An Ultrasensitive, Continuous Fluorometric Assay for Calpain Activity

Tompa, Péter; Schád, Éva; Baki, Andrea; Alexa, Anita; Batke, József; Friedrich, Péter

doi:10.1006/abio.1995.1352

Cited by 23 publications

(22 citation statements)

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“…Alternatively, the microtiter plate can be placed immediately in the Spectrafluor after the addition of BODIPY-FL-casein, and the time course of casein hydrolysis monitored (the Spectrafluor can record readings every 20 s even if all wells in a 96-well plate are read and in less time if only some of the wells are filled). Earlier studies using quenched fluorescent substrates have noted that the increase in fluorescence begins within seconds once the reaction has been initiated (24,25,32,33), and it is important to place the microtiter plate in the fluorometer as rapidly as possible after addition of the BODIPY-FL-casein. Every assay includes two blanks, an EDTA blank and a Ca 2ϩ blank.…”

Section: Methodsmentioning

confidence: 99%

A BODIPY Fluorescent Microplate Assay for Measuring Activity of Calpains and Other Proteases

Thompson

Saldaña

Cong

et al. 2000

Analytical Biochemistry

100

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Section: Methodsmentioning

confidence: 99%

A BODIPY Fluorescent Microplate Assay for Measuring Activity of Calpains and Other Proteases

Thompson

Saldaña

Cong

et al. 2000

Analytical Biochemistry

100

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“…A second assay of calpain activity used is the hydrolysis of microtubuleassociated protein 2 (MAP2)-dichlorotriazinylaminofluorescein (DTAF) by cell lysates (17,57). Briefly, MAP2 (Cytoskeleton, Denver, Colo.) was conjugated with DTAF by incubation in a pH 8.5 PIPES (piperazine ethanesulfonic acid) buffer for 30 min at 4°C.…”

Section: Methodsmentioning

confidence: 99%

Interferon-Inducible Protein 9 (CXCL11)-Induced Cell Motility in Keratinocytes Requires Calcium Flux-Dependent Activation of μ-Calpain

Satish

Blair

Glading

et al. 2005

Molecular and Cellular Biology

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Keratinocyte migration is critical to reepithelialization during wound repair. The motility response is promoted by growth factors, cytokines, and cytokines produced in the wound bed, including those that activate the epidermal growth factor (EGF) receptor. The Alu-Leu-Arg-negative CXC chemokine interferon-inducible protein 9 (IP-9; also known as CXCL11, I-TAC, beta-R1, and H-174) is produced by keratinocytes in response to injury. As keratinocytes also express the receptor, CXCR3, this prompted us to examine the role and molecular mechanism by which IP-9 regulates keratinocyte motility. Unexpectedly, as CXCR3 liganding blocks growth factor-induced motility in fibroblasts, IP-9 alone promoted motility in undifferentiated keratinocytes (37 ؎ 6% of the level of the highly motogenic EGF) as determined in a two-dimensional in vitro wound healing assay. IP-9 even enhanced EGF-induced motility in undifferentiated keratinocytes (116 ؎ 5%; P < 0.05 compared to EGF alone), suggesting two separate mechanisms of action. IP-9-increased motility and -decreased adhesiveness required the intracellular protease calpain. The increases in both motility and calpain activity by IP-9 were blocked by pharmacological and molecular inhibition of phospholipase C-␤3 and chelation of calcium, which prevented an intracellular calcium flux. Molecular downregulation or RNA interferencemediated depletion of -calpain (calpain 1) but not M-calpain (calpain 2) blocked IP-9-induced calpain activation and motility. In accord with elimination of IP-9-induced de-adhesion, RNA interference-mediated depletion of calpain 1 but not calpain 2 prevented cleavage of the focal adhesion component focal adhesion kinase and disassembly of vinculin aggregates. In comparison, EGF-induced motility of the same undifferentiated keratinocytes requires the previously described extracellular signal-regulated kinase to the M-calpain pathway. These data demonstrate that while both EGF-and IP-9-induced motility in keratinocytes requires calpain activity, the isoform of calpain triggered depends on the nature of the receptor for the particular ligand. Interestingly, physiological nonapoptotic calcium fluxes were capable of activating -calpain, implying that the calcium requirement of -calpain for activation is attained during cell signaling. This is also the first demonstration of differential activation of the two ubiquitous calpain isoforms in the same cell by different signals.

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“…After removing the cell debris by centrifugation, 0.9 g of DTAF-labeled MAP2 was added to the samples with either 0 or 0.1 mM free Ca 2ϩ concentration. Fluorescence was immediately measured by an Aminco-Bowman Series II spectrofluorimeter (Spectronic Instruments Inc., Rochester, NY), at excitation and emission wavelengths of 490 and 520 nm respectively, for 3 min at room temperature (5,33).…”

Section: Methodsmentioning

confidence: 99%

Membrane Proximal ERK Signaling Is Required for M-calpain Activation Downstream of Epidermal Growth Factor Receptor Signaling

Glading

Überall

Keyse

et al. 2001

Journal of Biological Chemistry

188

165

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Localization of signaling is critical in directing cellular outcomes, especially in pleiotropic signaling pathways. The extracellular signal-regulated kinase (ERK)/ microtubule-associated protein kinase, which promotes cell migration, proliferation, and differentiation is found in the nucleus and throughout the cytoplasm. Recently, it has been shown that nuclear translocation of ERK is required for transcriptional changes and cell proliferation. However, the cellular consequences, of cytoplasmic signaling have not been defined. We explored whether cytoplasmic, specifically membrane-proximal, ERK signaling is involved in growth factor-induced cell motility. We previously have demonstrated that increased M-calpain activity downstream of epidermal growth factor receptor (EGFR)-mediated ERK activation is necessary for epidermal growth factor (EGF)-induced motility. Calpain isoforms also have been found in nuclear, cytosolic, and plasma membrane-associated compartments in a variety of cell types. We now employ cell engineering approaches to control localization of the upstream EGFR and ERK activities to examine the spatial effect of upstream signal locale on downstream calpain activity. With differential ligand-induced internalization and trafficking-restricted receptor variants, we find that calpain activity is triggered only by plasma membrane-restricted activated EGFR, not by internalized (although still active) EGFR. Cells transfected with membrane-targeted ERK1 and ERK2, which sequester endogenous ERKs, exhibited normal EGF-induced calpain activity. Transfection of an inactive ERK phosphatase (MKP-3/Pyst1) that sequesters ERK in the cytoplasm prevented calpain activation as well as deadhesion. These data strongly suggest that EGF-induced calpain activity can be enhanced near sites of membrane-proximal EGFR-mediated ERK signaling, providing insights about how calpain activity might be regulated and targeted to enhance its effects on adhesionrelated substrates.

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An Ultrasensitive, Continuous Fluorometric Assay for Calpain Activity

Cited by 23 publications

References 0 publications

A BODIPY Fluorescent Microplate Assay for Measuring Activity of Calpains and Other Proteases

A BODIPY Fluorescent Microplate Assay for Measuring Activity of Calpains and Other Proteases

Interferon-Inducible Protein 9 (CXCL11)-Induced Cell Motility in Keratinocytes Requires Calcium Flux-Dependent Activation of μ-Calpain

Membrane Proximal ERK Signaling Is Required for M-calpain Activation Downstream of Epidermal Growth Factor Receptor Signaling

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