Immunolocalization of cytoplasmic and myelin mCalpain in transfected Schwann cells: I. effect of treatment with growth factors

Neuberger, Tim; Chakrabarti, Arun K.; Russell, Terry; DeVries, George H.; Hogan, Edward L.; Banik, Naren L.

doi:10.1002/(sici)1097-4547(19970301)47:5<521::aid-jnr7>3.0.co;2-j

Cited by 15 publications

(6 citation statements)

References 28 publications

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“…Currently, this is beyond our technical reach, as antibodies specific for active calpains or substrates that can be constrained to, or identified within, subcellular compartments are not available. Currently, only the distribution of total calpain protein has been demonstrated and is dependent on cell type (4,30,38). In our cell system, m-and -calpain proteins appear to be distributed evenly throughout the cell, as seen by immunofluorescence (data not shown).…”

Section: Vol 24 2004 Erk Phosphorylates and Activates M-calpain 2507mentioning

confidence: 60%

Epidermal Growth Factor Activates m-Calpain (Calpain II), at Least in Part, by Extracellular Signal-Regulated Kinase-Mediated Phosphorylation

Glading¹,

Bodnar²,

Reynolds³

et al. 2004

Molecular and Cellular Biology

248

148

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How m-calpain is activated in cells has challenged investigators because in vitro activation requires nearmillimolar calcium. Previously, we demonstrated that m-calpain activation by growth factors requires extracellular signal-regulated kinase (ERK); this enables tail deadhesion and allows productive motility. We now show that ERK directly phosphorylates and activates m-calpain both in vitro and in vivo. We identified serine 50 as required for epidermal growth factor (EGF)-induced calpain activation in vitro and in vivo. Replacing the serine with alanine limits activation by EGF and subsequent cell deadhesion and motility. A construct with the serine converted to glutamic acid displays constitutive activity in vivo; expression of an estrogen receptor fusion construct produces a tamoxifen-sensitive enzyme. Interestingly, EGF-induced m-calpain activation occurs in the absence of increased intracellular calcium levels; EGF triggers calpain even in the presence of intracellular calcium chelators and in calcium-free media. These data provide evidence that m-calpain can be activated through the ERK cascade via direct phosphorylation and that this activation may occur in the absence of cytosolic calcium fluxes.The calpain family of intracellular cysteine proteinases includes 13 known members, of which at least 2 are ubiquitously expressed (47, 48). These, m-and -calpain (calpain II and calpain I, respectively), are involved in cell migration and adhesion, being regulated downstream of both integrin and growth factor receptor activation (19). -Calpain has been implicated strongly in cell motility and adhesion primarily driven by integrin-mediated signals: calpains have been shown to be required during both cell spreading and adhesion (3,4,41) and for the release of the rear of migrating cells (26). On the other hand, m-calpain has been observed to be activated downstream of the epidermal growth factor (EGF) receptor (EGFR) and is required for growth factor-induced motility and deadhesion (18,44). This effect is specific to m-calpain, as antisense down-regulation of -calpain did not appreciably affect growth factor-induced motility and as EGF-induced calpain activity and motility were dependent on m-calpain (18). m-Calpain affects the migration of EGF-induced fibroblasts by promoting rear release during active motility (2, 43, 44). In general, functions of calpains in motility and adhesion apparently derive from their ability to cleave components of adhesion complexes in a limited manner, altering their function and leading to increased adhesion turnover (19,29,39). However, the molecular mechanism by which calpain activities are regulated during these events is not understood.The two ubiquitous isoforms, -and m-calpain, are presumed to be activated by intracellular calcium fluxes, since these enzymes require this divalent cation in vitro. Indeed mand -calpain are named for their relative requirement for calcium, with -calpain requiring micromolar, and m-calpain requiring near-millimolar, concentrations of calcium (16)....

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Section: Vol 24 2004 Erk Phosphorylates and Activates M-calpain 2507mentioning

confidence: 60%

Epidermal Growth Factor Activates m-Calpain (Calpain II), at Least in Part, by Extracellular Signal-Regulated Kinase-Mediated Phosphorylation

Glading¹,

Bodnar²,

Reynolds³

et al. 2004

Molecular and Cellular Biology

248

148

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“…Earlier findings of m‐calpain localization in myelin suggest that components of this membrane should be present in the myelin‐forming oligodendrocytes of central nervous system (CNS) and Schwann cells of peripheral nervous system (PNS). Calpain expression and activity in Schwann cells have been previously reported (Mata et al, 1991; Chakrabarti et al, 1997; Neuberger et al, 1997). Here we show that primary cultures of oligodendrocytes express μ‐calpain and m‐calpain as well as the endogenous inhibitor calpastatin at the mRNA level.…”

Section: Discussionmentioning

confidence: 82%

Calpain and calpastatin expression in primary oligodendrocyte culture: Preferential localization of membrane calpain in cell processes

Ray

Neuberger²,

Deadwyler

et al. 2002

J of Neuroscience Research

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The cellular localization of calpain is important in understanding the roles that calpain may play in physiological function. We, therefore, examined calpain expression, activity, and immunofluorescent localization in primary cultures of rat oligodendrocytes. The mRNA expression of m-calpain was 64.8% (P = 0.0033) and 50.5% (P = 0.0254) higher than that of mu-calpain and calpastatin, respectively, in primary culture oligodendrocytes. The levels of mRNA expression of mu-calpain and calpastatin were not significantly different. As revealed by Western blotting, cultured oligodendrocytes contained a 70 kD major band identified by membrane m-calpain antibody, a 80 kD band recognized by cytosolic m-calpain antibody, and calpastatin bands ranging from 45 to 100 kD detected by a calpastatin antibody. Calpain activity in oligodendrocytes was determined by Ca(2+)-dependent 71.2% degradation of endogenous myelin basic protein compared with control; this activity was inhibited significantly (P = 0.0111) by EGTA and also substantially by calpeptin. Localization of calpain in cultured oligodendrocytes revealed strong membrane m-calpain immunofluorescence in the oligodendrocyte cell body and its processes. In contrast, the cytosolic antibody stained primarily the oligodendrocyte cell body, whereas the processes were stained very weakly or not at all. These results indicate that the major form of calpain in glial cells is myelin (membrane) m-calpain. The dissimilar localization of cytosolic and membrane m-calpain may indicate that each isoform has a unique role in oligodendrocyte function.

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“…In some cases, nuclear localization of calpains is developmentally regulated or sensitive to changes in the environment. For example, both m-calpain and -calpain are predominantly nuclear in myoblasts, but become cytosolic as they differentiate into myotubes (38,62); and translocation of cytosolic calpains to the nucleus has been observed in Schwann cells treated with growth factors (63), in lens epithelial cell lines treated with ionomycin (64), and in hippocampal neurons exposed to ischemia (65). In A431 cells, nuclear translocation of calpain is ATP-dependent (37).…”

Section: Discussionmentioning

confidence: 99%

Nuclear Calpain Regulates Ca2+-dependent Signaling via Proteolysis of Nuclear Ca2+/Calmodulin-dependent Protein Kinase Type IV in Cultured Neurons

Tremper-Wells

Vallano

2005

Journal of Biological Chemistry

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CaMKIV is highly enriched in the nucleus and thought to be critical for improved survival. Here, we demonstrate by immunolocalization/ confocal microscopy and subcellular fractionation that the regulatory and catalytic subunits of m-calpain are enriched in GC nuclei, including GCs grown in medium containing 5 mM KCl. Calpain-mediated proteolysis of CaMKIV is selective, as several other nuclear and nonnuclear calpain substrates were not degraded under chronic depolarizing culture conditions. Depolarization and Ca 2؉ -dependent down-regulation of CaMKIV were associated with significant alterations in other components of the Ca 2؉ -CaMKIV signaling cascade: the ratio of phosphorylated to total cAMP response element-binding protein (a downstream CaMKIV substrate) was reduced by ϳ10-fold, and the amount of CaMK kinase (an upstream activator of CaMKIV) protein and mRNA was significantly reduced. We hypothesize that calpain-mediated CaMKIV proteolysis is an autoregulatory feedback response to sustained activation of a Ca 2؉ -CaMKIV signaling pathway, resulting from growth of cultures in medium containing 25 mM KCl. This study establishes nuclear m-calpain as a regulator of CaMKIV and associated signaling molecules under conditions of sustained Ca 2؉ influx.

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Immunolocalization of cytoplasmic and myelin mCalpain in transfected Schwann cells: I. effect of treatment with growth factors

Cited by 15 publications

References 28 publications

Epidermal Growth Factor Activates m-Calpain (Calpain II), at Least in Part, by Extracellular Signal-Regulated Kinase-Mediated Phosphorylation

Epidermal Growth Factor Activates m-Calpain (Calpain II), at Least in Part, by Extracellular Signal-Regulated Kinase-Mediated Phosphorylation

Calpain and calpastatin expression in primary oligodendrocyte culture: Preferential localization of membrane calpain in cell processes

Nuclear Calpain Regulates Ca2+-dependent Signaling via Proteolysis of Nuclear Ca2+/Calmodulin-dependent Protein Kinase Type IV in Cultured Neurons

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