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

Spizz, Gwendolyn; Blackshear, Perry J.

doi:10.1074/jbc.272.38.23833

Cited by 37 publications

(36 citation statements)

References 77 publications

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“…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).…”

Section: Mass Spectrometric Analysis Of Mrp Degradation Products-as Ssupporting

confidence: 79%

“…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.…”

Section: Inhibition Of Mrp Degradation By Effector Domain Peptides Ofmentioning

confidence: 94%

“…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).…”

Section: Mass Spectrometric Analysis Of Mrp Degradation Products-as Smentioning

confidence: 94%

“…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.…”

Section: Mass Spectrometric Analysis Of Mrp Degradation Products-as Smentioning

confidence: 99%

“…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.…”

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confidence: 99%

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

Corradin¹,

Ransijn²,

Corradin³

et al. 1999

Journal of Biological Chemistry

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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.Myristoylated alanine-rich C kinase substrate (MARCKS) 1 and MARCKS-related protein (MRP), also known as Mac-MARCKS, are members of a highly acidic myristoylated family of protein kinase C (PKC) substrates (1, 2). The primary structures of MARCKS and MRP exhibit significant homology, including a highly basic stretch of amino acid residues known as the effector domain (also as the phosphorylation site domain), which contains the serine residues subject to PKC-dependent phosphorylation as well as binding sites for calmodulin and actin. Whereas MARCKS is widely distributed in diverse cell types, MRP is present primarily in brain and reproductive tissue (3, 4) as well as in macrophages, where it was first characterized (5). The biologic functions of MARCKS proteins are unknown. Due to their high effector domain homology, it is also possible that MRP and MARCKS play overlapping roles in some cells. In macrophages, both proteins colocalize in the cytosol in association with components of the actin cytoskeleton (6 -9) and consequently are thought to participate in major cellular responses such as phagocytosis, motility, and membrane trafficking.The expression of MARCKS proteins appears to be highly regulated, and in vitro studies have demonstrated up-or downregulation of MARCKS at both the transcriptional and posttranscriptional levels (5, 10, 11). 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. At least one c...

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Section: Mass Spectrometric Analysis Of Mrp Degradation Products-as Ssupporting

confidence: 79%

Section: Inhibition Of Mrp Degradation By Effector Domain Peptides Ofmentioning

confidence: 94%

Section: Mass Spectrometric Analysis Of Mrp Degradation Products-as Smentioning

confidence: 94%

Section: Mass Spectrometric Analysis Of Mrp Degradation Products-as Smentioning

confidence: 99%

mentioning

confidence: 99%

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

Corradin¹,

Ransijn²,

Corradin³

et al. 1999

Journal of Biological Chemistry

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Myristoylation Alters Retinoic Acid-Induced Down-Regulation of MARCKS in Immortalized Hippocampal Cells

Wang

Watson

Lenox

2000

Biochemical and Biophysical Research Communications

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Transgenic Complementation of MARCKS Deficiency with a Nonmyristoylatable, Pseudo-Phosphorylated Form of MARCKS: Evidence for Simultaneous Positive and Dominant-Negative Effects on Central Nervous System Development

Kim

Swierczynski

Tuttle

et al. 1998

Developmental Biology

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MARCKS is a widely expressed protein kinase C substrate that is essential for normal prenatal development of the central nervous system in mice. MARCKS-deficient mice exhibit universal perinatal mortality and numerous developmental abnormalities of the brain and retina. To determine which domains of the protein were important in complementing these neurodevelopmental anomalies, we have interbred MARCKS knockout mice with transgenic mice expressing an epitope-tagged human MARCKS transgene that can completely correct the MARCKS-deficient phenotype. Previous structure-function studies showed that a nonmyristoylatable form of MARCKS could correct all of the neuroanatomical abnormalities, and resulted in approximately 25% viable pups that grew to adulthood and were fertile. The present experiment attempted a similar complementation strategy in which a nonmyristoylatable, "pseudo-phosphorylated" form of the protein was used, which has been shown to be almost completely cytosolic in cell expression studies. Surprisingly, this transgene was able to complement almost all of the cerebral anatomical abnormalities characteristic of the knockout mice. However, these mice also exhibited a universal, novel phenotype: profound retinal ectopia, in which retinal tissue was often found in the vitreous humor as well as extraocularly. Retrospective evaluation of the original MARCKS knockout phenotype revealed that this anomaly was present in about 43% of the knockout mice, and was clearly detectable as early as embryonic day 12.5, before retinal cell differentiation begins. These data suggest that a nonmyristoylatable, pseudo-phosphorylated form of MARCKS can complement most if not all cerebral aspects of the MARCKS-deficient phenotype, but that it appears to worsen a retinal phenotype, perhaps by exerting a dominant-negative effect on a coexpressed MARCKS homologue.

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

Cited by 37 publications

References 77 publications

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

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

Myristoylation Alters Retinoic Acid-Induced Down-Regulation of MARCKS in Immortalized Hippocampal Cells

Transgenic Complementation of MARCKS Deficiency with a Nonmyristoylatable, Pseudo-Phosphorylated Form of MARCKS: Evidence for Simultaneous Positive and Dominant-Negative Effects on Central Nervous System Development

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