Abstract
Objectives were to identify the PKC isoforms in cultured muscle cells, to examine roles of Ca 2+-dependent proteinases (calpains) in processing of various muscle PKC isozymes and to obtain a mechanistic description of the processing of PKCs by examining the temporal relationships between phorbol ester-dependent translocation of muscle PKCs and calpains between cytosolic and membrane compartments. Using six isoform (α, β, γ, σ, ε{lunate}, ζ )-specific polyclonal antibodies, PKC α, σ and ζ were detected in rat skeletal muscle and in L8 myoblasts and myotubes. PKC α and ζ were primarily localized in the cytosolic fraction of L8 myotubes whereas PKC σ was more abundant in the membrane fraction. Phorbol ester (TPA) caused rapid depletion of myotube PKC α and PKC σ isoforms from the cytosolic compartment and rapid appearance of these isoforms in the membrane fraction. However, long-term exposure of myotubes to TPA eventually caused down-regulation of PKCs in the membrane compartment. Down-regulation of PKCs in the membrane fraction was partially blocked by calpain inhibitor II. However, the rapid TPA-dependent cytosolic depletion of PKCs was unaffected by calpain inhibitor. This suggests that calpains may be responsible for membrane-associated down-regulation of PKCs but not for cytosolic depletion. In the final study we assessed the effects of phorbol ester on compartmentation of m-calpain with PKCs in muscle cells. Like the PKCs, TPA caused rapid association of m-calpain with the membrane fraction followed by down-regulation. This demonstrates that phorbol esters cause translocation of both PKCs and calpains to membranes where processing of PKCs may occur via the limited proteolysis exerted by calpains.
Original language | English (US) |
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Pages (from-to) | 45-54 |
Number of pages | 10 |
Journal | BBA - Molecular Cell Research |
Volume | 1267 |
Issue number | 1 |
DOIs | |
State | Published - May 29 1995 |
Externally published | Yes |
Keywords
- Calpain
- Muscle cell
- Phorbol ester
- Protein kinase C
ASJC Scopus subject areas
- Molecular Biology
- Cell Biology