Phosphorylation of human small heat shock protein HspB8 (Hsp22) by ERK1 protein kinase

For a successful human pregnancy, the phasic smooth muscle of the myometrium must remain quiescent until labor. Activation of cAMP/cAMP-dependent protein kinase A (PKA) pathways contributes to this quiescence. The small heat-shock protein 20 (HSP20) is a target of PKA, and phosphorylated HSP20 (pHSP20) modulates relaxation of tonic vascular smooth muscle via interaction with actin, independent of myosin dephosphorylation. Our objective was to determine whether relaxation in human myometrium is associated with changes in phosphorylation of HSP20. Myometrium was obtained at elective cesarean. Elevating cAMP with forskolin or rolipram (a phosphodiesterase inhibitor) caused substantial relaxation of spontaneously contracting human myometrial strips, of 92 ± 4% (mean ± sem, n = 10) and 84 ± 7% (n = 6), respectively. Subsequent two-dimensional electrophoresis with immunoblotting of strip extracts showed a significant 2.6- and 2.1-fold increase in phosphorylated HSP20 (pHSP20) after forskolin (P < 0.01; n = 5) or rolipram treatment (P < 0.05; n = 4). Noncyclic-nucleotide-mediated relaxation, induced by the calcium channel blocker nifedipine, did not alter pHSP20. Inhibition of PKA with H89 significantly attenuated rolipram-induced relaxation (P < 0.01; n = 4), and partially reduced rolipram-stimulated pHSP20. Total and pHSP20 protein was unchanged in term laboring and nonlaboring myometria. Coimmunoprecipitation studies revealed a specific association of HSP20 with α-smooth muscle actin and HSP27, a key regulator of actin filament dynamics. Finally, coimmunofluorescence demonstrated moderate colocalization of HSP20 with α-smooth muscle actin in the cytoplasm of laboring myometria. Our data support a novel role for pHSP20 in the modulation of cyclic-nucleotide-mediated myometrial relaxation, through interaction with actin. pHSP20 represents an important new target for future tocolytic therapy.

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Follicle Stimulating Hormone (FSH) Activates the p38 Mitogen-Activated Protein Kinase Pathway, Inducing Small Heat Shock Protein Phosphorylation and Cell Rounding in Immature Rat Ovarian Granulosa Cells

Endocrinology ◽

10.1210/endo.139.7.6188 ◽

1998 ◽

Vol 139 (7) ◽

pp. 3353-3356 ◽

Cited By ~ 70

Author(s):

Evelyn T. Maizels ◽

Joshua Cottom ◽

Jonathan C. R. Jones ◽

Mary Hunzicker-Dunn

Keyword(s):

Heat Shock ◽

Protein Kinase ◽

Heat Shock Protein ◽

Follicle Stimulating Hormone ◽

Small Heat Shock Protein ◽

Mitogen Activated Protein Kinase ◽

Immature Rat ◽

Ovarian Granulosa Cells ◽

Mitogen Activated Protein ◽

Kinase Pathway

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Heat shock protects cardiac cells from doxorubicin-induced toxicity by activating p38 MAPK and phosphorylation of small heat shock protein 27

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00395.2006 ◽

2006 ◽

Vol 291 (6) ◽

pp. H2680-H2691 ◽

Cited By ~ 56

Author(s):

C. D. Venkatakrishnan ◽

Arun K. Tewari ◽

Leni Moldovan ◽

Arturo J. Cardounel ◽

Jay L. Zweier ◽

...

Keyword(s):

Heat Shock ◽

Protein Kinase ◽

Heat Shock Protein ◽

Small Heat Shock Protein ◽

Heat Shock Protein 27 ◽

Cardiac Cells ◽

Two Dimensional Gel Electrophoresis ◽

Actin Remodeling ◽

Dilative Cardiomyopathy ◽

Endogenous Antioxidant

Doxorubicin (DOX) and its derivatives are used as chemotherapeutic drugs to treat cancer patients. However, production of DOX-mediated reactive oxygen species (ROS) by prolonged use of these drugs has been found to cause dilative cardiomyopathy and congestive heart failure. Thus various preventive modalities have been developed to avoid this side effect. We have found that the DOX-mediated oxidant-induced toxicity in cardiac cells could be minimized by hyperthermia-induced small heat shock protein 27 (HSP27); that is, this protein acts as an endogenous antioxidant against DOX-derived oxidants such as H2O2. Heat shock-induced HSP27 was found to act as an antiapoptotic protein (reducing ROS and Bax-to-Bcl2 ratio) against DOX, and its phosphorylated isoforms stabilized F-actin remodeling in DOX-treated cardiac cells and, hence, attenuated the toxicity. Protein kinase assays and proteomic analyses suggested that higher expression of HSP27 and its phosphorylation are responsible for the protection in heat-shocked cells. Two-dimensional gel electrophoresis showed six isoforms (nonphosphorylated and phosphorylated) of HSP27. Matrix-assisted laser desorption/ionization time of flight analyses showed α- and β-isoforms of HSP27, which are phosphorylated by various protein kinases. Ser15 and Ser85 phosphorylation of HSP27 by MAPK-assisted protein kinase 2 was found to be the key mechanism in reduction of apoptosis and facilitation of F-actin remodeling. The present study illustrates that hyperthermia protects cells from DOX-induced death through induction and phosphorylation of HSP27 and its antiapoptotic and actin-remodeling activities.

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The problem of protein kinase activity of small heat shock protein Hsp22 (H11 or HspB8)

Biochemical and Biophysical Research Communications ◽

10.1016/j.bbrc.2004.10.074 ◽

2004 ◽

Vol 325 (3) ◽

pp. 649-652 ◽

Cited By ~ 13

Author(s):

Maria V. Kim ◽

Alim S. Seit-Nebi ◽

Nikolai B. Gusev

Keyword(s):

Heat Shock ◽

Protein Kinase ◽

Heat Shock Protein ◽

Kinase Activity ◽

Small Heat Shock Protein ◽

Protein Kinase Activity

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Replacement of Arg in the conserved N-terminal RLFDQxFG motif affects physico-chemical properties and chaperone-like activity of human small heat shock protein HspB8 (Hsp22)

PLoS ONE ◽

10.1371/journal.pone.0253432 ◽

2021 ◽

Vol 16 (6) ◽

pp. e0253432

Author(s):

Vladislav M. Shatov ◽

Nikolai N. Sluchanko ◽

Nikolai B. Gusev

Keyword(s):

Heat Shock ◽

Heat Shock Protein ◽

Chemical Properties ◽

Small Heat Shock Protein ◽

Size Exclusion ◽

Terminal Domain ◽

Physico Chemical ◽

Exclusion Chromatography ◽

Erk1 Protein

The small heat shock protein (sHsp) called HspB8 (formerly, Hsp22) is one of the least typical sHsp members, whose oligomerization status remains debatable. Here we analyze the effect of mutations in a highly conservative sequence located in the N-terminal domain of human HspB8 on its physico-chemical properties and chaperone-like activity. According to size-exclusion chromatography coupled to multi-angle light scattering, the wild type (WT) HspB8 is present as dominating monomeric species (~24 kDa) and a small fraction of oligomers (~60 kDa). The R29A amino acid substitution leads to the predominant formation of 60-kDa oligomers, leaving only a small fraction of monomers. Deletion of the 28–32 pentapeptide (Δ mutant) results in the formation of minor quantities of dimers (~49 kDa) and large quantities of the 24-kDa monomers. Both the WT protein and its Δ mutant efficiently bind a hydrophobic probe bis-ANS and are relatively rapidly hydrolyzed by chymotrypsin, whereas the R29A mutant weakly binds bis-ANS and resists chymotrypsinolysis. In contrast to HspB8 WT and its Δ mutant, which are well phosphorylated by cAMP-dependent and ERK1 protein kinases, the R29A mutant is poorly phosphorylated. R29A mutation affects the chaperone-like activity of HspB8 measured in vitro. It is concluded that the irreplaceable Arg residue located in the only highly conservative motif in the N-terminal domain of all sHsp proteins affects the oligomeric structure and key properties of HspB8.

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