Calpain II colocalizes with detergent-insoluble rafts on human and Jurkat T-cells

Morford, Lorri A.; Forrest, Kathy; Logan, Barbara; Overstreet, L.Kevin; Goebel, Jens; Brooks, William H.; Roszman, Thomas L.

doi:10.1016/s0006-291x(02)00676-9

Cited by 33 publications

(21 citation statements)

References 46 publications

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“…When the cells were extracted with detergent, calpain 1 was found mostly in Triton X-100-soluble fractions, whereas calpain 2 also was present in Triton X-100-insoluble fractions (Fig. 5B), as has been described previously (14,19,25). The intracellular localization of calpain 1 also was studied by flotation gradient centrifugation, and it was not found in detergent-resistant membrane fractions, in contrast to the GPI-APs or caveolin-1 (not shown).…”

Section: Ev1 Infection Increases Calpain Activitysupporting

confidence: 82%

Calpain 1 and 2 Are Required for RNA Replication of Echovirus 1

Upla

Marjomäki

Nissinen

et al. 2008

J Virol

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Calpains are calcium-dependent cysteine proteases that degrade cytoskeletal and cytoplasmic proteins. We have studied the role of calpains in the life cycle of human echovirus 1 (EV1). The calpain inhibitors, including calpeptin, calpain inhibitor 1, and calpain inhibitor 2 as well as calpain 1 and calpain 2 short interfering RNAs, completely blocked EV1 infection in the host cells. The effect of the inhibitors was not specific for EV1, because they also inhibited infection by other picornaviruses, namely, human parechovirus 1 and coxsackievirus B3. The importance of the calpains in EV1 infection also was supported by the fact that EV1 increased calpain activity 3 h postinfection. Confocal microscopy and immunoelectron microscopy showed that the EV1/caveolin-1-positive vesicles also contain calpain 1 and 2. Our results indicate that calpains are not required for virus entry but that they are important at a later stage of infection. Calpain inhibitors blocked the production of EV1 particles after microinjection of EV1 RNA into the cells, and they effectively inhibited the synthesis of viral RNA in the host cells. Thus, both calpain 1 and calpain 2 are essential for the replication of EV1 RNA.

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Section: Ev1 Infection Increases Calpain Activitysupporting

confidence: 82%

Calpain 1 and 2 Are Required for RNA Replication of Echovirus 1

Upla

Marjomäki

Nissinen

et al. 2008

J Virol

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“…Moreover, the relationship between membrane trafficking and the calpains has been confirmed recently, e.g. calpain localization in the endoplasmic reticulum and Golgi (55), in lipid rafts (56), and in multivesicular bodies (57) etc., suggesting a general involvement of various calpains in a variety of membrane trafficking processes. Continued anterograde protein transport in the secretory pathway is tightly coupled with COPImediated retrograde, retrieval transport system.…”

Section: Discussionmentioning

confidence: 82%

Stomach-specific Calpain, nCL-2, Localizes in Mucus Cells and Proteolyzes the β-Subunit of Coatomer Complex, β-COP

Hata

Koyama

Kawahara

et al. 2006

Journal of Biological Chemistry

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Calpain is a Ca2؉ -regulated cytosolic protease. Mammals have 14 calpain genes, half of which are predominantly expressed in specific organ(s); the rest are expressed ubiquitously. A defect in calpains causes lethality/pathogenicity, indicating their physiological indispensability. nCL-2/calpain-8a was identified as a stomach-specific calpain, whose physiological functions are unclear. To elucidate these, we characterized nCL-2 in detail. Unexpectedly, nCL-2 was localized strictly to the surface mucus cells in the gastric epithelium and the mucus-secreting goblet cells in the duodenum. Yeast twohybrid screening identified several nCL-2-intracting molecules. Of these, the ␤-subunit of coatomer complex (␤-COP) occurs in the stomach pit cells and is proteolyzed by nCL-2 in vitro. Furthermore, ␤-COP and nCL-2 co-expressed in COS7 cells co-localized in the Golgi, and Ca 2؉ -ionophore stimulation caused the proteolysis of ␤-COP near the linker region, resulting in the dissociation of ␤-COP from the Golgi. These results strongly suggest novel functions for nCL-2 that involve the membrane trafficking of mucus cells via interactions with coat protein.Calpain (EC 3.4.22.17) is an intracellular Ca 2ϩ -regulated cysteine protease, comprising a superfamily in most organisms, including some bacteria, budding yeasts, fungi, plants, and animals. Calpains proteolyze specific substrates at very limited sites to irreversibly modify/modulate their functions, structures, and activities, and are thus called "modulator proteases." Fourteen calpain genes have been identified in humans and mice and can be classified into two categories according to their expression, i.e. ubiquitous or tissue-specific (1-3). Ubiquitous -and m-calpains, two major calpains in mammals, form a heterodimer composed of a distinct 80-kDa catalytic large subunit (abbreviated to "CL" and "mCL," respectively) 2 and a common 30-kDa regulatory small subunit (30K). Calpain three-dimensional structures define four (I-IV) and two (V and VI) domains in the large and small subunits, respectively: the regulatory N-terminal domain (I), the protease domain (II), the C2-domain-like Ca 2ϩ /phospholipid-binding domain (III), the penta-EF-hand domains (IV and VI), and the Glyclustering domain (V) (4, 5). In the absence of Ca 2ϩ , the protease domain (II) is further divided into two subdomains, IIa and IIb, which form a single active domain upon binding to Ca 2ϩ (6, 7). Genetic studies in mammals and other organisms have clearly shown the physiological indispensability of the calpains. A gene knock-out mouse with a mutation in the gene for 30K, Capn4, exhibits embryonic lethality (8); mutations in the human gene for skeletal-muscle-specific p94/calpain 3, CAPN3, are responsible for limb-girdle muscular dystrophy type 2A (9); and a single nucleotide polymorphism in intron 3 of the human gene for calpain 10, CAPN10, is associated with type 2 diabetes (10). Mutations in the calpain genes of various other organisms, including yeasts, plants, nematodes, and Drosophila, result in sign...

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“…Since force-induced exposure of vinculin-binding sites on talin through a conformational change leads to the reinforcement of the focal adhesions, talin is also considered as a key mechanotransduced molecule in focal adhesions (5,20,28,35). Furthermore, previous evidence suggested that talin is functionally associated with the lipid rafts (33,41,45). Together, the retardation of the lipid rafts as well as talin externalization may lead to decreased ␤ 1 -integrin activation under low substratum rigidity.…”

Section: Discussionmentioning

confidence: 99%

Mechanosensing machinery for cells under low substratum rigidity

Wei¹,

Lin²,

Shen³

et al. 2008

American Journal of Physiology-Cell Physiology

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Wei WC, Lin HH, Shen MR, Tang MJ. Mechanosensing machinery for cells under low substratum rigidity. Am J Physiol Cell Physiol 295: C1579 -C1589, 2008. First published October 15, 2008 doi:10.1152/ajpcell.00223.2008.-Mechanical stimuli are essential during development and tumorigenesis. However, how cells sense their physical environment under low rigidity is still unknown. Here we show that low rigidity of collagen gel downregulates ␤ 1 -integrin activation, clustering, and focal adhesion kinase (FAK) Y397 phosphorylation, which is mediated by delayed raft formation. Moreover, overexpression of autoclustered ␤ 1-integrin (V737N), but not constitutively active ␤ 1-integrin (G429N), rescues FAKY397 phosphorylation level suppressed by low substratum rigidity. Using fluorescence resonance energy transfer to assess ␤ 1-integrin clustering, we have found that substratum rigidity between 58 and 386 Pa triggers ␤ 1-integrin clustering in a dose-dependent manner, which is highly dependent on actin filaments but not microtubules. Furthermore, augmentation of ␤ 1-integrin clustering enhances the interaction between ␤ 1-integrin, FAK, and talin. Our results indicate that contact with collagen fibrils is not sufficient for integrin activation. However, substratum rigidity is required for integrin clustering and activation. Together, our findings provide new insight into the mechanosensing machinery and the mode of action for epithelial cells in response to their physical environment under low rigidity.focal adhesion kinase; ␤ 1-integrin activation; ␤1-integrin clustering; lipid raft; actin cytoskeleton EXTRACELLULAR MATRIX (ECM) has an active and complex role in regulating the behavior of the cells that contact it, influencing their survival, adhesion, spreading, migration, proliferation, apoptosis, and differentiation (32). The major receptors of ECM are integrins, which are transmembrane receptors composed of two subunits, ␣ and ␤, that form connections between the cytoskeleton and ECM. Each ␣␤ heterodimer has its own binding and signal specificity (25). The initiation of integrinmediated activities involves two steps, namely, integrin activation and clustering. Integrins are locked in the inactive form without stimulation of extracellular ligands. After binding to its ligand with the extracellular domain (outside-in) (19) or stimulation by intracellular signals (inside-out) (38), an integrin is activated and its extracellular domain undergoes conformational changes, which lead to exposure of several ligandinduced binding sites (LIBS) (49). In addition to conformation changes, ligand binding also induces elevation of lateral mobility and clustering of integrins in the plasma membrane (31). Both ligand binding and clustering of integrins are critically important to activate intracellular signals. However, the regulatory mechanisms of controlling integrin activation and clustering are still unclear.Activation of integrin leads to the recruitment and organization of a number of different cytoskeletal proteins (␣-actinin, tal...

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Calpain II colocalizes with detergent-insoluble rafts on human and Jurkat T-cells

Cited by 33 publications

References 46 publications

Calpain 1 and 2 Are Required for RNA Replication of Echovirus 1

Calpain 1 and 2 Are Required for RNA Replication of Echovirus 1

Stomach-specific Calpain, nCL-2, Localizes in Mucus Cells and Proteolyzes the β-Subunit of Coatomer Complex, β-COP

Mechanosensing machinery for cells under low substratum rigidity

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