PKA-induced F-actin rearrangement requires phosphorylation of Hsp27 by the MAPKAP kinase MK5

Cellular Signalling 21 (2009) 712–718

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Cellular Signalling j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / c e l l s i g

PKA-induced F-actin rearrangement requires phosphorylation of Hsp27 by the MAPKAP kinase MK5 Sergiy Kostenko, Mona Johannessen, Ugo Moens ⁎ University of Tromsø, Faculty of Medicine, Department of Microbiology and Virology, N-9037 Tromsø, Norway

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Article history: Received 10 December 2008 Accepted 3 January 2009 Available online 8 January 2009 Keywords: F-actin Hsp27 MAPKAP-kinase PRAK MK2 MK5

a b s t r a c t Mitogen-activated protein kinase (MAPK) pathways can play a role in F-actin dynamics. In particular, the p38 MAPK/MAPK-activated protein kinase 2 (MK2)/heat shock protein 27 (Hsp27) pathway is involved in F-actin alternations. Previously, we showed that MK5 is implicated in F-actin rearrangement induced by the cAMP/ cAMP-dependent protein kinase pathway in PC12 cells, while others found Hsp27 to be a good in vitro MK5 substrate. Here we demonstrate that MK5 can specifically interact with Hsp27 in vivo and can induce phosphorylation at serine residues 78 and 82 in cells. siRNA-mediated depletion of Hsp27 protein levels, as well as overexpression of the non-phosphorylatable Hsp27-3A mutant prevented forskolin-induced F-actin reorganization. While ectopic expression of a constitutive active MK5 mutant was sufficient to induce F-actin rearrangement in PC12 cells, co-expression of Hsp27-3A could ablate this process. Our results imply that MK5 is involved in Hsp27-controlled F-actin dynamics in response to activation of the cAMP-dependent protein kinase pathway. These findings render the MK5/Hsp27 connection into a putative therapeutic target for conditions with aberrant Hsp27 phosphorylation such as metastasis, cardiovascular diseases, muscle atrophy, autoimmune skin disease and neuropathology. © 2009 Elsevier Inc. All rights reserved.

1. Introduction Mitogen-activated protein kinase (MAPK) signalling pathways are implicated in several cellular processes, including cell shape changes and motility. In particular, the p38 MAPK pathway was shown to be involved in actin dynamics. p38 MAPK-induced F-actin reorganization and cell migration is mediated through MAPK-activated protein kinase 2 (MK2) and the murine heat shock protein 25 (Hsp25) or its human homologue Hsp27 in different cell lines [1–5]. Non-phosphorylated Hsp27 acts as a potent inhibitor of F-actin polymerization and it has been postulated that Hsp27 in its non-phosphorylated form binds to the barbed growing ends of F-actin filaments, stabilizes them, and inhibits further actin polymerization. Phosphorylation of Hsp27 will trigger release of Hsp27 from the barbed ends and allow F-actin remodelling [6–9]. MAPK-activated protein kinase 5 (MK5) or its human homologue p38-regulated/activated kinase (PRAK) is closely related to MK2 with an overall amino acid sequence identity of 42%. Despite their high homology, MK2 and MK5 possess different functions. While MK2 participates in several biological processes including cytokine production, endocytosis, reorganization of the cytoskeleton, cell migration, cell cycle control, chromatin remodelling and transcriptional regulation (reviewed in [10] and in [11]), less is known about the functions of MK5. A recent study with MK5 deficient mice revealed a role for MK5 as tumour suppressor [12], while mice expressing a constitutive active ⁎ Corresponding author. Tel.: +47 776 4622; fax: +47 776 45350. E-mail address: [email protected] (U. Moens). 0898-6568/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.cellsig.2009.01.009

MK5 mutant demonstrated anxiety-related traits and locomotor differences compared to their control littermates. This phenotype may implicate a role for MK5 in neurological processes [13]. Moreover, others and we reported that MK5 is involved in microfilament rearrangement and cell migration ([14,15]; reviewed in [16]). Our previous studies demonstrated that MK5 mediates forskolin-induced F-actin rearrangement in PC12 cells. PKA activated MK5 and triggered nuclear export of MK5. In fact, cytoplasmic expression of a constitutive active MK5 mutant mimicked forskolin-induced F-actin remodelling in these cells [14]. Previous studies have shown that human Hsp27 or its murine homologue Hsp25 is a good in vitro substrate for MK5/PRAK [12,15,17,18]. Hence, we wanted to establish whether cAMP/PKAinduced F-actin rearrangement could be mediated through MK5dependent phosphorylation of Hsp27. Our results reveal that Hsp27 is an in vivo substrate for MK5 and that depletion of Hsp27 or overexpression of a non-phosphorylatable Hsp27 mutant ablates PKA/MK5triggered F-actin remodelling in PC12 cells. Thus besides p38 MAPK/ MK2/Hsp27, PKA/MK5/Hsp27 may represent an alternative route employed by MAPK signalling pathways to control F-actin dynamics. 2. Materials and methods 2.1. Materials Forskolin was purchased from Sigma Aldrich (St. Louis, MO, USA). 32P γ-ATP was obtained from PerkinElmer (Waltham, MA, USA), while recombinant active MK5 was from Invitrogen (Carlsbad, CA, USA). Anti-

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Fig. 1. MK5 phosphorylates Hsp27 in vitro and in vivo. (A) Top panel. In vitro kinase assay was performed with purified Hsp27 and activated MK5 protein. The proteins were run on SDS PAGE and phosphorylation was detected by autoradiography. Lane 1: purified activated MK5; lane 2: purified Hsp27; lane 3: purified MK5 and Hsp27. The week slower migrating band in lane 1 and lane 3 represent phosphorylated MK5 due to autophosphorylation. The positions of phosphoMK5 and phosphoHsp27 are indicated by an arrow. Middle panel and lower panel: Western blotting was performed with anti-MK5 and anti-Hsp27 antibodies, respectively to ensure that equal amounts of proteins were used in the in vitro kinase assay. (B) HEK293 cells were co-transfected with expression plasmid for Flag-tagged Hsp27 and with plasmids encoding different EGFP–MK5 fusion proteins. Lane 1: lysates from cells transfected only with Flag–Hsp27 expression vector; lane 2: lysates from cells co-transfection with a plasmid EGFP–NES–MK5 L337A encoding a cytoplasmic located constitutive active MK5 mutant; lanes 3: lysates from cells co-transfected with an expression plasmid for a kinase dead MK5 mutant (EGFP-NES-MK5 T182A); lane 4: as in lane 2 but the constitutive active MK5 variant lacks a NES (EGFP–MK5 L337A); lane 5: lysates from cells co-transfected with the EGFP expression vector. Cell lysates were monitored for the phosphoserine 15 (left panel), 78 (middle panel) and 82 (right panel) levels of Hsp27 with phosphospecific antibodies. Equal loading was ensured by stripping the membrane and reprobing with anti-actin antibodies. Similar results were obtained in an independent experiment.

NuPage 4–12% Bis–Tris SDS-PAGE (Invitrogen) for 50 min at 200 V, and then subjected to autoradiography.

PRAK antibody (A-7), which cross-react with MK5, was from Santa Cruz Biotechnology Inc (Santa Cruz, CA, USA), while anti-phospho-p38 (#9211) and anti-phosphoERK1/2 (#9106) were obtained from Cell Signaling (Beverly, MA). Anti-GFP (ab290) was purchased from AbCam (Cambridge, UK), and Alexa Fluor 488 antibody was from Invitrogen. The alkaline phosphatase-conjugated secondary antibodies sheep antimouse IgG and anti-rabbit IgG were from Sigma Aldrich. Oligonucleotides were purchased from Sigma Aldrich or Eurogentec (Seraing, Belgium). Phospho-hsp27 antibodies and anti-Hsp27 were obtained from Millipore (Temecula, CA, USA). Anti-actin antibodies were from Sigma, Saint Louis, MO, USA), and the anti-Flag M2 antibody was purchased from Stratagene (La Jolla, CA, USA). The EGFP fusion proteins with wild-type MK5, and the MK5 mutants T182A and L337A, and pEGFP–NES–MK5 T182A and pEGFP–NES–MK5 L337A have been previously described [14]. The plasmid Hsp27-3A was a kind gift of Dr. Jonathan Dean [19], and the Flag-tagged Hsp27 was generously provided by Dr. Kuy-Jin Park [20].

PC12 cells, a kind gift from Dr. Jaakko Saraste (University of Bergen, Norway) were maintained in F-12K Medium (Kaighn's Modification of Ham's F-12 Medium) supplemented with 2 mM L-glutamine, 1.5 g/l sodium bicarbonate, 15% Horse serum (Gibco) and 2.5% fetal calf serum, penicillin (110 units/ml) and streptomycin (100 µg/ml). HEK293 cells were purchased from the European Collection of Cell cultures (cat. no. 85120602; Salisbury, Wiltshire, UK) and kept in Eagle's Minimum Essential Medium supplemented with 10% fetal calf serum, 2 mM L-glutamine, penicillin (110 units/ml) and streptomycin (100 µg/ml). Cells were transfected with Lipofectamine 2000 (Invitrogen) or using the Nucleofection kit (Amaxa) according to the manufacturers instructions.

2.2. In vitro kinase assay

2.4. Western blotting

Phosphorylation of purified Hsp27 (SignalChem, Richmond, Canada) with MK5 was performed in 25 mM Tris∙HCl pH 7.5, 10 mM MgCl2, 0.05 mg/ml BSA, 2.5 mM DTT, 0.15 mM cold ATP and 0.3 µl 32P γ-ATP(3000 Ci/mmol; PerkinElmer) in a total volume of 40 µl at 30 °C for 1 h. The reaction was stopped in 4XLDS Sample buffer and proteins denatured at 70 °C for 10 min. The phosphorylation was analyzed on

Cell lysates or protein samples were analyzed by SDS-PAGE NuPage 4–12% Bis–Tris SDS-PAGE (Invitrogen) according to the manufacturers protocol and blotted onto a 0.45 µm PVDF membrane (Millipore, Billerica, MA, USA). Immunoblotting was performed by first blocking the membrane with PBS-T (PBS with 0.1% Tween-20 (Sigma Aldrich) containing 10% (w/v) dried skimmed milk for 1 h and probed with

2.3. Cell culture and transfection

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Fig. 2. MK5 interacts with Hsp27 in vivo. HEK293 cells were transfected with expression plasmids for flag-tagged Hsp27 (Flag–Hsp27) and EGFP-tagged MK5 (EGFP–MK5) or EGFP (EGFP-C1). Hsp27–MK5 complexes were co-immunoprecipitation and the immunoprecipitated proteins were separated by SDS-PAGE and analyzed by western blot. (A) Top panel: immunocomplexes were precipitated with anti-Flag antibodies and the presence of EGFP-tagged MK5 was assayed with anti-GPF antibodies. Bottom panel: the presence of Hsp27 in input controls and co-immunoprecipitates was monitored by western blot with antibodies against the Flag tag of ectopically expressed Hsp27. (B) Top panel: immunocomplexes were precipitated with antibodies directed against the human homologue of MK5, i.e. PRAK. These antibodies cross-react with MK5. The presence of Flag–Hsp27 was assayed with antiHsp27 antibodies. Bottom panel: control western blot with anti-PRAK antibodies was performed to verify the presence of MK5 in the input controls and in the immunoprecipitates. (C) Whole cell extracts of PC12 cells were immunoprecipitated with anti-Hsp27 antibodies. The presence of endogenous MK5 in the immunoprecipitate was examined by anti-PRAK antibodies. The molecular masses (kD) of the protein standard are indicated in each panel.

primary antibody. After 4 washes, the membrane was incubated with the appropriate secondary antibody for 1 h. Visualization of proteins was achieved by using CDP Star (Tropix, Bedford, MA, USA) substrate and Lumi-Imager F1 from Roche (Basel, Switzerland). 2.5. Immunoprecipitation HEK293 extracts were harvested and lysed in buffer containing 20 mM Tris∙HCl pH 7.5, 1% Triton X-100, 5 mM sodium pyrophosphate, 50 mM sodium fluoride, 1 mM EDTA, 1 mM EGTA, 1 mM sodium orthovanadate, 0.27 M sucrose, 10 mM β-glycerophosphate, and Complete protease inhibitor cocktail (Roche). Lysates were cleared by centrifugation at 4° for 10 min at 15,000 g. Lysates were incubated with the appropriate antibody for at least 1 h at 4 °C, before addition of 60 µl slurry (i.e. 30 µl protein G-agarose (Amersham/GE Healthcare) equilibrated with 30 µl lysis buffer) and incubated for an additional hour. The immunoprecipitates were then washed three times in lysis buffer and twice in 50 mM Tris–Cl pH 8.0. Twenty µl 2XLDS sample buffer was added to the beads before denaturation at 70 °C for 10 min. The immunoprecipitates were either analyzed by Western blotting.

antibody followed by incubation either with Alexa Fluor 647 goat antimouse IgG antibody (#A21235; Molecular Probes, Eugene, OR) or with Alexa Fluor 488 goat anti-mouse IgG antibody (#A11001; Molecular Probes, Eugene, OR). For F-actin staining, the cells were pre-incubated with PBS containing 1% BSA for 20–30 min and stained with Alexa Fluor 594 phalloidin (#A12381; Molecular Probes, Eugene, OR) for 20 min. The cells were then washed with PBS and examined using confocal laser-scanning Zeiss LSM 510 META and Leica SP5 microscopes. Several hundred cells were monitored and representative pictures are presented. 2.7. Small interfering RNA (siRNA) Validated MK5-directed siRNA and Hsp27 siRNA, purchased from Ambion Inc. (Austin, TX), was transfected by Nucleofection (Amaxa) into PC12 cells according to the manufacturers instructions using 100 nM siRNA/2 × 106 cells. The levels of MK5 protein or Hsp27 in untreated and siRNA treated cells were monitored 48 h after transfection by western blotting using anti-PRAK antibody (Santa Cruz) or Hsp27 antibody (Millipore) to verify the efficiency of reduction in MK5 expression and Hsp27 expression respectively.

2.6. Cell staining and microscopy 3. Results Cells were rinsed twice with phosphate-buffered saline (PBS) and fixed for 10 min with 4% formaldehyde. Next, the cells were washed twice with PBS and then permeabilized for 10 min with 0.1% Triton X100. For detection of ectopically expressed Hsp27 3A mutant, the cells were stained for 1 h at room temperature with anti-Flag primary

3.1. MK5 phosphorylates Hsp27 in vitro and in vivo Although several groups have demonstrated that Hsp27 is a good in vitro substrate for MK5 [12,15,17,18], it is questioned whether

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Fig. 3. Depletion of either MK5 or Hsp27 protein levels by siRNA disrupts forskolin-induced F-actin rearrangement in PC12 cells. Cells were transfected with 100 nM siRNA/106 cells and were stained for F-actin 48 h after transfection. PC12 cells treated with siRNA-directed against MK5 are shown in panels F–J, while cells transfected with siRNA against Hsp27 are depicted in panels K–O. Panels A–E represent cells transfected with scrambled siRNA. SiRNA-mediated knockdown of MK5 and Hsp27 protein levels was verified by western blotting (bottom part of this figure). Monitoring ERK2 or α-actin protein levels confirmed the specificity of the siRNA treatment.

MK5 is a bona fide Hsp27 kinase because both arsenite and phorbol esters induced Hsp27 phosphorylation in MK5−/−, but not in MK2−/− mouse embryonic fibroblasts [21]. It is, however, plausible that MK5 mediates phosphorylation of Hsp27 provoked by other stimuli. To address this, we explored whether active MK5 could phosphorylate Hsp27 in vitro. Recombinant active MK5 protein was incubated with purified Hsp27 in the presence of radioactive ATP. A readily phosphorylation of Hsp27 was observed (Fig. 1A). Tryptic phosphopeptide mapping studies by New and colleagues revealed that Hsp27 serine residues 15, 78 and 82 are in vitro phosphoacceptor sites for MK5 [17]. Therefore, we tested whether Hsp27 becomes phosphorylated at these sites in cells using phosphospecific antibodies. We transfected cells with a nuclear localized constitutive active (EGFP– MK5 L337A) mutant [22], or with cytoplasmic residing variants of either a kinase dead (EGFP–NES–MK5 T182A) or a constitutive active (EGFP–NES–MK5 L337A) MK5 mutant [14]. Our results show that overexpression of a constitutive active MK5 mutant induced phosphorylation of serine 78 and 82, while we were unable to detect

changes in the phosphorylation status of serine residue 15. The kinase dead MK5 mutant could not trigger Hsp27 phosphorylation at any of the three sites tested (Fig. 1B). 3.2. MK5 interacts specifically with Hsp27 The ability of activated MK5 to trigger phosphorylation of Hsp27 in cells suggests that Hsp27 may indeed be a genuine substrate for MK5. To seek further proof, we examined whether these two proteins can form stable complexes within the cell. Thereto, cells were cotransfected with expression plasmids for Flag-tagged Hsp27 and EGFP-tagged MK5. Protein complexes were immunoprecipitated with either anti-Flag antibodies (Fig. 2A) or anti-MK5 antibodies (Fig. 2B) and the immunoprecipitated proteins were separated by SDS-PAGE and analyzed by western blotting using anti-GFP (Fig. 2A) or antiHsp27 (Fig. 2B) antibodies. These studies confirmed the presence of EGFP–MK5, respectively Flag-Hsp27 in the immunoprecipitates. Next, we wondered whether endogenous Hsp27 and MK5 could interact.

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Fig. 4. Overexpression of the non-phosphorylatable Hsp27-3A mutant inhibited forskolin-induced F-actin reorganization in PC12 cells. Cells transfected with an expression vector for Flag-tagged Hsp27-3A were exposed to 10 µM forskolin for 30 min. Expression of this mutant was visualized with fluorescence labeled antibodies against the Flag tag (green channel), while F-actin was visualized with Alexa Fluor 594 phalloidin (red channel). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Thereto, we immunoprecipitated endogenous Hsp27 from PC12 cells and found that endogenous MK5 co-immunoprecipitated (Fig. 2C). Thus our results clearly demonstrate that both proteins can be immunoprecipitated as complexes from cells, indicating that MK5 and Hsp27 specifically interact in vivo.

3.3. Hsp27 is involved in PKA-mediated F-actin rearrangement It has been postulated that Hsp27 in its non-phosphorylated form binds to the barbed growing ends of F-actin filaments, stabilizes them, and inhibit further actin polymerization. Phosphorylation of

Fig. 5. Overexpression of the Hsp27-3A mutant ablated F-actin rearrangement induced by constitutive active MK5. PC12 cells were co-transfected with an EGFP–NES–MK5 L337A encoding vector and an empty vector (top panels) or with EGFP–NES–MK5 L337A and Flag-tagged Hsp27-3A expression vectors (bottom panels). The expression of Hsp27-3A was visualized anti-Flag antibody and Alexa 647-coupled secondary antibody (colored magenta), while F-actin was visualized with Alexa Fluor 594 phalloidin (red channel). Constitutive active MK5 that resides in the cytoplasm could induce F-actin rearrangements (speckle-like structures in the cells), while the non-phosphorylatable Hsp27 mutant abrogated these rearrangements. No such speckle-like structures are observed in cells co-expressing the MK5 and Hsp27 mutants (indicated by arrows). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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prevented when non-phosphorylatable Hsp27-3A mutant was overexpressed (Fig. 5). Taken together, our data show that cAMP/ PKA-triggered F-actin remodelling can be mediated by MK5/Hsp27 and that phosphorylation of Hsp27 by MK5 is necessary. 3.5. The p38 MAPK/MK2 pathway is not involved in forskolin-induced F-actin remodelling

Fig. 6. The p38 MAPK/MK2 pathway is not involved in forskolin-induced F-actin rearrangements in PC12 cells. PC12 cells were treated with 10 µM forskolin for the time points indicated. The activation of p38 MAPK was assayed by western blotting with phospho-specific antibodies. Top panel: no phosphorylation of p38 MAPK was observed after forskolin treatment. An unspecific band of ~ 90 kD with similar intensity is observed in all lanes. Bottom panel: the membrane was stripped and the activation of ERK1/2 by forskolin was tested with specific phospho-ERK1/2 antibodies. The molecular masses of the protein marker are shown in kD.

Numerous stimuli that trigger F-actin rearrangement seem to operate through the p38 MAPK/MK2 signalling pathway [1–5]. To explore a possible role of this pathway in forskolin-proved F-actin reorganization, we monitored the activation state of p38MAPK in untreated and forskolin-treated PC12 cells. Western blot analysis failed to detect phosphorylated p38 in untreated cells, but also in forskolin-treated cells, even after 6 h of stimulation (Fig. 6). We have previously demonstrated that at this time-point, clear F-actin rearrangements can be observed [14]. To ensure that forskolin was functional, we verified phosphoERK1/2 levels, since these MAP kinases are activated by forskolin in PC12 cells [23]. These results combined with our previous findings that PKA does not interact nor phosphorylate MK2 [14], strongly suggest that p38 MAPK/MK2 is not implicated in forskolin-provoked F-actin remodelling. 4. Discussion

Hsp27, however, will trigger release of Hsp27 from the barbed ends and allow F-actin remodelling [7,9]. Because MK2 can mediate cytoskeleton changes induced by stress through phosphorylation of Hsp27, we reasoned that MK5 might exert a similar role in forskolininduced F-actin rearrangement as our previous studies had shown that MK5 is implicated in F-actin reorganization induced by activation of the cAMP/PKA signalling pathway [14]. To elucidate whether Hsp27 is involved in forskolin-triggered F-actin remodelling we applied two different experimental approaches. In the first type of experiments, cells were transfected with either scrambled siRNA or with siRNA that targets Hsp27 transcripts and were then exposed to forskolin. Cells treated with scrambled siRNA still displayed F-actin rearrangement (noticeable by speckle-like structures) upon exposure to forskolin (Fig. 3, top panel), yet depletion of Hsp27 protein levels by siRNA prevented forskolin-induced F-actin remodelling (Fig. 3, bottom panel). We also confirmed our previous findings that knocking down MK5 expression also impaired forskolin-provoked F-actin remodelling (Fig. 3, middle panel). As a second strategy, we examined forskolin-induced F-actin rearrangement in cell overexpressing the non-phosphorylatable Hsp27-3A mutant. This mutant contains substitution of the serine residues 15, 78 and 82 into alanine, and prevents its role in F-actin polymerization [1]. Merging the green and red channels demonstrated no F-actin rearrangement after forskolin treatment in cells expressing Hsp27-3A (cells on the right in Fig. 4), while cells that lack Hsp27-3A expression (e.g. cell on the left in Fig. 4) displayed F-actin remodeling after forskolin treatment. These findings clearly demonstrate that ectopically expressed Hsp273A abrogated changes in the F-actin structures caused by forskolin and sustain the involvement of phosphoHsp27 in F-actin dynamics induced by the cAMP/PKA signalling pathway. 3.4. Phosphorylation of Hsp27 by MK5 is required for PKA-induced F-actin rearrangement We have previously shown that cytoplasmic expression of an activated MK5 mutant suffices to induce reorganization of F-actin [14]. We reasoned that if phosphorylation of Hsp27 is required to mediate MK5-induced F-actin rearrangement, overexpression of the non-phosphorylatable Hsp27-3A mutant might interfere with this process. Thereto, we ectopically expressed EGFP–NES–MK5L337A in the presence or absence of Hsp27-3A. Cytoplasmic localized active MK5 could induce F-actin rearrangement, a property that was

Although several studies in the past had shown that Hsp27 is a good in vitro substrate for MK5 [12,15,17,18], this is the first report that proofs that Hsp27 and MK5 can form complexes in cells and that MK5 can induce phosphorylation of Hsp27 at serine 78 and 82 in vivo. Moreover, we attribute a biological relevance for this MK5– Hsp27 interaction, i.e. MK5-mediated phosphorylation of Hsp27 contributes to the dynamics of F-actin triggered by the cAMP/PKA pathway. A recent study by Tak and co-workers showed that the 14–3–3ε protein interacts with MK5 in vivo, and that ectopic expression of MK5 stimulated phosphorylation of Hsp27 at serine82. MK5-provoked Hsp27 phosphorylation was reduced by overexpression of 14–3–3ε. MK5 immunoprecipitated from cells transfected with an MK5 expression vector was also able to phosphorylate Hsp27 in vitro, while reduced kinase activity of MK5 towards Hsp27 was monitored with MK5 immunoprecipitated from cells co-transfected with vectors encoding MK5 and 14–3–3ε. The authors also demonstrated that 14–3–3ε prevented MK5mediated F-actin reorganization and cell migration. They concluded that 14–3–3ε disrupts MK5-mediated Hsp27 phosphorylation, which is required for F-actin polymerization and cell migration [15]. Our findings confirm and extend the results of Tak and colleagues. However, in contrary to our studies, they did not present direct proof that MK5 and Hsp27 interact. This is important since 14–3–3 proteins can also interact with protein kinase D (PKD) [24,25] and PKD can phosphorylate Hsp27 at serine residue 82 ([26–29]; S.K., unpublished results). Therefore, it cannot be completely excluded that some of the effects of 14–3–3 on F-actin remodelling and cell migration are implemented through PKD. Although both our study and the study by Tak et al. suggest that Hsp27 is a genuine substrate for MK5, studies in MK5-deficient mouse embryonic fibroblasts jeopardized the role of MK5 as an Hsp27 kinase. PhosphoHsp25 was detected in MK5−/− MEF cells stimulated with arsenite or phorbol ester, but not in MK2−/− MEF cells, suggesting that MK2 rather than MK5 is an Hsp25/27 kinase. The authors also failed to detect Hsp25 phosphorylation with immunoprecipitated endogenous MK5 in vitro [21]. An obvious explanation for this discrepancy is that the nature of the stimulus determines which signalling pathway and hence which MK is activated and can phosphorylate Hsp27. Another observation also supports a role for MK5 as Hsp27 kinase. Hsp25 kinase activity isolated from different cells was associated with two protein bands with molecular masses of approximately 45 and 50 kDa [17,30–

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32]. These correspond well with the reported molecular masses for one of the isoforms of MK2 (43kD), and for MK5 (54 kD) [10]. Studies with synthetic peptides demonstrated that the synthetic peptide with sequence KKKALNRQLGVAA preferentially inhibited Hsp25 kinase activity. Interestingly, this sequence shows identity with the artificial MK5 substrate PRAKtide (KKLNRTLSVA; the identical amino acids are underlined). The peptide SRVLKEDKERWEDVK derived from the autoinhibitory domain of MK2 did not inhibit Hsp25 kinase activity [32]. Phosphotryptic mapping had identified Hsp27 serine residues 15, 78 and 82 as in vitro phosphoacceptor sites for MK5 [17]. We found that MK5 could induce phosphorylation of serines 78 and 82 in cell culture, while Tak and colleagues only investigated and confirmed serine 82 as a target for MK5 in vivo [15]. We cannot exclude that other sites may become phosphorylated by MK5 in vivo, but overexpressing the triple mutant Hsp27-3A in which the serines 15, 78, and 82 are substituted by alanine abrogated MK5-induced F-actin interaction. This finding indicates that at least one of these sites is a crucial regulator for Hsp27's function in modulating F-actin rearrangements by MK5. Besides its role as chaperone, Hsp27 accomplishes additional functions including effects on cell movement, embryogenesis, and the apoptotic pathway. It is tempting to speculate that MK5, through phosphorylation of Hsp27, may also participate in these processes. Indeed, inhibition of MK5 kinase activity by 14–3–3ε abrogated Hsp27 phosphorylation and inhibited cell migration [15], while MK5 deficient mice on a C57/B6 genetic background displayed developmental defects and embryonic lethality [33]. Perturbed Hsp27 phosphorylation may be associated with several disorders such as vascular disease, cancer, muscle atrophy, autoimmune skin disease and neuropathological conditions [34–39]. Although a role for MK5mediated Hsp27 phosphorylation in these pathologies remains to be established, modulating the MK5/Hsp27 association offers an attractive putative therapeutic target for diseases with perturbed regulation of Hsp27 phosphorylation. Recently, a specific Hsp27 phosphorylation inhibitor, KRIBB3, which induced mitotic arrest and apoptosis in human cancer cells was described [40,41]. This makes KRIBB3 a good drug candidate for treating diseases with perturbed Hsp27 phosphorylation. 5. Conclusions This study demonstrates that MK5 can mediate cAMP/PKAinduced F-actin rearrangements in PC12 cells through modulating phosphorylation of Hsp27 because: – MK5 can phosphorylate Hsp27 at Ser-78 and 82, – MK5 forms a complex with Hsp27 in cells, – depletion of either MK5 or Hsp27 protein levels abrogate F-actin remodelling caused by the cAMP/PKA pathway, – the non-phosphorylatable Hsp27-3A mutant ablates MK5-triggered F-actin rearrangements.

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.cellsig.2009.01.009. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12]

[13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] [33] [34] [35] [36] [37]

[38]

Acknowledgements

[39] [40]

The authors wish to thank Drs. Jonathan Dean and Kuy-Jin Park for the kind gift of the expression plasmid Hsp27-3A and Flag-Hsp27, respectively. This work was supported by grants for the Norwegain Cancer Society (Kreftforeningen A5308 and A5313) and the Mohn Grant (Forskningsstiftelsen Tromsø).

[41]

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