Degradative inactivation of cyclic AMP-dependent protein kinase by a membranal proteinase is restricted to the free catalytic subunit in its native conformation.

Abstract

A membranal proteinase from brush-border epithelial cells of the rat small intestine was shown to bring about a restricted and limited degradation of the free catalytic subunit (C) of cyclic AMP-dependent protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37) with concomitant inactivation of the kinase. This membranal proteinase exhibits a remarkable specificity. (i) It degrades C in its native conformation, but not after it has been heat-denatured. (ii) The degradation of C (Mr 40,000) does not proceed further, once a distinct clipped product (Mr 34,000) is formed. (iii) The undissociated ("stored") form of the enzyme (R2C2) is not attacked by the membranal proteinase, preserving both its potential catalytic activity and its molecular integrity. Only upon addition of cyclic AMP to release free C does the proteinase attack it. (iv) The membranal proteinase does not degrade the regulatory subunit (R), released by cyclic AMP from R2C2, although R is quite susceptible to degradation by other proteolytic enzymes. None of these features of the membranal proteinase could be reproduced with trypsin, chymotrypsin, clostripain, or papain. The specific, restricted, and limited action of this membranal enzyme raises the possibility that it may have a distinct physiological assignment associated with the bioregulation of cyclic AMP-dependent protein kinase.Cyclic AMP (cAMP)-dependent protein kinase (cAMPdPKase; ATP:protein phosphotransferase, EC 2.7.1.37) (1) can be regarded as a major intracellular sensor ofchanges in cAMP levels, which take place in response to extracellular hormonal stimuli. As such, it is in charge of implementing the instructions given by a hormone to its target cell. This enzyme has been found in quite a few mammalian tissues (2) and is composed of two types of subunits, one being catalytically active (C) and the other having a regulatory function (R) (3, 4). These two subunits are assembled together to yield the inactive form of the enzyme, R2C2, which is now believed to be activated by cAMP according to the following equation (5-7):R2C2 + 4cAMP = R2(cAMP)4C2± R2(cAMP)4 + 2C. One of the intriguing questions regarding the physiological function of cAMPdPKase arises from the fact that this enzyme can phosphorylate various proteins, at least in vitro and probably even in intact cells (8). Therefore, it becomes imperative to elucidate the mechanism(s) by which the kinase activity may be channeled in vivo to bring about a specific phosphorylation at a given time and a given locus within the cell in response to a distinct metabolic signal. Some reports have suggested that different hormones that function through cAMP may trigger different chains of metabolic events within the same cell (9). This raises the possibility that there may be additional regulatory devices for modulating the cellular response to the hormonal stimulus.cAMPdPKase activity in brush-border membranes (from the rat small intestine) vanishes within a few minutes upon addition of cAMP (10). The inactivation was shown to be ...

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

“…As seen in Fig. 4, KSMP cleaves the peptides between acidic amino acids in positions that correspond either to the E 332 -E 333 bond, or to the D 328 -D 329 bond in C. The cleavage 2 The C-subunit expressed in E. coli is not myristylated and does not undergo the same post-translational modifications that occur in the mammalian enzyme. The lack of cleavage in spite of full catalytic activity is intriguing and is currently being studied in our laboratory.…”

“…The first step toward this analysis was to set up an adequate expression system for monitoring the cleavage of the C-subunit and its mutants since we found out that the wildtype C-subunit itself, though fully active as a protein kinase (38), is not cleaved if expressed in bacteria (data not shown) 2 . The rabbit reticulocyte lysate translation system using [ 35 S]methionine was found to be appropriate for that purpose.…”

“…This kind of "shortened rope" effect may, of course, have a detrimental structural outcome in many deletion studies except when the deletion involves a loop that does not interact with the core of the protein. 2 To avoid some of the difficulties mentioned above and to obtain a more accurate assessment of the individual contribution of each amino acid residue to the KSMP recognition and C-cleavage, we carried out an alanine scanning of the glutamic acid residues within the cluster of acidics in the C-subunit (Scheme 1). Quantitative analysis of the initial rates of cleavage of these mutants showed that there is only a ϳ20% reduction in the cleavage rate when a single glutamic acid residue is mutated into an alanine, even if the Glu to Ala mutation results in a discontinuity in the sequence of the negatively charged residues.…”

“…(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).…”

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

“…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.…”