Hood Joshua L, Logan Barbara B, Sinai Anthony P, Brooks William H, Roszman Thomas L
Department of Microbiology, Immunology and Molecular Genetics, University of Kentucky Medical Center, Lexington, KY 40536-0298, USA.
Biochem Biophys Res Commun. 2003 Oct 31;310(4):1200-12. doi: 10.1016/j.bbrc.2003.09.142.
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.
钙激活半胱氨酸蛋白酶钙蛋白酶与调节细胞黏附和迁移密切相关。这种蛋白酶的两种普遍存在的同工型，钙蛋白酶I和II，被认为是胞质蛋白，可转运至粘着斑复合体/黏附点或质膜。利用共聚焦显微镜和等密度离心法，结果表明钙蛋白酶I和II、30kDa调节亚基以及钙蛋白酶抑制蛋白与内质网和高尔基体相关。共聚焦显微镜显示，在与层粘连蛋白结合的细胞中，钙蛋白酶II与亚细胞蛋白钙连蛋白和Rab6共定位。为了进一步验证这种关联，对从层粘连蛋白刺激和未刺激的细胞中制备的细胞裂解物进行等密度离心。结果显示，层粘连蛋白刺激后，钙蛋白酶I、II、钙蛋白酶抑制蛋白和30kDa调节亚基与内质网和高尔基体的关联增加，这可通过它们在梯度中相对于钙连蛋白、Rab6、小窝蛋白和β1整合素的位置来证明。这与内质网-高尔基体界面处可诱导的钙蛋白酶活性积累相关。进一步的实验证实，钙蛋白酶II与磷脂酰肌醇4,5-二磷酸共定位。最后，钙蛋白酶II与膜脂筏相关。这些结果为钙蛋白酶/钙蛋白酶抑制蛋白网络在与细胞外基质结合的细胞中如何进行空间和时间调节提供了新的见解。
