Affinity labeling of the catalytic subunit of cyclic AMP-dependent protein kinase by N alpha-tosyl-L-lysine chloromethyl ketone.

Kupfer, Abraham; Gani, Viviane; Jiménez, Juan S.; Shaltiel, Shmuel

doi:10.1073/pnas.76.7.3073

Cited by 67 publications

(41 citation statements)

References 28 publications

(13 reference statements)

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“…As a chloromethyl ketone with alkylating activity, TPCK also inhibits other enzymes, an activity which can be attributed to its reactivity to nucleophiles such as thiol groups. 44,50,51 The irreversible inactivation of the catalytic subunit of cAMP-dependent protein kinase 44 and the inactivation of the transcription factor TFIIIC by TPCK occur solely through thiol group modification. 52 Importantly, TPCK shows considerable reactivity to the thiol group of glutathione 51 and can impair the glutathione redox system which regulates several cellular processes including apoptosis.…”

Section: Discussionmentioning

confidence: 99%

“…44 The possibility therefore exists that the sulphydryl group of NAC functions as a target for TPCK, thus potentially protecting thiol groups of proteins with a function in cellular signalling from alkylation. Alternatively, NAC, in its capacity as a glutathione precursor, could protect the cell by increasing the levels of intracellular glutathione.…”

Section: Effects Of Tpck and Nac On Ikb Phosphorylation In Tpi T-cellsmentioning

confidence: 99%

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N-acetylcysteine blocks apoptosis induced by N-α-tosyl-L-phenylalanine chloromethyl ketone in transformed T-cells

et al. 1999

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The serine protease inhibitor N-a-tosyl-L-phenylalanine chloromethyl ketone (TPCK) can interfere with cell-cycle progression and has also been shown either to protect cells from apoptosis or to induce apoptosis. We tested the effect of TPCK on two transformed T-cell lines. Both Jurkat T-cells and Theileria parva-transformed T-cells were shown to be highly sensitive to TPCK-induced growth arrest and apoptosis. Surprisingly, we found that the thiol antioxidant, Nacetylcysteine (NAC), as well as L-or D-cysteine blocked TPCK-induced growth arrest and apoptosis. TPCK inhibited constitutive NF-kB activation in T. parva-transformed T-cells, with phosphorylation of IkBa and IkBb being inhibited with different kinetics. TPCK-mediated inhibition of IkB phosphorylation, NF-kB DNA binding and transcriptional activity were also prevented by NAC or cysteine. Our observations indicate that apoptosis and NF-kB inhibition induced by TPCK result from modifications of sulphydryl groups on proteins involved in regulating cell survival and the NF-kB activation pathway(s).

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Section: Discussionmentioning

confidence: 99%

Section: Effects Of Tpck and Nac On Ikb Phosphorylation In Tpi T-cellsmentioning

confidence: 99%

N-acetylcysteine blocks apoptosis induced by N-α-tosyl-L-phenylalanine chloromethyl ketone in transformed T-cells

et al. 1999

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“…Pure catalytic subunit of PKA was prepared according to Beavo et al [18] and assayed as described by Kupfer et al [19]. The resulting In both lanes C and D, the reaction was allowed to proceed for 30 min at 30°C then arrested by addition of sample buffer and boiling (3 min at 95°C).…”

Section: The Catalytic Subunit Of Pkamentioning

confidence: 99%

The phosphorylation of the two‐chain form of vitronectin by protein kinase A is heparin dependent

et al. 1990

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In circulating blood, vi&one&in occurs in two forms: a single-chain (75 kDa) and an endogenously clipped two-chain form (65 kDa and 10 kDa) held together by a disulfide bridge. The 75 kDa form was previously shown to be phosphorylated at Seti78 by protein kinase A, released by physiologically stimulated platelets. By contrast, at pH 7.5 the two-chain form is not phosphorylated at all. Heparin or heparan sulfate are shown here to modulate the conformation of clipped vitronectin at physiological pH, exposing Se2's and allowing its stoichiometric phosphorylation by the kinase. At this pH the two-chain form of vitronectin in plasma exhibits a higher affinity for heparin, and behaves as a flexible molecule, which can conformationally respond to heparin and heparan sulfate, effecters involved in vitronectin function.

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“…granule preparation, in an islet sonicate several proteins (61,000, 49,000, 40,000, 38,000, and 31,500) were affinity labeled with 1`I-Tyr-Ala-Lys-ArgCH2Cl (data not shown). It is not known in which cellular compartment these proteins are located or whether they are all proteases, bearing in mind that Tos-LysCH2CI has been shown to affinity label the catalytic subunit of cAMP-dependent protein kinase (26). DISCUSSION The results reported in this paper suggest that a 31,500 molecular weight thiol protease may be involved in the conversion ofproinsulin to insulin.…”

Section: Resultsmentioning

confidence: 68%

Conversion of proinsulin to insulin: involvement of a 31,500 molecular weight thiol protease.

Docherty¹,

Carroll²,

Steiner³

1982

Proc. Natl. Acad. Sci. U.S.A.

123

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A lysed crude secretory granule fraction from rat islets of Langerhans was shown to process endogenous proinsulin to insulin with a pH optimum of 5.0-6.0. The converting activity in the lysed fraction was not inhibited by serine protease inhibitors (diisopropyl fluorophosphate, soybean trypsin inhibitor, and aprotinin) or metalloprotease inhibitors (EDTA and o-phenanthroline) but was inhibited by some thiol protease reagents (p-chloromercuribenzenesulfonic acid, antipain, and leupeptin) but not by others (N-ethylmaleimide and iodoacetamide). NG-p-Tosyl-L-lysyl chloromethyl ketone only mildly inhibited at higher concentrations, whereas L-alanyl-L-lysyl-L-arginyl chloromethyl ketone was a powerful inhibitor. L-Alanyl-L-lysyl-L-arginyl chloromethyl ketone was ['"I]iodotyrosylated and used as an affinity labeling agent for the converting activity. Because the crude granule preparation contained contaminating lysosomes the affinity labeling of the granule preparation proteins was compared with that in liver lysosomes purified from rats injected with Triton WR1339. In the crude granule fraction the affinity label bound in a cysteine-enhanced manner to a single 31,500 molecular weight protein, but in purified liver lysosomes the major affinity-labeled protein had a molecular weight of 25,000 and minor 31,500 and 35,000 molecular weight proteins were also labeled. Evidence suggests that these proteins are thiol proteases and that in islets the 31,500 molecular weight thiol protease is involved in the conversion of proinsulin to insulin.Insulin is synthesized as a larger precursor, preproinsulin (1), which is rapidly cleaved to proinsulin (2). The conversion of proinsulin to insulin is then a relatively slow process, which is thought to commence in the Golgi apparatus and continue into the maturing secretory granule (3). The unequivocal identification of the converting activities present in these organelles has proven to be an elusive problem. Conversion is thought to be mediated by two enzymes: one with trypsin-like specificity and the other similar to carboxypeptidase B. Evidence for this is based on the known structure of the cleavage products and intermediate forms (4), the ability of these exocrine pancreatic enzymes to correctly process proinsulin in vitro (5), and the detection ofsimilar activities in the appropriate subcellular fractions of rat islets (6). Previous studies have demonstrated that both activities have a slightly acidic pH optimum and that the trypsin-like activity is activated by thiols and inhibited by thiol reagents (6-8). This suggests that this enzyme may be related to the lysosomal cathepsins. Alternatively, serine proteases [kallikrein (9) and plasminogen activator (10)] have also been implicated in the conversion process (see ref. 11 for review).In the experiments reported here, we have further characterized the inhibitor profile of the trypsin-like converting activity associated with a crude secretory granule preparation from rat islets, and on the basis of this information have at...

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Affinity labeling of the catalytic subunit of cyclic AMP-dependent protein kinase by N alpha-tosyl-L-lysine chloromethyl ketone.

Cited by 67 publications

References 28 publications

N-acetylcysteine blocks apoptosis induced by N-α-tosyl-L-phenylalanine chloromethyl ketone in transformed T-cells

N-acetylcysteine blocks apoptosis induced by N-α-tosyl-L-phenylalanine chloromethyl ketone in transformed T-cells

The phosphorylation of the two‐chain form of vitronectin by protein kinase A is heparin dependent

Conversion of proinsulin to insulin: involvement of a 31,500 molecular weight thiol protease.

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