MARCKS-related protein (MRP) is a substrate for the Leishmania major surface protease leishmanolysin (gp63)

Myristoylated alanine-rich C kinase substrate (MARCKS) and MARCKS-related p rotein (MRP; MacMARCKS) are protein kinase C substrates in diverse cell typ es. Activation of murine macrophages by cytokines increases MRP expression, but infection with Leishmania promastigotes during activation results in M RP depletion, We therefore examined the effect of Leishmania major LV39 on recombinant MRP, Both live promastigotes and a soluble fraction of LV39 lys ates degraded MRP to yield lower molecular weight fragments. Degradation wa s independent of MRP myristoylation and was inhibited by protein kinase C-d ependent phosphorylation of MRP, MRP was similarly degraded by purified lei shmanolysin (gp63), a Leishmania surface metalloprotease. Degradation was e vident at low enzyme/substrate ratios, over a broad pH range, and was inhib ited by 1,10-phenanthroline and by a hydroxamate dipeptide inhibitor of lei shmanoiysin, Using mass spectrometric analysis, cleavage was shown to occur within the effector domain of MRP between Ser(92) and Phe(93), in accordan ce with the substrate specificity of leishmanolysin, Moreover, an MRP const ruct in which the effector domain had been deleted was resistant to cleavag e. Thus, Leishmania infection may result in leishmanolysin-dependent hydrol ysis of MRP, a major protein kinase C substrate in macrophages.