Analysis of Properties of Small Heat Shock Protein Hsp25 in MAPK-activated Protein Kinase 2 (MK2)-deficient Cells

Abstract

Small heat shock proteins (sHsps) exist in dynamic oligomeric complexes and display diverse biological functions ranging from chaperone properties to modulator of apoptosis. So far, the role of stress-dependent phosphorylation of mammalian sHsps for its structure and function has been analyzed by using various phosphorylation site mutants overexpressed in different cell types as well as by non-exclusive inhibitors of the p38 MAPK cascade. Here we investigate the role of phosphorylation of endogenous sHsp in a genetic model lacking the major Hsp25 kinase, the MAP kinase-activated protein kinase MK2. We demonstrate that in MK2-deficient fibroblasts, where no stress-dependent phosphorylation of Hsp25 at Ser 86 and no in vitro binding to 14-3-3 was detectable, stress-dependent disaggregation of endogenous Hsp25 complexes is impared and kinetics of arsenite-dependent, H 2 O 2 -dependent, and sublethal heat shock-induced insolubilization of Hsp25 is delayed. Similarly, green fluorescent protein-tagged Hsp25 shows retarded subcellular accumulation into stress granules in MK2-deficient cells after arsenite treatment. Decreased insolubilization of Hsp25 in MK2-deficient cells correlates with increased resistance against arsenite, H 2 O 2 , and sublethal heat shock treatment and with decreased apoptosis. In contrast, after severe, lethal heat shock MK2-deficient embryonic fibroblasts cells show fast and complete insolubilization of Hsp25 independent of MK2 and no increased stress resistance. Hence, MK2-dependent formation of insoluble stress granules and irreversible cell damage by oxidative stresses and sublethal heat shock correlate and only upon severe, lethal heat shock MK2-independent processes could determine insolubilization of Hsp25 and are more relevant for cellular stress damage.

AKT-dependent HspB1 (Hsp27) Activity in Epidermal Differentiation

“…Additional kinases are also known to regulate HspB1 (reviewed in Ref. 41), and we suspect that these kinases are active developmentally and in adult epidermis. The p38 MAPK pathway resulting in HspB1 phosphorylation is active in keratinocytes subject to stress (31,42).…”

The p38 MAPK stress pathway as a tumor suppressor or more?

“…In human colon cancer cells, however, the p38 activation only leads to a cell-death response when K-Ras gene is mutated, and this selective K-Ras dependent apoptotic effect was shown to be due to a down-regulation of anti-apoptotic vitamin D receptor (VDR) protein expression (94). Knockout studies further revealed a proapoptotic property of the p38 pathway by demonstrating a decreased cell death in cells lacking MKK6 (95), p38alpha (64,96) and MK2 (97). These results together suggest a sufficient role of the p38 pathway in inducing cell death.…”

Cross-regulation of cytokine signalling: pro-inflammatory cytokines restrict IL-6 signalling through receptor internalisation and degradation

“…Phenylarsine oxide (PAO) was from Sigma-Aldrich. The MK2-inhibitor (2,4-diamino-5H-chromeno-[2,3-b]pyridine-3-carbonitrile; U.S. patent 2004/0127511) was used as described by Vertii et al (Vertii et al, 2006). Poly(vinylidene difluoride) (PVDF) membrane was from PALL (Dreieich, Germany).…”