Myristoylated alanine-rich C kinase substrate (MARCKS) and MARCKS-related
protein (MRP; MacMARCKS) are
protein kinase C substrates in diverse cell types. Activation of murine macrophages by
cytokines increases MRP expression, but
infection with Leishmania promastigotes during activation results in MRP depletion. We therefore examined the effect of Leishmania major LV39 on recombinant MRP. Both live promastigotes and a soluble fraction of LV39 lysates degraded MRP to yield lower molecular weight fragments. Degradation was independent of MRP myristoylation and was inhibited by
protein kinase C-dependent phosphorylation of MRP. MRP was similarly degraded by purified
leishmanolysin (gp63), a Leishmania surface
metalloprotease. Degradation was evident at low
enzyme/substrate ratios, over a broad pH range, and was inhibited by
1,10-phenanthroline and by a hydroxamate
dipeptide inhibitor of
leishmanolysin. Using mass spectrometric analysis, cleavage was shown to occur within the effector domain of MRP between Ser(92) and Phe(93), in accordance with the substrate specificity of
leishmanolysin. Moreover, an MRP construct in which the effector domain had been deleted was resistant to cleavage. Thus,
Leishmania infection may result in
leishmanolysin-dependent hydrolysis of MRP, a major
protein kinase C substrate in macrophages.