Limited proteolysis of the catalytic subunit of cAMP-dependent protein kinase — A membranal regulatory device?

Alhanaty, Eytan; Shaltiel, Shmuel

doi:10.1016/0006-291x(79)90633-8

Cited by 55 publications

(43 citation statements)

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“…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.…”

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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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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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“…We have shown [7] that upon exposure of the free catalytic subunit (C) of CAMP-dependent protein kinase to brush border membranes from the rat small intestine, the kinase loses its catalytic activity in a time-dependent process. In addition the factor c --*occo IlrwmB---deCg."'…”

Section: Resultsmentioning

confidence: 99%

“…------- Fig.1. Timedependent degradation of C by an enzyme extracted from brush border membranes through solubihzation in octyl-fi-Dglucopyranoside [7]. A reaction mixture containing pure C (final cont.…”

Section: Resultsmentioning

confidence: 99%

“…At the indicated times, aliquots (40 ~1) were removed and mixed with 10 ~1 of a solution containing SDS (10%) and 2-mercaptoethanol (7.5 M), and boiled for 5 min at 100°C. The samples were then subjected to SDS-polyacrylamide gradient gel electrophoresis as in section 2. responsible for this inactivation can be further purified and solubilized in octyl-fi-D-glucopyranoside [7].…”

Section: Resultsmentioning

confidence: 99%

“…These membranes were further purified by the method in [6]. The C-subunit inactivating factor which brings about degradation of C was solubilized as in [7]. The low M, calibration kit of proteins for SDS-polyacrylamide gel electrophoresis was purchased from Pharmacia (Uppsala); fluorescamine was obtained from Sigma.…”

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Monitoring the resolution of proteins and peptides in the course of their electrophoresis

1981

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Cytofluorographic evidence that thymocyte dipeptidyl peptidase IV (CD26) activity is altered with stage of ontogeny and apoptotic status

et al. 1996

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CD26 is a multifunctional molecule implied to have a variety of roles in the immune response including its activity as a membrane exopeptidase (Dipeptidyl peptidase IV) which cleaves several protein molecules. In order to further define the expression and functional activity of CD26 in the developing thymus, we utilized a nondisruptive, cytofluorogenic assay which allowed simultaneous measurement of DPP IV activity with a fluorochrome‐conjugated peptide substrate and surface staining of the T lymphocyte lineage antigens CD4 and CD8. Neonatal and adult murine thymi were examined using the three‐color assay and significant differences in DPP IV activity were found among the thymocyte subsets defined by their CD4/CD8 phenotype. Single‐positive cells bore higher activity than CD4−/CD8− cells and neonates had higher activity than adults. Thymocytes with characteristics consistent with apoptotic cells expressed higher DPP IV activity. Thus, DPP IV appears to be upregulated both as thymocytes mature and among thymocytes which are undergoing programmed cell death. These results suggest that CD26 is ontogenically controlled during T cell maturation and may play a role in thymic deletion of emerging clones. © 1996 Wiley‐Liss, Inc.

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Limited proteolysis of the catalytic subunit of cAMP-dependent protein kinase — A membranal regulatory device?

Cited by 55 publications

References 19 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

Monitoring the resolution of proteins and peptides in the course of their electrophoresis

Cytofluorographic evidence that thymocyte dipeptidyl peptidase IV (CD26) activity is altered with stage of ontogeny and apoptotic status

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