Unveiling the Substrate Specificity of Meprin β on the Basis of the Site in Protein Kinase A Cleaved by the Kinase Splitting Membranal Proteinase

Abstract

The kinase splitting membranal proteinase (KSMP) is a metalloendopeptidase that inactivates the catalytic (C) subunit of protein kinase A (PKA) by clipping off its carboxyl terminal tail. Here we show that this cleavage occurs at Glu 332 -Glu 333 , within the cluster of acidic amino acids (Asp 328 -Glu 334 ) of the kinase. The K m values of KSMP and of meprin ␤ (which reproduces KSMP activity) for the C-subunit are below 1 M. The K m for peptides containing a stretch of four Glu residues are in the micromolar range, illustrating the significant contribution of this cluster to the substrate recognition of meprin ␤. This conclusion is supported by a systematic study using a series of the C-subunit mutants with deletions and mutations in the cluster of acidics. Hydrophobic amino acids vicinal to the cleavage site increase the K cat of the proteinase. These studies unveil a new specificity for meprin ␤, suggesting new substrates that are 1-2 orders of magnitude better in their K m and K cat than those commonly used for meprin assay. A search for substrates having such a cluster of acidics and hydrophobics, which are accessible to meprin under physiological conditions, point at gastrin as a potential target. Indeed, meprin ␤ is shown to cleave gastrin at its cluster of five glutamic acid residues and also at the M-D bond within its WMDF-NH 2 sequence, which is indispensable for all the known biological activities of gastrins. The latter meprin cleavage will lead to the inactivation of gastrin and thus to the control of its activity.The presence of a kinase splitting membranal proteinase (KSMP) 1 in the brush-border membranes of the rat small intestine was demonstrated as early as 1979 (1). This proteinase was shown to clip the catalytic (C) subunit of PKA, yielding a distinct cleavage product (CЈ) that was found to be devoid of the kinase activity. The biochemical characterization of KSMP as a proteinase revealed that it is an intriguing enzyme with a combination of the following unique features. (a) KSMP cleaves the C-subunit when it is free but not when inhibited by its regulatory (R) subunits, as in the R 2 C 2 complex (1, 2). (b) This cleavage could not be simulated by other proteinases (trypsin, chymotrypsin, clostripain, and papain (2)), suggesting that its specificity is not due merely to an interdomain exposure in C. (c) The proteinase was found to single out and selectively cleave the C-subunit in the presence of the large number of other proteins found in crude extracts of different tissues (brain, liver, or muscle) (3). (d) KSMP was found to cleave the Csubunit in its native conformation but not if the kinase is pre-denatured (2). (e) It distinguishes between the "open" and "closed" conformations of the C-subunit (4) that were recently identified by x-ray crystallography of this kinase (5).The cleavage of the C-subunit by KSMP leads to the removal of the carboxyl terminus tail of this kinase and seemed to occur at a distinct site (6 -8). Interestingly, two other kinases, the EGF-and the insulin-receptor ki...

Role of the COOH-terminal Domains of Meprin A in Folding, Secretion, and Activity of the Metalloendopeptidase

“…Both samples were subjected to SDS-PAGE (7.5% gels with ␤-mercaptoethanol), followed by immunoblotting using anti-mouse meprin ␣ polyclonal antibodies. B, the amount of meprin ␣ protein per microliter in both the cell lysates and the media was determined by densitometric scans of immunoblots using laser densitometry as described previously (12). The data are shown as the mean Ϯ S.E.…”

“…They are capable of degrading proteins such as collagen and gelatin, hormones such as parathyroid hormone, luteinizing hormone-releasing hormone, and melanocyte-stimulating hormone, and small peptides such as bradykinin, angiotensins, and gastrin (11,12). The meprins may, therefore, be involved in activation or inactivation of important extracellular proteins and peptides, and are highly regulated at transcriptional and posttranscriptional levels themselves.…”

N-Linked Oligosaccharides on the Meprin A Metalloprotease Are Important for Secretion and Enzymatic Activity, but Not for Apical Targeting

“…Meprins are capable of degrading proteins such as collagens and gelatin; hormones such as parathyroid hormone, luteinizing hormone-releasing hormone, and melanocyte-stimulating hormone; and small peptides such as bradykinin, angiotensins, and gastrin (3,11). The meprins may therefore be involved in activation or inactivation of important extracellular proteins and peptides and are highly regulated at transcriptional and post-transcriptional levels themselves.…”

Marked Differences between Metalloproteases Meprin A and B in Substrate and Peptide Bond Specificity

“…Previous studies have shown that a variety of biologically active peptides and proteins are hydrolyzed by meprins in vitro. For example, meprins cleave blood pressure regulators such as bradykinin, metabolism mediators such as parathyroid hormone, signaling molecules such as protein kinase A, and basement membrane proteins such as entactin (1,2,16,17). There has been no systematic study, however, of the enzymatic differences between meprin A and B from any species or of the contributions of the individual subunits to activity.…”