Modulation of cellular thermoresistance and actin filament stability accompanies phosphorylation-induced changes in the oligomeric structure of heat shock protein 27.

Phosphorylation of heat shock protein 27 (HSP27) can modulate actin filament dynamics in response to growth factors. During heat shock, HSP27 is phosphorylated at the same sites and by the same protein kinase as during mitogenic stimulation. This suggests that the same function of the protein may be activated during growth factor stimulation and the stress response. To determine the role of HSP27 phosphorylation in the heat shock response, several stable Chinese hamster cell lines that constitutively express various levels of the wild-type HSP27 (HU27 cells) or a nonphosphorylatable form of human HSP27 (HU27pm3 cells) were developed. In contrast to HU27 cells, which showed increased survival after heat shock, HU27pm3 cells showed only slightly enhanced survival. Evidence is presented that stabilization of microfilaments is a major target of the protective function of HSP27. In the HU27pm3 cells, the microfilaments were thermosensitized compared with those in the control cells, whereas wild-type HSP27 caused an increased stability of these structures in HU27 cells. HU27 but not HU27pm3 cells were highly resistant to cytochalasin D treatment compared with control cells. Moreover, in cells treated with cytochalasin D, wild-type HSP27 but not the phosphorylated form of HSP27 accelerated the reappearance of actin filaments. The mutations in human HSP27 had no effect on heat shock-induced change in solubility and cellular localization of the protein, indicating that phosphorylation was not involved in these processes. However, induction of HSP27 phosphorylation by stressing agents or mitogens caused a reduction in the multimeric size of the wild-type protein, an effect which was not observed with the mutant protein. We propose that early during stress, phosphorylation-induced conformational changes in the HSP27 oligomers regulate the activity of the protein at the level of microfilament dynamics, resulting in both enhanced stability and accelerated recovery of the filaments. The level of protection provided by HSP27 during heat shock may thus represent the contribution of better maintenance of actin filament integrity to overall cell survival.

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Regulation of actin filament dynamics by p38 map kinase-mediated phosphorylation of heat shock protein 27

Journal of Cell Science ◽

10.1242/jcs.110.3.357 ◽

1997 ◽

Vol 110 (3) ◽

pp. 357-368 ◽

Cited By ~ 6

Author(s):

J. Guay ◽

H. Lambert ◽

G. Gingras-Breton ◽

J.N. Lavoie ◽

J. Huot ◽

...

Keyword(s):

Heat Shock ◽

Protein Kinase ◽

Heat Shock Protein ◽

Chinese Hamster ◽

Strong Support ◽

Specific Inhibitor ◽

Heat Shock Protein 27 ◽

Cell Extracts ◽

Hsp27 Phosphorylation ◽

Homeostatic Function

We have studied the contribution of the individual kinases of the MAP (mitogen-activated protein) kinase family, including ERK (extracellular-signal regulated kinase), JNK/SAPK (c-JUN NH2-terminal kinase/stress-activated protein kinase) and p38, to activation of the HSP27 (heat shock protein 27) kinase MAPKAP kinase-2/3 and to HSP27 phosphorylation in Chinese hamster CCL39 cells stimulated by either growth factors, cytokines or stressing agents. In vitro assays using fractionated cell extracts or immunoprecipitates indicated that only fractions containing ERK or p38, and not those containing JNK/SAPK, had the capacity to activate MAPKAP kinase-2/3. In vivo, however, it appeared that only p38 is an upstream activator of HSP27 phosphorylation after both stress or growth factor stimulation: expression of an interfering mutant of ras, which blocked the activation of ERK by both types of inducers, had no effect on HSP27 phosphorylation and p38 activation; and the cell-permeant specific inhibitor of 038, SB203580, blocked MAPKAP-kinase2/3 activation and HSP27 phosphorylation. HSP27 has been suggested to have a phosphorylation-activated homeostatic function at the actin cytoskeleton level. This raises the possibility that p38 might be directly involved in mediating actin responses to external stimuli. Accordingly, we observed that a prior activation of p38 increased the stability of the actin microfilaments in cells exposed to cytochalasin D. The effect was dependent on the expression of HSP27 and was totally annihilated by blocking the p38 activity with SB203580. The results provide strong support to the idea that activation of p38 during adverse environmental conditions serves a homeostatic function aimed at regulating actin dynamics that would otherwise be destabilized during stress. Its activation during normal agonist stimulation may constitute an additional actin signaling pathway, the importance of which depends on the level of expression of HSP27.

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Inhibition of Daxx-Mediated Apoptosis by Heat Shock Protein 27

Molecular and Cellular Biology ◽

10.1128/mcb.20.20.7602-7612.2000 ◽

2000 ◽

Vol 20 (20) ◽

pp. 7602-7612 ◽

Cited By ~ 299

Author(s):

Steve J. Charette ◽

Josée N. Lavoie ◽

Herman Lambert ◽

Jacques Landry

Keyword(s):

Heat Shock ◽

Heat Shock Protein ◽

Major Change ◽

Heat Shock Protein 27 ◽

Supramolecular Organization ◽

Protective Function ◽

Cellular Protection ◽

Hsp27 Phosphorylation ◽

Fas Pathway ◽

Induced Apoptosis

ABSTRACT Heat shock protein 27 (HSP27) confers cellular protection against a variety of cytotoxic stresses and also against physiological stresses associated with growth arrest or receptor-mediated apoptosis. Phosphorylation modulates the activity of HSP27 by causing a major change in the supramolecular organization of the protein, which shifts from oligomers to dimers. Here we show that phosphorylated dimers of HSP27 interact with Daxx, a mediator of Fas-induced apoptosis, preventing the interaction of Daxx with both Ask1 and Fas and blocking Daxx-mediated apoptosis. No such inhibition was observed with an HSP27 phosphorylation mutant that is only expressed as oligomers or when apoptosis was induced by transfection of a Daxx mutant lacking its HSP27 binding domain. HSP27 expression had no effect on Fas-induced FADD- and caspase-dependent apoptosis. However, HSP27 blocked Fas-induced translocation of Daxx from the nucleus to the cytoplasm and Fas-induced Daxx- and Ask1-dependent apoptosis. The observations revealed a new level of regulation of the Fas pathway and suggest a mechanism for the phosphorylation-dependent protective function of HSP27 during stress and differentiation.

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Identification of a protein altered in mutants resistant to microtubule inhibitors as a member of the major heat shock protein (hsp70) family

Molecular and Cellular Biology ◽

10.1128/mcb.10.10.5160-5165.1990 ◽

1990 ◽

Vol 10 (10) ◽

pp. 5160-5165

Author(s):

S Ahmad ◽

R Ahuja ◽

T J Venner ◽

R S Gupta

Keyword(s):

Heat Shock ◽

Heat Shock Protein ◽

Cho Cells ◽

Chinese Hamster ◽

Immunofluorescent Staining ◽

Mutant Form ◽

Two Dimensional ◽

Wild Type ◽

Cho Cell ◽

Microtubule Inhibitors

A major cellular protein (P2; approximately 70 kilodaltons) which is altered in Chinese hamster ovary (CHO) cell mutants resistant to the microtubule inhibitors colchicine and podophyllotoxin has been shown to correspond to the constitutive form of the 70-kilodalton heat shock protein (hsc70). The inference that P2 and hsc70 are the same protein is based on the following observations: (i) migration of P2 in two-dimensional polyacrylamide gels in the same position as that reported for hsc70; (ii) cross-reactivity of a monoclonal antibody which reacts with both the constitutive and induced forms of hsp70 with the P2 spot from wild-type CHO cells and with both P2 and a mutant form of P2 in a CHO cell mutant; (iii) specific reactivity of a polyclonal antibody to P2 with both the constitutive and heat-induced forms of hsp70 in human cells; (iv) identical immunofluorescent staining of dot/patchlike structures with both P2 and hsp70 antibodies in human and CHO cells; and (v) a cDNA clone for hsc70 has been isolated and sequenced from wild-type CHO cells. The in vitro transcription and translation product of this cDNA has been shown to comigrate with the P2 protein spot in two-dimensional gels, indicating their identity. The fact that there is an alteration in hsc70 in mutants resistant to antimitotic drugs suggests a role for this protein in the in vivo assembly and function of microtubules.

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Identification of a protein altered in mutants resistant to microtubule inhibitors as a member of the major heat shock protein (hsp70) family.

Molecular and Cellular Biology ◽

10.1128/mcb.10.10.5160 ◽

1990 ◽

Vol 10 (10) ◽

pp. 5160-5165 ◽

Cited By ~ 37

Author(s):

S Ahmad ◽

R Ahuja ◽

T J Venner ◽

R S Gupta

Keyword(s):

Heat Shock ◽

Heat Shock Protein ◽

Cho Cells ◽

Chinese Hamster ◽

Immunofluorescent Staining ◽

Mutant Form ◽

Two Dimensional ◽

Wild Type ◽

Cho Cell ◽

Microtubule Inhibitors

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Cilia‐mediated heat shock protein 27 (hsp27) suppression modulates actin polymerization and organization in wild type mouse and oak ridge polycystic kidney (orpk) cells

The FASEB Journal ◽

10.1096/fasebj.22.1_supplement.1177.2 ◽

2008 ◽

Vol 22 (S1) ◽

Author(s):

Thomas Joseph Jones ◽

Blair M Mell ◽

Linda A Dokas ◽

Bradley K Yoder ◽

Surya M Nauli

Keyword(s):

Heat Shock ◽

Heat Shock Protein ◽

Actin Polymerization ◽

Wild Type Mouse ◽

Heat Shock Protein 27 ◽

Wild Type ◽

Polycystic Kidney ◽

Oak Ridge

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Neflamapimod Induces Vasodilation in Resistance Mesenteric Arteries by Inhibiting p38 Mitogen-Activated Protein Kinase Alpha (MAPKα) and Heat-Shock Protein 27 (Hsp27) Phosphorylation

10.21203/rs.3.rs-1094202/v1 ◽

2021 ◽

Author(s):

Ajay K. Pandey ◽

Farzana Zerin ◽

Sreelakshmi N. Menon ◽

Khue P. Nguyen ◽

Tran Vo ◽

...

Keyword(s):

Smooth Muscle ◽

Heat Shock ◽

Heat Shock Protein ◽

Clinical Investigation ◽

Lewy Bodies ◽

Systemic Blood Pressure ◽

Heat Shock Protein 27 ◽

Mesenteric Arteries ◽

Mitogen Activated Protein Kinase ◽

Hsp27 Phosphorylation

Abstract Neflamapimod, a selective inhibitor of p38 MAPKα, is under clinical investigation for its efficacy in Alzheimer’s disease (AD) and dementia with Lewy Bodies (DLB). Here, we investigated if neflamapimod-mediated acute inhibition of p38 MAPKα could induce vasodilation in resistance-size rat mesenteric arteries. Our pressure myography data demonstrated that neflamapimod produced a dose-dependent vasodilation in mesenteric arteries. Our Western blotting data revealed that acute neflamapimod treatment significantly reduced the phosphorylation of p38 MAPKα and its downstream target heat-shock protein 27 (Hsp27) that is involved in cytoskeletal reorganization and smooth muscle contraction. Likewise, non-selective inhibition of p38 MAPK by SB203580 attenuated p38 MAPKα and Hsp27 phosphorylation, and induced vasodilation. Endothelium denudation or pharmacological inhibition of endothelium-derived vasodilators such as nitric oxide (NO) and prostacyclin (PGI2) had no effect on such vasodilation. Neflamapimod-evoked vasorelaxation remained unaltered by the inhibition of smooth muscle cell K+ channels. Altogether, our data for the first time demonstrates that in resistance mesenteric arteries, neflamapimod inhibits p38 MAPKα and phosphorylation of its downstream actin-associated protein Hsp27, leading to vasodilation. This novel finding may be clinically significant and is likely to improve systemic blood pressure and cognitive deficits in AD and DLB patients for which neflamapimod is being investigated.

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