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 Ser86 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, H2O2-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, H2O2, 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. 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 Ser86 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, H2O2-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, H2O2, 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. sHsps 2The abbreviations used are: Hsp, heat shock protein; sHsp, small Hsp; GA, glutaraldehyde; MAPK, mitogen-activated protein kinase; MEF, mouse embryonic fibroblast; MK, MAPK-activated protein kinase; PK, protein kinase; WT, wild type; GFP, green fluorescent protein; EGFP, enhanced GFP; GST, glutathione S-transferase; PBS, phosphate-buffered saline. contain the well conserved α-crystallin domain and are present as a family of ten structurally related proteins in humans (1Kappe G. Franck E. Verschuure P. Boelens W.C. Leunissen J.A. de Jong W.W. Cell Stress Chaperones. 2003; 8: 53-61Crossref PubMed Scopus (383) Google Scholar). 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In cells, Hsp25 Ser86 phosphorylation and stress-dependent In addition, insolubilization of Hsp25 is in to arsenite, H2O2, and sublethal heat shock the consequences in stress resistance are and were from p38 and its were from was from A. for Hsp25 were from which can of Hsp25, was a from of Hsp25 were from against MK2 and were from Cell and were from was from was from was from was from was from were from for wild Hsp27 and was by of T. M. Kotlyarov A. G. C. C. M. A.P. Buchner J. Gaestel M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google with and and into for 14-3-3 proteins and were a from of and and were a from of Cell and mouse embryonic fibroblasts from wild and MK2-deficient were by with large Mol. 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Full Text Full Text PDF PubMed Scopus (275) Google and of cell were with of in for at the was by of and were to Hsp25 was by with against cells were and to cells were three with and with was with with arsenite cells were three with and were with of 14-3-3 or protein and of glutathione at were with and to by Hsp25 was by are as between wild and MK2-deficient cells were using of was to be and MK2-dependent Phosphorylation of sHsps upon Stress by the role of phosphorylation of endogenous sHsp we the of for of in from mouse and human phosphorylation of serine of the two MK2 phosphorylation sites in Hsp25 (10Stokoe D. Engel K. Campbell D.G. Cohen P. Gaestel M. FEBS Lett. 1992; 313: 307-313Crossref PubMed Scopus (472) Google Scholar), can be phosphorylation of Hsp25 at site can be by the for of the p38 MAPK pathway J.C. McDonnell P.C. Kumar S. D. D. M.J. M.M. Livi G.P. Young P.R. Nature. 1994; PubMed Scopus Google Scholar), structurally related against the human of the MK2 phosphorylation site in Hsp25, serine to the from mouse cells are in stress-dependent phosphorylation of two sites of we analyzed Hsp27 phosphorylation at serine 15 and serine the site to Ser86 in in human cells can be that sites are phosphorylated in to arsenite treatment and that phosphorylation is by Hence, we the that of be for stress-dependent phosphorylation of sHsps at In cells, we used a against MK2 and the S. as In a of 15 which has of to a of Hsp27 phosphorylation at sites the role of the p38 MAPK the kinases MK2 MK3 in sHsp that the is well for Hsp25 phosphorylation in we to in which lack the protein kinase MK2 A. A. C. R. C. Gaestel M. Nat. Cell Biol. PubMed Scopus Google Scholar). While in cells phosphorylation of Hsp25 can be is in MK2-deficient cells, p38 is in cell to a the that MK2 is the major kinase that endogenous Hsp25 after arsenite treatment in mouse embryonic of Hsp25 in and upon phosphorylation of Hsp25 is in cell be well to the role of phosphorylation of endogenous Hsp25 for its in of from and and cells was We were to two different for Hsp25 as to with the of oligomeric molecular at to and as or in a between and In cells, Hsp25 is between and molecular and is no between and after arsenite treatment the of Hsp25 in molecular complexes is decreased in cells, whereas cells show no from as from of three independent in that is a and that its is we demonstrate that of Hsp25 in cells is molecular complexes after arsenite whereas only are in in In the between and molecular after with we as as dimers of Hsp25 can be at a of in and cells, dimers and to large arsenite treatment large can be in at of dimers are the disaggregation of large In contrast, only of dimers are at of or in cells, and large the that the disaggregation of Hsp25 is in MK2-deficient We used to in As phosphorylated Hsp25 can only be in cells after arsenite with Hsp25, to be in dimers that phosphorylation disaggregation of Hsp25 to dimers that phosphorylation with MK2-dependent in proteins are are in of processes in the cell and and with more after phosphorylation J. Cell 2004; PubMed Scopus Google Scholar, M. Campbell D.G. N. C. Biochem. J. 2004; PubMed Scopus Google Scholar). of sHsps family been recently as protein P. L. M. N. Y. A. J. 2005; PubMed Scopus Google Scholar), and MK2 phosphorylation can 14-3-3 binding sites in of MK2 M. Nat. Mol. Cell. Biol. PubMed Scopus Google Scholar), we Hsp25 with 14-3-3 proteins in a MK2-dependent 14-3-3 proteins been used for were or with arsenite for and with Hsp25 with after of the cell with arsenite in cells is decreased Hsp25 binding to is no Hsp25 was in with 14-3-3 using from Hsp25 been in with in vitro in a MK2-dependent as well that is no for at in in the in vitro binding of phosphorylated Hsp25 is to the proteins present in and the to be in of and in and and their phosphorylation been demonstrated to a role in actin J.N. Hickey E. Weber L.A. J. J. Biol. Chem. Full Text PDF PubMed Google Scholar, J. Lambert H. G. Lavoie J.N. J. J. J. Cell PubMed Google Scholar, T. K. Vandekerckhove J. M. J. 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