Studying the structure of the intracellular moiety of the insulin receptor with a kinase-splitting membranal proteinase.

Seger, Rony; Zick, Yehiel; Shaltiel, Shmuel

doi:10.1002/j.1460-2075.1989.tb03395.x

Cited by 9 publications

(9 citation statements)

References 37 publications

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“…It attracted our attention not merely because it selectively inactivates PKA but also because of the specificity with which it clips off the carboxyl terminus tail of the C subunit of PKA. In addition, KSMP was found to clip off the carboxyl terminus tail of two other important kinases: the epidermal growth factor receptor kinase (9) and the insulin receptor kinase (10). In all three kinases, the cleavage was shown to be conformation-dependent (2, 7, 9, 10).…”

Section: Discussionmentioning

confidence: 99%

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The Cleavage of Protein Kinase A by the Kinase-splitting Membranal Proteinase Is Reproduced by Meprin β

Chestukhin

Muradov

Litovchick

et al. 1996

Journal of Biological Chemistry

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The Kinase-Splitting Membranal Proteinase (KSMP) is a metallo-endoproteinase that clips off the carboxyl terminus tail of the catalytic (C) subunit of protein kinase A to yield a truncated, catalytically inactive protein (C). Here we report (a) a new procedure for the purification of KSMP, yielding a major protein band in SDS-polyacrylamide gel electrophoresis that correlates with the characteristic KSMP activity; (b) the sequence of tryptic peptides obtained from this band, suggesting an identity between this protein and meprin ␤; (c) the immuno-detection by specific anti-peptide antibodies of both the ␣ and the ␤ subunits of meprin in KSMP preparations; (d) the stable expression of meprin ␤ in a mammalian cell line (293) to establish a clone that constitutively expresses the full-length precursor of meprin ␤; and (e) the optimalization of the proteolytic activation of this precursor to obtain an enzyme exhibiting the specific KSMP cleavage, suggesting that KSMP is either derived from, or identical with, the meprin ␤ gene product. It is hoped that these results will shed light on the possible physiological role of KSMP and the way it may affect protein kinase A-mediated processes.The Kinase-Splitting Membranal Proteinase (KSMP) 1 is a brush-border metallo-endoproteinase originally discovered in our laboratory (1) as an enzyme that selectively clips the C subunit of protein kinase A (PKA) (40 kDa) to yield a truncated form denoted CЈ (34 -36 kDa), which is devoid of catalytic activity (1-3). The biochemical properties of KSMP were found to be quite intriguing: (a) it cleaves the C subunit at a distinct site in the carboxyl terminus tail of the kinase (4 -8); (b) it cleaves the C subunit when it is free and active, not when inhibited by its regulatory (R) subunits, as in the R 2 C 2 complex (2); (c) it singles out and selectively cleaves the C subunit in the presence of the large number of proteins in crude cell extracts of different tissues (3); (d) it cleaves the C subunit in its native conformation, but not if the kinase is pre-denatured (2); and (e) it distinguishes between the "open" and "closed" conformations of the C subunit (7).In the course of these studies, it was found that KSMP also clips off specifically the carboxyl-terminal end of the epidermal growth factor receptor and the insulin receptor (9, 10), again in a conformation-dependent manner. Somewhat unexpectedly at the time, it was found that KSMP is an ecto-enzyme, i.e. that its active site is oriented toward the cell exterior (11), raising the question whether protein kinases, which were known to regulate biological processes within the cell, are indeed the physiological targets of KSMP. However, studies in our laboratory which were prompted by these findings showed that protein kinase A may well have an extra-cellular regulatory role, for example in blood (12, 13).Until recently, we could not establish unequivocally the molecular identity of KSMP, since its purified preparations were found to vary in their specific activity, to be labile, to ...

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Section: Discussionmentioning

confidence: 99%

“…In the course of these studies, it was found that KSMP also clips off specifically the carboxyl-terminal end of the epidermal growth factor receptor and the insulin receptor (9,10), again in a conformation-dependent manner. Somewhat unexpectedly at the time, it was found that KSMP is an ecto-enzyme, i.e.…”

mentioning

confidence: 99%

The Cleavage of Protein Kinase A by the Kinase-splitting Membranal Proteinase Is Reproduced by Meprin β

Chestukhin

Muradov

Litovchick

et al. 1996

Journal of Biological Chemistry

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“…The evidence supporting the importance of hydrophobic amino acid residues is summarized in the following. (i) The protein substrate (C-subunit) with which KSMP was originally discovered (1-3), as well as the EGF-and insulin-receptor kinases (7,10,11), contain hydrophobic amino acid residues within or adjacent to their cluster of acidic amino acids. (ii) The dye 1-anilino-8-naphthalenesulfonate, which is known to bind to hydrophobic sites in proteins, inhibits the cleavage of the Csubunit by KSMP (45).…”

Section: Use Of Synthetic Peptides Derived From the C-subunit To Estamentioning

confidence: 99%

“…Interestingly, two other kinases, the EGF-and the insulin-receptor kinases (which share certain sequence homology with the C-subunit (9)) were also shown to undergo a specific and conformation-dependent cleavage by KSMP (6,[10][11][12]. In both receptor kinases, it was shown that the KSMP cleavage occurs at the carboxyl-terminal part of the molecules.…”

mentioning

confidence: 99%

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

Chestukhin¹,

Litovchick²,

Muradov

et al. 1997

Journal of Biological Chemistry

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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...

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“…boxy-terminus tail of C (327FDDYEEEEI335) is the major biorecognition element for KSMP, since antiidiotypic monoclonal antibodies against a copolymer containing clusters of E and Y were found to inhibit KSMP [12]. A similar conformation-dependent cleavage by KSMP was shown to occur also in two other protein kinases which contain such clusters of acidic amino acids at their carboxy-terminus tail --the EGF receptor kinase, and the insulin receptor kinase [13,14], raising the possibility that the malleable tails at the carboxy terminus of these kinases may play an important role in their biorecognition and regulation [13][14][15][16]. This paper describes the preparation and characterization of anti-head and anti-tail antibodies against distinct epitopes in C, and their use in the specific identification of PKA and in the assay of KSMP by the determination of C and C'.…”

Section: Introductionmentioning

confidence: 99%

Anti‐head and anti‐tail antibodies against distinct epitopes in the catalytic subunit of protein kinase A Use in the study of the kinase splitting membranal proteinase KSMP

et al. 1996

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Protein kinases share a considerable sequence homology in their catalytic core (residues 40-300 in PKA). Each core is flanked by "head" and "tail" segments at its amino-and carhoxy-termini, which are different in the various kinases. These end segments may play an important role in creating the preferential affinity of each kinase for its physiological substrates or regulatory ligands. Here we describe three antipeptide antibodies (~P-1, o.P-2, and c¢P-3) that specifically recognize the head and tail segments of the catalytic subunit (C) of PKA. (i) otP-I (against 6A-Kt3) react with C when denatured but not when in its native structure; (ii) ffP-2 (against 319K-I33S), bind to the site in C cleaved by the kinase splitting membranai proteinase (KSMP) and inhibit this cleavage of C; (iii) otP-3 (against 33SS-F3S°) react with C but not with the KSMP cleavage product C', useful for detecting a KSMP-like activity in different tissues and subcellular loci. The combined use of the antibodies described here provides a strict definition of C, and thus a high degree of fidelity in its biorecognition.

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Studying the structure of the intracellular moiety of the insulin receptor with a kinase-splitting membranal proteinase.

Cited by 9 publications

References 37 publications

The Cleavage of Protein Kinase A by the Kinase-splitting Membranal Proteinase Is Reproduced by Meprin β

The Cleavage of Protein Kinase A by the Kinase-splitting Membranal Proteinase Is Reproduced by Meprin β

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

Anti‐head and anti‐tail antibodies against distinct epitopes in the catalytic subunit of protein kinase A Use in the study of the kinase splitting membranal proteinase KSMP

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