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Transducible heat shock protein 20 (HSP20) phosphopeptide alters cytoskeletal dynamics

Catherine M. Dreiza^*, Colleen M. Brophy^*,,,1, Padmini Komalavilas^*,,, Elizabeth J. Furnish^*,, Lokesh Joshi^*,, Manuel A. Pallero, Joanne E. Murphy-Ullrich, Moritz von Rechenberg^||, Yew-seng J. Ho^||, Bonnie Richardson^||, Nafei Xu^||, Yuejun Zhen^||, John M. Peltier^|| and Alyssa Panitch^*,

^* The Biodesign Institute at Arizona State University, Tempe, Arizona, USA;
Carl T. Hayden VAMC, Phoenix, Arizona, USA;
The Harrington Department of Bioengineering, Arizona State University, Tempe, Arizona, USA;
Department of Pathology, Division of Molecular and Cellular Pathology, University of Alabama at Birmingham, Birmingham, Alabama, USA; and
^|| Prolexys Pharmaceuticals Inc., Salt Lake City, Utah, USA

¹Correspondence: Department of Bioengineering, ECG # 334, PO Box 879709, Tempe, AZ 85287-9709, USA. E-mail: colleen.brophy{at}asu.edu

SPECIFIC AIM

To determine the effects of phosphorylated HSP20 on the actin cytoskeleton, a phosphopeptide analog of HSP20 was synthesized. This peptide contained 1) the amino acid sequence surrounding the phosphorylation site of HSP20, 2) a phosphoserine, and 3) a protein transduction domain. Swiss 3T3 cells were treated with HSP20 phosphopeptide and HSP20 control peptide analogs. We hypothesized that cells treated with HSP20 phosphopeptides would disrupt actin stress fibers consistent with the effects of cAMP activation. We examined the effects of HSP20 phosphopeptide on focal adhesion complexes. We hypothesized that HSP20-induced cytoskeletal disruption would lead to focal adhesion disassembly.

PRINCIPAL FINDINGS

1. HSP20 phosphopeptide causes disruption of the actin cytoskeleton
Cultured cells were treated with FITC-labeled HSP20 phosphopeptides, stained with phalloidin, and visualized using confocal microscopy (Fig. 1 A). Untreated cells displayed robust stress fibers (a), similar to cells treated with 10 µM LPA (b). In contrast, cells treated with 10 µM forskolin or 25 µM HSP20 phosphopeptide displayed disrupted stress fibers and a stellate morphology (c–f). Cells treated with the control HSP20 peptide, did not show stress fiber disruption (g–i). The peptides were localized throughout the cytoplasm and nucleus of treated cells (e, h).

View larger version (38K): [in this window] [in a new window]   Figure 1. FITC-labeled HSP20 phosphopeptide disrupts the actin cytoskeleton. A) 3T3 cells were untreated (a) or treated for 30 min with 10 µM LPA (b), 10 µM forskolin (c), 25 µM FITC-labeled pHSP20 peptide (d–f), or 25 µM FITC-labeled control HSP20 peptide (g–i). Cells were fixed and stained for f-actin using Alexa 568 phalloidin (a–d, g). FITC-peptide fluorescence (e, h) is overlaid with actin staining to show colocalization (f, i). Scale bar: 50 µm. B) 3T3 cells were cultured and treated as indicated. % of monomeric g-actin with respect to total protein was biochemically quantitated using a DNase 1 inhibition assay. The level of g-actin in the cell extract that caused 50% inhibition of DNase 1 was estimated from a standard actin curve. *P < 0.05 compared with untreated and LPA-treated cells.

To confirm that the loss of stress fibers is associated with the loss of filamentous (f-) actin and commensurate increases in globular (g-) actin, a DNase 1 inhibition assay was performed (Fig. 1B ). There was a statistically significant increase in g-actin content when cells were treated with 25 µM HSP20 phosphopeptide (40.2%±1.35) or 10 µM forkolin (34.9%±3.68) compared with untreated (20.1%±0.82) or LPA-treated (14.5%±0.25) groups. Thus, transducible HSP20 phosphopeptides and activation of the upstream adenylate cyclase activator forskolin led to similar changes in actin filament dynamics and cellular morphology.

2. HSP20 phosphopeptide alters focal adhesion protein localization and induces focal adhesion disassembly
To determine whether HSP20 phosphopeptide-induced actin depolymerization was associated with disruption of focal adhesion complexes, immunoreactive localization of specific focal adhesion proteins was studied using confocal microscopy (Fig. 2 A). Cells were treated with FITC-labeled HSP20 phosphopeptides and localization of -actinin was examined. Untreated cells displayed distinct punctate localization of -actinin (a) comparable to cells treated with 10 µM LPA (b). Cells treated with 25 µM HSP20 phosphopeptide (d–f) or 10 µM forskolin (c) displayed decreased punctate localization of -actinin, indicative of focal adhesion disruption. Cells treated with nonphosphorylated control HSP20 peptide showed no change in localization of -actinin (g–i) compared with untreated cells. Peptides were localized throughout the cytoplasm and nucleus of treated cells (e, h).

View larger version (38K): [in this window] [in a new window]   Figure 2. FITC-labeled HSP20 phosphopeptide disrupts focal adhesions. A) 3T3 cells were untreated (a) or treated for 30 min with 10 µM LPA (b), 10 µM forskolin (c), 25 µM FITC-labeled HSP20 phosphopeptide (d–f), or 25 µM FITC-labeled control HSP20 peptide (g–i), and immunostained for -actinin (a–d, g). FITC-peptide fluorescence (e, h) is overlaid with focal adhesion protein staining to show colocalization (f, i). Scale bar: 50 µm. B) Results from the focal adhesion assay using interference reflection microscopy to determine numbers of cells positive for focal adhesions. 3T3 cells were treated as shown in the graph. Hep I peptide was used as a positive control. *P < 0.05 compared with untreated cells.

Quantitation of changes in focal adhesion complexes was determined using interference reflection microscopy (Fig. 2B ). Cells treated with 10 µM or 25 µM HSP20 phosphopeptide demonstrated a statistically significant decrease in cells positive for focal adhesions compared with untreated control cells. Similarly, cells treated with hep I showed a statistically significant decrease in cells positive for focal adhesions. Cells treated with nonphosphorylated control HSP20 peptides demonstrated no decrease in focal adhesions. These data show that HSP20 phosphopeptide leads to qualitative and quantitative decreases in focal adhesions and suggests that transducible HSP20 phosphopeptide may act as a downstream effector protein of the cyclic nucleotide pathways.

3. HSP20 phosphopeptide binds to 14-3-3 and leads to dephosphorylation of cofilin
A proteomic approach was used to identify specific binding proteins of the HSP20 phosphopeptide and clarify the mechanism of HSP20 phosphopeptide-induced cytoskeletal disruption. Pull-down analysis of immobilized HSP20 peptide demonstrated that 14-3-3 specifically associated with HSP20 phosphopeptide. The immobilized HSP20 phosphopeptide lane exhibited a band at 31 kDa, suggestive of 14-3-3 proteins. Mass spectrometric analysis of the various pull-down products indicated that the only proteins identified with high confidence in the HSP20 phosphopeptide pull-down experiments were various isoforms of 14-3-3, which did not associate with HSP20 control peptides or the ethanolamine-coated beads.

Pull-down analysis of immobilized 14-3-3 demonstrated that phospho-cofilin (p-cofilin) associated with 14-3-3. Phospho-cofilin did not associate with 14-3-3 in the presence of HSP20 phosphopeptide, but p-cofilin bound to 14-3-3 in the presence of HSP20 control peptide, suggesting that HSP20 phosphopeptide prevents the binding of p-cofilin to 14-3-3.

The effect of HSP20 phosphopeptide on the phosphorylation state of cofilin was examined. Cells were untreated or treated with 10 µM LPA, 10 µM forskolin, or 25 µM HSP20 phosphopeptide. Immunoblot analysis of cell lysates demonstrated that cofilin expression levels remained constant in all treatment groups, but there was a decrease in immunoreactive p-cofilin in cells treated with 25 µM HSP20 phosphopeptide or 10 µM forskolin compared with untreated and LPA-treated groups. A similar decrease in p-cofilin has been observed with hep I treatment (A. W. Orr and J. E. Murphy-Ullrich, unpublished data). These data suggest that association between 14-3-3 and HSP20 phosphopeptide are sequence and phosphorylation state specific. Cofilin and HSP20 phosphopeptide may share a 14-3-3 binding site as HSP20 phosphopeptide prevented interaction between 14-3-3 and p-cofilin, and led to cofilin dephosphorylation (i.e., activation). Activated cofilin catalyzes the depolymerization of f-actin.

CONCLUSIONS AND SIGNIFICANCE

Data presented here demonstrate that protein transduction of a biologically active domain from the small heat shock-related protein HSP20 alters cytoskeletal dynamics. Treatment of 3T3 cells with a phosphopeptide analog of HSP20 led to loss of actin stress fibers and focal adhesion complexes. Treatment with phosphopeptide analog of HSP20 also led to dephosphorylation of the actin-depolymerizing protein cofilin. Pull-down assays demonstrated that 14-3-3 proteins associated with a phosphopeptide analog of HSP20. Binding of 14-3-3 protein to a phosphopeptide analog of HSP20 prevented the association of cofilin with 14-3-3. We speculate that HSP20 may modulate actin cytoskeletal dynamics by competing with cofilin for binding to the scaffolding protein 14-3-3.

Since the HSP20 peptide has little or no conformational structure, these data suggest that post-translational modifications of proteins alter biologic behavior through mechanisms independent of conformational changes. The phosphopeptide analog of HSP20 does not modulate responses based on interactions with receptors or signaling pathways, but more likely modulates downstream protein-protein interactions. This represents a potentially novel "proteomic" approach to drug development with significant implications for treating human disease.

View larger version (27K): [in this window] [in a new window]   Figure 3. Proposed mechanism of HSP20 phosphopeptide-induced actin depolymerization. Forskolin-induced activation of the cyclic nucleotide signaling pathway causes an increase in cAMP levels, which in turn causes activation of PKA and downstream phosphorylation of HSP20. The data suggest that HSP20 phosphopeptide disrupts the actin cytoskeleton and focal adhesion complexes. Phospho-HSP20 peptide prevents the associated of p-cofilin and 14-3-3 leading to decreased immunoreactive p-cofilin. The proposed mechanism for HSP20 phosphopeptide-induced actin remodeling requires that HSP20 phosphopeptide binds to 14-3-3. Phosphorylated cofilin is released from a complex with 14-3-3 and is susceptible to phosphatases such as slingshot. Unbound cofilin catalyzes the depolymerization of actin filaments.

FOOTNOTES

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