An uncommon multiphosphorylated lipid in Neurospora crassa whose formation is catalyzed in vitro by a calcium- and phospholipid-dependent kinase

Abstract

The presence of a calcium- and phospholipid-dependent kinase activity in the soluble extracts of Neurospora crassa has been reported earlier. This activity phosphorylated in vitro two endogenous substrates, one of apparent molecular weight ( M r) 85 000, the other much lower than 14 000. This paper reports that the smaller substrate is a lipid. The lower M r phosphorylated compound could be extracted from proteins by organic solvents. Various thin-layer chromatographic analyses revealed that it did not comigrate with any of the phospholipids tested. Chemical and enzymatic structural analyses indicated that the phosphorylated compound was sensitive to mild alkaline methanolysis, alkaline phosphate and phospholipase A 1 but resistant to phospholipases A 2, C and D or nitrous acid treatment. A similar compound was detected in the organic phase after extraction of N. crassa mycelia labelled in vivo with either 32P i or [ 3H]glycerol. It could not be detected when [ 3H]inositol was used as label. The addition of in vivo 32P-labelled lipids in phosphorylation reactions revealed that the low M r substrate of the calcium- and phospholipid-dependent kinase already contained some phosphate which was itself sensitive to alkaline phosphatase. An identical apolar component was generated by alkaline phosphatase treatment of both the substrate of the kinase and the hosphorylated substrate. From these data it is concluded that the low M r phosphorylated compound is a lipid, presumably consisting of a diacylglycerol backbone with an as yet undetermined structure linked to the glycerol moiety. This structure contains at least two phosphate groups.