Identification and Characterization of Cathepsin B as the Cellular MARCKS Cleaving Enzyme

Abstract

The importance of regulating the cellular concentrations of the myristoylated alanine-rich C kinase substrate (MARCKS), a major cellular substrate of protein kinase C, is indicated by the fact that mice lacking MARCKS exhibit gross abnormalities of central nervous system development and die shortly after birth. We previously identified a novel means of regulating cellular MARCKS concentrations that involved a specific proteolytic cleavage of the protein and implicated a cysteine protease in this process (Spizz, G., and Blackshear, P. J. (1996) J. Biol. Chem. 271, 553-562). Here we show that p40, the carboxyl-terminal fragment resulting from this cleavage of MARCKS, was associated with the mitochondrial/lysosomal pellet fraction of human diploid fibroblasts and that its generation in cells was sensitive to treatment with NH 4 Cl. These data suggest the involvement of lysosomes in the generation and/or stability of p40. The MARCKS-cleaving enzyme (MCE) activity was peripherally associated with a 10,000 ؋ g pellet fraction from bovine liver, and it co-purified with the activity and immunoreactivity of a lysosomal protease, cathepsin B. Cathepsin B catalyzed the generation of p40 from MARCKS in a cell-free system and behaved similarly to the MCE with respect to mutants of MARCKS previously shown to be poor substrates for the MCE. Treatment of fibroblasts with a cell-permeable, specific inhibitor of cathepsin B, CA074-Me, resulted in parallel time-and concentration-dependent inhibition of cathepsin B and MCE activity. Incubation of a synthetic MARCKS phosphorylation site domain peptide with purified cathepsin B resulted in cleavage of the peptide at sites consistent with preferred cathepsin B substrate sites. These data provide evidence for the identity of the MCE as cathepsin B and suggest that this cleavage most likely takes place within lysosomes, perhaps as a result of specific lysosomal targeting sequences within the MARCKS primary sequence. The data also suggest a direct interaction between MARCKS and cathepsin B in cells and leave open the possibility that MARCKS may in some way regulate the protease for which it is a substrate.The myristoylated alanine-rich C kinase substrate (MARCKS) 1 is a prominent cellular substrate for protein kinase C (PKC) (1, 2). Expression of this heat-stable, acidic protein is essential for life, as demonstrated by the perinatal death of mice that are completely deficient in MARCKS (3). Complete lack of expression leads to gross abnormalities of central nervous system development; however, heterozygous mice, which express MARCKS at 50% wild-type levels, appear to be normal.Cellular levels of MARCKS are regulated by both transcriptional and translational mechanisms (4 -14). In addition, we recently demonstrated that the cellular concentrations of MARCKS can also be regulated by a proteolytic event. This proteolytic cleavage results in amino-and carboxyl-terminal fragments of MARCKS that co-exist in cells with the full-length protein. This cleavage of MARCKS was inhibited in intact fi...

MARCKS-related Protein (MRP) Is a Substrate for the Leishmania major Surface Protease Leishmanolysin (gp63)

“…Moreover, MRP subjected to PKC-dependent phosphorylation was resistant to leishmanolysin, strongly suggesting that the unphosphorylated serine residues of the effector domain are part of the leishmanolysin recognition sequence. These data are in line with those of Spizz and Blackshear (12,13), who found that PKC-phosphorylated MARCKS was a poor substrate for cathepsin B. Similarly, decreased susceptibility of MARCKS to cathepsin L after phosphorylation by PKC has been reported (33), and regulation of proteolysis by substrate phosphorylation was also described for the related GAP-43 protein (34).…”

“…8B) were consistently observed that could represent the N-and C-terminal peptides resulting from cleavage at site b (calculated molecular mass [M ϩ H] ϩ of the N-terminal fragment ϭ 9941.8 Da; expected value for the C-terminal fragment ϭ 12,252.0 Da based on the intact MRP peak at 22,175.2 Da). This site is analogous to a site described earlier for cathepsin B cleavage of MARCKS (12). Although some other minor peaks were observed, peptides that might result from cleavage at site c (molecular mass [M ϩ H] ϩ ϭ 10,399 and 11,803 Da) were not detected.…”

“…As shown in ϩ ϭ 1305.8 Da) resulting from cleavage at site b. As mentioned above, this site corresponds to an earlier described cathepsin B cleavage site in MARCKS (12). DISCUSSION We previously demonstrated that macrophages infected with Leishmania promastigotes express very low levels of MRP (14).…”

“…Such a mechanism appeared particularly interesting in light of recent studies by Spizz and Blackshear (13) demonstrating that cellular concentrations of MARCKS are regulated by proteolysis. A cellular MARCKScleaving enzyme was identified as cathepsin B, and the purified enzyme was later shown to cleave MARCKS within its basic effector domain in a cell-free system (12). In the present study, we investigated whether a proteolytic activity associated with Leishmania might similarly exhibit MRP-cleaving activity.…”

“…One mechanism of post-transcriptional regulation involves proteolytic degradation. Spizz and Blackshear (12) recently identified cathepsin B as a cellular MARCKS-cleaving enzyme in fibroblasts. They suggested that cleavage might occur within lysosomes as a result of specific lysosomal targeting sequences identified within the MARCKS primary sequence.…”