-protein produced 15.5-, 5.4-, 4.2-, and 1.6-fold increase in activity of these enzymes, respectively, in the presence of 1 mM free calcium ion.