Association of the calpain/calpastatin network with subcellular organelles.
The calcium-activated cysteine protease calpain is intimately involved in modulating cell adhesion and migration. The two ubiquitous isoforms of this protease, calpain I and II, are considered to be cytosolic proteins that can translocate to both focal complexes/adhesions or the plasma membrane. Using confocal microscopy and isopycnic density centrifugation, the results demonstrate that calpain I and II, the 30kDa regulatory subunit, and calpastatin associate with the endoplasmic reticulum and Golgi apparatus. Confocal microscopy reveals that calpain II colocalizes with the subcellular proteins calnexin and Rab6 in cells bound to laminin. To further verify this association, cell lysates prepared from laminin stimulated and unstimulated cells were subjected to isopycnic density centrifugation. The results reveal an increased association of calpain I, II, calpastatin, and the 30kDa regulatory subunit with the endoplasmic reticulum and Golgi apparatus as evidenced by their position in the gradient relative to calnexin, Rab6, caveolin, and beta1 integrin after laminin stimulation. This correlates with the accumulation of inducible calpain activity at the endoplasmic reticulum-Golgi apparatus interface. Further experiments established that calpain II colocalizes with phosphatidylinositol 4,5-bisphosphate. Finally, calpain II associates with membrane lipid rafts. These results provide new insights into how the calpain/calpastatin network is spatially and temporally regulated in cells binding to the extracellular matrix.
Hood
Joshua L
JL
Department of Microbiology, Immunology and Molecular Genetics, University of Kentucky Medical Center, Lexington, KY 40536-0298, USA.
Logan
Barbara B
BB
Sinai
Anthony P
AP
Brooks
William H
WH
Roszman
Thomas L
TL
D015153:Blotting, Western; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D002154:Calpain / Q000378:metabolism*; D045744:Cell Line, Tumor; D048429:Cell Size; D002478:Cells, Cultured; D002498:Centrifugation; D006801:Humans; D008563:Membrane Lipids / Q000378:metabolism; D018613:Microscopy, Confocal; D015388:Organelles / Q000378:metabolism*; D013347:Subcellular Fractions / Q000378:metabolism*; D020691:rab GTP-Binding Proteins / Q000378:metabolism
D002135:Calcium-Binding Proteins; D008563:Membrane Lipids; C094135:Rab6 protein; C033668:calpastatin; D002154:Calpain; D020691:rab GTP-Binding Proteins