cAMP-Triggered Proteolysis of cAMP-Dependent Protein Kinase in Brush Border Membranes

Alhanaty, Eytan; Tauber-Finkelstein, Miriam; Schmeeda, Hilary; Shaltiel, Shmuel

doi:10.1016/b978-0-12-152827-0.50030-x

Cited by 25 publications

(27 citation statements)

References 17 publications

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“…(iii) While a copolymer composed of Glu and Tyr is cleaved by KSMP, a polymer of Glu amino acid residues alone is not cleaved by it and acts as a competitive inhibitor of this proteinase (7,45). (iv) The hydrophobic proteinase inhibitor chymostatin inhibits the cleavage of the Csubunit by KSMP though at relatively high concentrations (Ն 10 Ϫ3 M) (3). (v) In general, members of the astacin family of peptidases have been shown to cleave peptides containing hydrophobic amino acid residues and to possess an arylamidase activity (26).…”

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

confidence: 99%

“…(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).…”

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confidence: 99%

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

confidence: 99%

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 cleavage was shown to occur in the native structure of C, but not if C is pre-denatured [10,11], suggesting that KSMP recognizes the conformation of the kinase. More recently we have shown that the cluster of acidic amino acids at the car-*Corresponding author.…”

Section: Introductionmentioning

confidence: 99%

“…This procedure yields a membrane preparation enriched with KSMP. This membrane preparation was solubilized with 1% octyl-l]-o-glucopyranoside [10,11,23], the insoluble particles were removed by centrifugation at 100 000 ×g for 30 rain, and the resulting supernatant (protein concentration, 1 mg/ml) was used in the present study.…”

Section: Preparation Of the Kinase Splitting Membranal Proteinase (Ksmp)mentioning

confidence: 99%

“…We have previously identified a kinase splitting membranal proteinase (KSMP), which specifically cleaves C to yield a distinct clipped product C' devoid of kinase activity [9][10][11]. The cleavage was shown to occur in the native structure of C, but not if C is pre-denatured [10,11], suggesting that KSMP recognizes the conformation of the kinase.…”

Section: Introductionmentioning

confidence: 99%

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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.

1989

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A kinase‐splitting membranal proteinase specifically clips the cytoplasmic moiety of the insulin receptor beta‐subunit (95 kd) to yield an 84‐kd fragment. Using antibodies against different domains in the receptor, cleavage is shown to remove an 11‐kd ‘tail’ (rooted at the C‐terminal end of the kinase domain) which includes tyrosines 1316 and 1322. This cleavage impairs the ability of the clustered tyrosines 1146, 1150 and 1151 to undergo autophosphorylation. Nevertheless, the clipped beta‐subunit is as active as the intact subunit if its kinase activity is measured at high exogenous substrate concentrations (greater than or equal to 2 mg/ml) indicating that autophosphorylation is not obligatory for insulin‐dependent phosphotransferase activity. With low substrate concentrations (e.g. 0.2 mg/ml) a severe damage to the kinase activity is detected, which may reflect an important structural contribution of the ‘tail’ and/or the clustered phosphotyrosines in creating the preferential affinity of the kinase for its in vivo substrate(s). The membranal proteinase strictly recognizes the native conformation of the kinase domain, and fails to cleave it after denaturation. Since such a conformation‐dependent cleavage occurs also in the case of the cytoplasmic moiety of the EGF receptor and the catalytic subunit of cAMP‐dependent protein kinase, it is suggested that the similarity between these three kinase domains extends beyond their reported sequence homology to reflect a similarity in conformation.

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cAMP-Triggered Proteolysis of cAMP-Dependent Protein Kinase in Brush Border Membranes

Cited by 25 publications

References 17 publications

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

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

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

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