Proteolysis of the tumour suppressor hDlg in response to osmotic stress is mediated by caspases and independent of phosphorylation

Abstract

Human disc‐large (hDlg) is a scaffold protein critical for the maintenance of cell polarity and adhesion. hDlg is a component of the p38γ MAP kinase pathway, which is important for the adaptation of mammalian cells to changes in environmental osmolarity. Here we report a strong decrease in the levels of hDlg protein in the human epithelial cell line HeLa when exposed to osmotic shock. This is independent of the phosphorylation state of hDlg, is prevented by preincubating the cell with the caspase inhibitor z‐VAD and is part of the apoptotic process triggered by cellular stress. Although, both caspase 3 and caspase 6 are strongly activated by osmotic shock, the time course of caspase 6 activation parallels hDlg degradation, suggesting that this caspase may be responsible for the proteolysis. Mutating hDlg Asp747 to Ala abolishes caspase‐induced cleavage, but does not affect the early stage of apoptosis or cell attachment. Our findings show that osmotic stress triggers hDlg degradation through a mechanism different from the one mediated by proteasomes, and we identify hDlg as a caspase substrate during the apoptotic process, although its proteolysis may not be implicated in the progression of early apoptosis.

Hyperosmotic Stress Induces Tau Proteolysis by Caspase‐3 Activation in SH‐SY5Y Cells

“…Our results show that sorbitol increases the activity of these proteases in a time-dependent manner. The ability of hyperosmotic stress to induce proteasome, m-calpain and caspase-3 activation has been documented in different neuronal and non-neuronal cell lines [Stoothoff and Johnson, 2001;Inesta-Vaquera et al, 2009;Nakaya et al, 2009].…”

p38γ and p38δ modulate innate immune response by regulating MEF2D activation

“…Lysates were centrifuged at 20,800 g for 15 min at 4°C, the supernatants removed, quick frozen in liquid nitrogen and stored at -80°C until used. Transfection of MEF was carried out using poly(ethylenimine), each 6 cm dish of cells was transfected with 2-12 μg of poly(ethylenimine) (Polysciences, Eppelheim, Germany) and 1-6 μg of plasmid DNA, as described previously (Iñesta-Vaquera et al ., 2009). Cells were lysed 24-36 h after transfection.…”

“…Transfection of MEF was carried out using poly(ethylenimine), each 6 cm dish of cells was transfected with 2-12 µg of poly(ethylenimine) (Polysciences, Eppelheim, Germany) and 1-6 µg of plasmid DNA, as described previously (Iñesta-Vaquera et al, 2009). Cells were lysed 24-36 h after transfection.…”

Cellulose alters the expression of nuclear factor kappa B-related genes and Toll-like receptor-related genes in human peripheral blood mononuclear cells

“…MAPK13 is a kinase in the p38 MAP kinase family, which can be activated by proinflammatory cytokines and cellular stress (Yang et al, 2014;Zhong et al, 2014). MAPK13 substrates include the transcription factor ATF2 and the microtubule dynamics regulator stathmin, and it is reported to play a role in differentiation, apoptosis, and inflammatory diseases (Inesta-Vaquera et al, 2009;O'Callaghan, Fanning, & Barry, 2014). Upregulation of these genes within TLR-related pathways demonstrates that cellulose not only exerts effects on genes which are directly related to innate immune reactivity but also impacts on genes involved in cell cycle regulation and metabolic processes of immune cells.…”