Rapid down-regulation of protein kinase C in (chediak-higashi syndrome) beige mouse by phorbol ester

Abnormal down-regulation of PKC is responsible for giant granule formation in fibroblasts from CHS (beige) mice—a thiol proteinase inhibitor, E-64-d, prevents giant granule formation in beige fibroblasts

“…The cells were disrupted by sonication for 10 s three times at 4°C in 20 mM Tris-HCl (pH7.5), 0.25 M sucrose, 2 mM EDTA, 5 mM EGTA, 2 mM phenylmethylsulfonyl fluoride, 0.01% leupeptin, and 50 mM 2-mercaptoethanol. The cytosolic and the membrane fractions were prepared as described previously [9]. When total PKC activity was measured, the cells were disrupted by sonication in the same buffer containing 0.1% Triton X-100.…”

“…Because we have previously reported that the membrane-bound PKC activity was rapidly down-regulated by PMA stimulation in PMNs and NK cells [9,10], we examined whether the abnormality in PKC activity would occur in fibroblasts from beige mice. In unstimulated cells, the PKC activity in beige fibroblasts was lower than that in normal fibroblasts, both in the cytosolic and the membrane-bound fractions (Fig.…”

“…We have previously reported that the abnormally rapid down-regulation of protein kinase C (PKC) activity in polymorphonuclear leukocytes (PMNs) and natural killer (NK) cells from beige mice is responsible for the impaired immune functions [9,10]. The abnormal PKC down-regulation is eliminated by the treatment of cells with inhibitors of calpain, which is a thiol proteinase and proteolyzes PKC to an inactive form [9], and in addition, calpain inhibitors correct the abnormalities in concanavalin A (Con A)-induced cap formation and NK activity in beige mice [10,11].…”

“…The abnormal PKC down-regulation is eliminated by the treatment of cells with inhibitors of calpain, which is a thiol proteinase and proteolyzes PKC to an inactive form [9], and in addition, calpain inhibitors correct the abnormalities in concanavalin A (Con A)-induced cap formation and NK activity in beige mice [10,11]. It is well known that the lysosomal enzyme activity of elastase and cathepsin G is selectively decreased in PMNs from beige mice [12,13].…”

Mast Cell Abnormalities in the Chédiak-Higashi Syndrome

“…Thus, neutrophils exhibit abnormal patterns of cytoplasmic granule fusion during develop ment [17][18][19][20][21], as well as augmented aggregation of surface molecules, high rates of metabolic activity, impaired ad herence and reduced chemotaxis [22][23][24][25][26]; both leukocytes [27] and erythrocytes [28] exhibit increased membrane fluidity. Abnormalities of microtubule function have also been described in the CHS [22,29,30], Finally, analysis of beige and normal mouse leukocytes indicates that the beige mutation results in enhanced ATP-depcndent Ca2+ uptake by cytoplasmic granules [31] and a markedly accel erated and enhanced downrcgulation of membrane-asso ciated protein kinase C activity in response to TPA stim ulation [32], a defect which can be corrected in vitro by thiol proteinase inhibitors [33].…”