Publications
Presentations/Lectures



Modulation of F-actin rearrangements by the cyclic AMP/PKA pathway is mediated by MAPKAP Kinase 5 and requires PKA-induced nuclear export of MK5

Nancy Gerits*, Theresa Mikalsen*, Sergiy Kostenko, Alexey Shiryaev, Mona Johannessen and Ugo Moens#

Department of Microbiology and Virology, Faculty of Medicine, University of Tromsø, N-9037 Tromsø, Norway.

#Corresponding author: Ugo Moens, Department of Microbiology and Virology, Faculty of Medicine, University of Tromsø, N-9037 Tromsø, Norway, Phone:+47-776 44622, Fax: +47-776 45350, e-mail: ugom@fagmed.uit.no. * Both authors contributed equally. ``Journal of Biological Chemistry'' October 2007. In Press.


 

Abstract

The mitogen-activated protein kinase (MAPK)-activated protein kinases belong to the Ca2+/calmodulin-dependent

protein kinases. Within this group, MK2, MK3 and MK5 constitute three structurally related enzymes with distinct functions. Few genuine substrates for MK5 have been identified and the only known biological role is in ras-induced senescence and in tumour suppression. Here, we demonstrate that activation of PKA or ectopic expression of the catalytic subunit Cα in PC12 cells results in transient nuclear export of MK5, which requires the kinase activity of both Cα and MK5 and the ability of Cα to enter the nucleus. Cα and MK5, but not MK2, interact in vivo and Cα increased the kinase activity of MK5. Moreover, Cα augments MK5 phosphorylation, but not MK2, while MK5 does not seem to phosphorylate Cα. Activation of PKA can induce actin filament accumulation at the plasma membrane and formation of actin-based filopodia. We demonstrate that siRNA-triggered depletion of MK5 interferes with PKA-induced F-actin rearrangements. Moreover, cytoplasmic expression of an activated MK5 variant is sufficient to mimic PKA provoked F-actin remodelling. Our results describe a novel interaction between the PKA pathway and MAPK signalling cascades, and suggest that MK5, but not MK2 is implicated in PKA-induced microfilament rearrangements.

Introduction

Mitogen activated protein (MAP)kinase signalling pathways play important roles in several cellular processes, including proliferation, differentiation, apoptosis, development, gene transcription, and motility. A typical MAP kinase module acts through subsequent phosphorylation and activation of a MAP kinase kinase kinase, a MAP kinase kinase, and a MAP kinase. The MAP kinase may phosphorylate non-protein kinase substrates or yet other protein kinases, referred to as MAPK-activating protein kinases (MAPKAPK) [1-5]. Based on the sequence homology of the kinase domain, the MAPKAPK belong to the superfamily of Ca2+/calmodulin-dependent protein kinases [6]. The mammalian MAPK APK include the ribosomal-S6-kinases (RSK1-4 or MAPKAPK1a-d), the mitogen-and stress-activated kinases (MSK1 and 2), the MAPK-interacting kinases (MNK1 and 2) and the MAPKAPK MK2, MK3 and MK5. MK2 was isolated 15 years ago in an attempt to identify protein kinases involved in adrenergic control of glycogen synthase activity, while MK3 was first described as a protein encoded by a gene located in the small cell lung cancer tumour suppressor gene region 3p21.3 [7-8]. The murine MAPKAPK5 (MK5) or its human homologue PRAK was originally described as a downstream target of p38 MAPK [9-10]. MK5 shares approximately 45% sequence homology with MK2 and MK3, while MK2 and MK3 display 75% sequence identity. All three kinases contain the same conserved regulatory phosphorylation site (Thr182, Thr222, Thr201, respectively). Despite their high similarity, gene deletion experiments reveal that these three kinases possess separate functions [11-12]. MK2 is involved in many cellular processes like cell cycle regulation, inflammation and cell migration [13-18], whereas the biological roles of MK3 and MK5 remain unclear. MK3 may be involved in transcription regulation through modulating the activity of the transcription factor E47 and the transcriptional repressor Polycomb group Bmi-1 protein. Moreover, MK3 may function as a tumour suppressor [19-21]. The biological function of MK5 is poorly understood, as MK5 knockout mice bred onto different backgrounds displayed either no obvious phenotype or embryonic lethality. The reason for this lethality, however, remains unknown. Furthermore, the atypical MAP kinases ERK3 and ERK4 are the only identified in vivo MK5-interacting partners, but the physiological relevance of the interaction remains elusive [11,22-26]. However, a recent study demonstrated that MK5 could phosphorylate p53 at serine residue 37 in vivo, that MK5 deficiency enhanced mutagen-induced skin carcinogenesis in mice, and that MK5-/- primary cells were more susceptibility to oncogenic transformation. These findings point to a role for MK5 in tumour suppression [27]. Our group, in collaboration with others, found that increased levels of ERK3 during nerve growth factor-induced differentiation of PC12 cells was accompanied by increased MK5 activity, hence suggesting a role for MK5 in neuronal differentiation [24].

Cyclic AMP (cAMP) functions as an intracellular second messenger to transmit extracellular signals, such as hormones and neurotransmitters, to the intracellular environment. The major target of cAMP is the cAMP-dependent protein kinase (PKA), a tetrameric holoenzyme that consists of two regulatory (R) and two catalytic (C) subunits. After cAMP binds the R subunits, the C subunits dissociate and phosphorylate their substrates [28]. The cAMP/PKA signalling pathway regulates a variety of cellular processes such as cell proliferation, differentiation, actin cytoskeletal rearrangements, and gene transcription (reviewed in [29-31]). In order to optimise the responsiveness to a wide variety of signals, the PKA pathway interacts with other signalling pathways, including the MAPK pathways. In fact, PKA has been shown to modulate the activity of several MAPK cascades [32-35]. Recently, the MAPKAP kinase, RSK1, was found to either bind the catalytic C or regulatory R subunit of PKA, depending on the phosphorylation status of RSK1, but the physiological relevance of this interaction is not completely understood [36].

A role for PKA in actin cytoskeletal changes is well illustrated, and it has been suggested that MK2/3/5 may be implicated in actin remodelling (reviewed in [11] and [13]). However, whether there is a specific role for a certain MK in actin redistribution and whether PKA may recruit MKs to mediate actin rearrangement remains unsolved. This prompted us to examine whether MK5 might contribute in cAMP/PKA-provoked modification of the microfilament architecture in the rat pheochromocytoma cell line PC12. Our results demonstrate a new interaction between the cAMP/PKA and MK5, and recognise a novel role of MK5 as a mediator of cAMP-induced F-actin rearrangements.

Experimental Procedures

Materials

Forskolin, PKA-C and PKA-R were purchased from Sigma Aldrich (St. Louis, MO, USA). 32P δ-ATP was obtained from Amersham/GE Healthcare (Buckinghamshire, UK), while recombinant active and inactive His-MK5/PRAK were from Invitrogen (Carlsbad, CA, USA). The PRAKtide MK5 substrate peptide was purchased from Upstate (Charlottesville, VA, USA). The monoclonal antibody against the regulatory RIα of PKA was a kind gift from Dr. Skålhegg (University of Oslo). Anti-PRAK (A-7), anti-ERK2 (C-14) and anti-PKAcat (C-20) were all from Santa Cruz Biotechnology Inc (Santa Cruz, CA, USA). Anti-phospho-CREB (#9190) and anti-MK2 (#3042) were obtained from Cell signalling (Beverly, MA), while anti-GFP (ab290) was purchased from AbCam (Cambridge, UK). Anti-ERK3 antibodies have been described before [24]. The monoclonal anti-Ras clone RAS10 antibody was from Upstate (cat. no. 05-516; Charlottesville, VA, USA). Alexa Fluor 488 antibody was from Invitrogen. The alkalic phosphatase-conjugated secondary antibodies sheep anti-mouse IgG and anti-rabbit IgG were from Sigma Aldrich. Oligonucleotides were purchased from Sigma Aldrich or Eurogentec (Seraing, Belgium).

Plasmids

The expression vectors for p38β and the activated MKK6E/E mutant, as well as for the EGFP fusion proteins with wild-type MK5, and the MK5 mutants K51E, T182A and L337A, and truncated MK5 1-423 have been previously described [24,37]. Dr. Maurer generously provided the expression plasmid of the heat-stable inhibitor of PKA (pCMVPKI) [38]. The Cα-NLS plasmid was a kind gift of Dr. Thiel [39]. The kinase dead Cα L72H was generated by site-directed mutagenesis using the primer (only one strand is shown): 5'-GAA-CAA-CTA-CGC-CAT-GCA-CAT-CTT-AGA-CAA-G-3'. The expression plasmids for cytoplasmic located MK5 variants were generated by cloning the NES signal of the Rev protein of human immunodeficiency virus into
pEGFP-MK5. Tagging a protein with this sequence will direct it to the cytoplasm [40]. Thereto, the complementary oligonucleotides 5'-CCGGAGACGCTCTACCACCGCTTGAGAGACTTACTCTTGACCGAGCT-3' and 5'-CGGTCAAGAGTAAGTCTCTCAAGCGGTGGTA GAGCGTCT-3' were ligated into the BspEI/Sac I sites of pEGFP-MK5, pEGFP-MK5 T182A, and pEGFP-MK5 L337A, respectively. To construct the RFP-Cα-NLS and RFP-Cα-NES
expression plasmids, the BspEI and BamHI fragment of GPKAnls, respectively GPKAnes, was ligated into the corresponding sites of pAsRed2-C1 (Clontech, Mountain View, CA, USA). The GPKAnls and GPKAnes plasmids were
generously provided by Dr. S.H. Green [41]. The plasmid pCRE-LUC was from Clontech. All newly generated plasmids and mutations were confirmed by sequencing.

In vitro kinase assay

Phosphorylation of purified MK2 (Upstate Millipore, Billerica, MA, USA), MK5 (Upstate Millipore), and ERK3 [24] by 2.5 units PKA-C was performed in 25 mM Tris.HCl pH7.5, 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; Amersham/GE Healthcare) in a total volume of 40 μl at 30°C for 1 hour. The reaction was stopped in 4xLDS Sample buffer and proteins denatured at 70°C for 10 minutes. The phosphorylation was analysed on NuPage 4-12% Bis-Tris SDS-PAGE (Invitrogen) for 50 minutes at 200V, then subjected to autoradiography.

Cell culture and transfection

PC12 cells, a kind gift from Dr. Jaakko Saraste (University of Bergen, Norway), and PKA-deficient PC12A123.7 cells (generously provide by Dr. J. Yao) were maintained in RPMI-1640, supplemented with 10% horse serum (Gibco) and 5% foetal bovine serum, 2 mM L-glutamine, penicillin (110 units/ml) and streptomycin (100 μg/ml). HeLa cells were maintained in Eagle's Minimum Essential Medium supplemented with 1x non-essential amino acids, 10% foetal calf serum, 2 mM L-glutamine, 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% foetal 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. We routinely obtained >75% transfection efficiency in the different cell lines with these transfection agents as judged by green fluorescent positive cells when using the pEGFP plasmid as a marker.

Preparation of nuclear and cytoplasmic extracts

The NE-PER® Nuclear and Cytoplasmic Extraction Reagents (Pierce, Rockford, IL, USA; cat. no. 78833) was used according to the instructions of the manufacturer.

Western blotting

For detection of co-immunoprecipitates and MK5, samples were analysed 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 hour and probed either by 4D7, anti-PRAK, anti-MK2, anti-ERK2 or anti-PKA cat. After 4 washes, the membrane was incubated with the appropriate secondary antibody for 1 hour. Visualisation of proteins was achieved by using CDP Star (Tropix, Bedford, MA, USA) substrate and Lumi-Imager F1 from Roche (Basel, Switzerland).

Immunoprecipitation

HeLa 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°C for 10 minutes at 15,000g. Lysates were incubated with the appropriate antibody for at least 1 hour 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 pH8.0. Twenty μl 2xLDS sample buffer was added to the beads before denaturation at 70°C for 10 minutes. The immunoprecipitates were either analysed on Western blots or by means of kinase assays.

Cell staining and microscopy

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 permeabilised for 10 min with 0.1% Triton X-100. 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. Cell nuclei were visualised by DRAQ5 staining (Biostatus Limited, Leicestershire, United Kingdom). We routinely examined 50 cells or more and representative pictures are shown in the results.

Transient transfection and luciferase assay

HEK293 cells were plated 3x105 cells per well in a 6-wells plate. Cells were transfected using Lipofectamine 2000 according to the manufacturer's instruction. Luciferase assays were as previously described [42].

Immune complex kinase assay

For the immune complex kinase assays, HeLa cells were transfected with expression plasmids encoding EGFP fusion proteins with wild-type MK5 or mutants and Cα. Corresponding empty vector control plasmids (pEGFP and pRcCMV, respectively) were used as control. As a positive control for the assay, cells were either transfected with pEGFP-MK5L337A, encoding an activated MK5 variant, or pEGFP-MK5 plus expression plasmids for the activated MKK6E/E mutant and p38β MAPK. MK5 fusion proteins were immunoprecipitated using anti-GFP antibody and the kinase activity was monitored against 30 mu M PRAKtide substrate peptide at 30°C for 20 minutes in 50 mu l buffer containing 40 mM TrisCl pH 7.5, 0.4 mg/ml BSA, 15 mM MgCl2, 0.1 mM cold ATP, and 1 μCi 32P gamma
-ATP (3000Ci/mmol). Following incubation, 20 mu l aliquots were spotted onto phosphocellulose discs (Upstate) and
washed extensively with 1% phosphoric acid before measurement of radioactive incorporation by scintillation counting.

Quantitation of F-actin

The amount of globular (G-actin) and filamentous actin (F-actin) was determined using the G-actin/F-actin In Vivo Assay Kit from Cytoskeleton (Denver, CO, USA) according to the instructions of the manufacturer. Briefly, cells were lysed and the lysate was centrifugated at 100,000g for 1h at 37°C. The supernatant contained G-actin, while the pellet contained F-actin. As a control, cell lysates were either treated with F-actin enhancing solution (phalloidin) or F-actin polymerisation solution (cytochalasin) before ultracentrifugation. The amount of G- and F-actin was determined by western blotting using an anti-actin specific antibody.

 Small interfering RNA (siRNA)

Validated MK5-directed siRNA, purchased from Ambion Inc. (Austin, TX), was transfected by Nucleofection (Amaxa) into PC12 cells according to the manufacturers instructions using 100 mu M siRNA/106cells. The levels of MK5 protein in untreated and siRNA treated cells were monitored 48 hours after transfection by western blotting using anti-PRAK antibody (Santa Cruz) to verify the efficiency of reduction in MK5 expression.

Results

The cAMP/PKA pathway induces nuclear export of MK5

Both endogenous and ectopically expressed MK5 as green fluorescent protein (EGFP)-, cyan fluorescent protein-, and HA-tagged proteins reside predominantly in the nucleus in different cells [23, 37, 43]. Studies have proven that the cAMP/PKA signalling pathway participates in actin remodelling, and that MKs may regulate the cytoskeletal architecture through modulating the activity of cytoplasmic proteins involved in F-actin organisation (reviewed in [11] and [30]). Because the putative involvement of MK5 in stimulus-induced microfilament rearrangement raises the likelihood of cytoplasmic distribution of MK5, we tested whether activation of PKA triggered subcellular redistribution of MK5 in PC12 cells. Thereto cells transfected with an expression plasmid for EGFP-MK5 fusion protein were treated with the cAMP-elevating agent forskolin. Consistent with previous findings of both ectopic and endogenous MK5, we observed MK5 mainly in the nucleus of unstimulated cells [23, 37, 43]. A transient nuclear export of MK5 was observed in cells treated with forskolin, but not in control cells treated with the vehicle DMSO. Increased cytoplasmic MK5 residence was observed 15 min after forskolin exposure and maintained elevated for at least 1 hour, after which EGFP-MK5 relocalised mainly to the nucleus. After 30 minutes of forskolin stimulation, the amount of cells displaying both nuclear and cytoplasmic EGFP-MK5 versus nuclear EGFP-MK5 alone increased from 6% to 84% (50 cells were counted). After 6 hours, the subcellular distribution of MK5 was comparable to untreated cells (Figure 1A). In 95% (+/- 2%; 50 cells were counted in several independent experiments) of the DMSO-treated cells, MK5 was predominantly nuclear. Forskolin treatment had no effect on the subcellular localisation of the EGFP protein (results not shown). Endogenous MK5 was also exported from the nucleus in cells with the PKA signalling pathway activated with the same kinetics as EGFP-MK5 (Figure 1B). These findings confirm that MK5 and EGFP-MK5 fusion protein behave identically in their subcellular redistribution in response to forskolin. Monitoring the amount of MK5 in the cytoplasmic and nuclear fraction of untreated and forskolin-treated cells confirmed an increased nuclear export of MK5 after forskolin exposure. A 55% decrease of nuclear MK5 was observed after 30 min forskolin treatment, while a 41% increase in cytoplasmic MK5 was monitored in forskolin-exposed cells (Figure 1C).

Next, we tested whether overexpression of the catalytic subunit of PKA tagged with a nuclear localisation signal (Calpha -NLS) was sufficient to induce nuclear export of MK5. Similar to forskolin-exposed cells, MK5 was located in the cytoplasm of cells that ectopically expressed Calpha -NLS (Supplementary data, Figure 1). To elaborate the contribution of PKA in forskolin-induced nuclear export of MK5, we used PC12A123.7 cells, which are deficient in PKA [44]. No subcellular redistribution of MK5 was monitored in these cells after forskolin treatment (Supplementary data, Figure 2). In conclusion, these results demonstrate that activation of the cAMP/PKA signalling pathway provokes nuclear export of MK5 in a time-dependent manner.

 

The catalytic Cα subunit of PKA phosphorylates MK5 and increases the kinase activity of MK5

Previous studies by our group and others have shown that nuclear export of MK5 can be achieved by overexpression of the atypical MAP kinases ERK3 and ERK4. This nuclear exclusion requires the direct interaction between MK5 and ERK3 or ERK4, and leads to activation of MK5 [23-26]. To find out whether PKA can interact with MK5, HeLa cells were transfected with EGFP-MK5 and Calpha expression plasmids and reciprocal co-immunoprecipitation studies were performed with anti-Calpha antibody followed by western blotting with MK5 antibody, or co-immunoprecipitation with anti-EGFP antibody followed by western blotting with anti-Calpha antibody. A specific interaction was monitored between these two proteins (Figure 2A). The positive signal in Fig. 2A, lane 6 (lysates of cells ectopically expressing EGFP-MK5 but not Calpha ) is probably the result of interaction between ectopically expressed MK5 and endogenous Calpha . No interactions were detected in the control pull-down assays using empty expression vectors. Because of the sequence similarity between MK2 and MK5, we investigated whether Calpha also associates with MK2. Cells were co-transfected with EGFP-MK2 and Calpha expression vectors or empty vectors as controls and lysates were immunoprecipitated with anti-Calpha or anti-EGFP antibodies. The presence of the co-immunoprecipitated partner was detected by western blot using anti-MK2 or anti-Calpha antibodies, respectively. However, under these experimental conditions no interaction between MK2 and Calpha was detected (Figure 2B). The MAPKAP kinase RSK1 was reported to interact with both the Calpha and the regulatory subunit of PKA [36]. This prompted us to test whether such an interaction also occurred with MK5. No association between MK5 and the regulatory RIalpha subunit of PKA was observed (results not shown). Next, we examined whether PKA, in agreement with the MK5-interacting protein kinases ERK3 and ERK4, possessed the ability to phosphorylate MK5 and activate its intrinsic kinase activity. An in vitro kinase assay demonstrated that both Calpha and MK5 possessed autophosphorylation activity, and that incubation of purified Calpha with purified MK5 increased the phosphorylation levels of MK5 approximately 2.5-fold, but not of Cα (Figure 3A). Calpha did not phosphorylate MK2, but phosphorylated the myelin basic protein (MBP), which was used as a positive control (Figure 3B). The inability of Cα to phosphorylate MK2 is in concordance with our previous findings that MK2 and Calpha did not interact in vivo (Figure 2B). To monitor the effect of PKA on the enzymatic activity of MK5, we performed an in vitro kinase assay. ERK3 was selected as a substrate for MK5. ERK3 displayed low levels of autophosphorylation activity. Cα alone or inactive MK5 alone did not induce phosphorylation of ERK3. However, incubation of Cα plus inactive MK5 strongly increased the phosphorylation levels of ERK3 (Figure 4A, top panel). Western blotting with anti-ERK3 antibodies ensured that equal amounts of ERK3 substrate were used in the samples (Figure 4A, bottom panel). These results suggest that Calpha enhanced the kinase activity of MK5 towards ERK3. Subsequently, we tested the effect of PKA on the in vivo kinase activity of MK5 using an immune complex kinase assays. Thereto, a series of co-transfections with EGFP-MK5 and Cα-NLS expression plasmids or empty vectors (pEGFP-C1 and pRcCMV, respectively) were performed and the protein kinase activity of MK5 towards the synthetic PRAKtide substrate was measured. While EGFP alone or in the presence of Cα had no kinase activity towards PRAKtide (results not shown), EGFP-MK5 phosphorylated PRAKtide. Co-expression of Cα enhanced the kinase activity of MK5 approximately 2-fold in most experiments (n=6), although occasionally we did not observe an increase of MK5 activity in the presence of Cα (n=2). The constitutive active MK5 mutant L337A [37] and an activated MK5 by co-expression of active MKK6 E/E and p38 MAPK possessed a 3- to 5-fold increased kinase activity compared to wild-type MK5 (Figure 4B). Forskolin stimulation alone also induced the kinase activity of MK5 (results not shown). In conclusion, Cα physically interacts with and stimulates the kinase activity of MK5 in vivo.

PKA-induced nuclear export of MK5 requires nuclear localisation of Cα

The co-immunoprecipitation studies suggest an in vivo interaction between Cα and MK5, but they do not establish whether Cα is directly involved in the subcellular redistribution of MK5. We therefore wanted to determine whether nuclear entrance of Cα is required to induce nuclear export of MK5. We monitored the subcellular localisation of MK5 in forskolin-treated cells that overexpress PKI, a peptide that contains a strong nuclear export signal and that can export Calpha out of the nucleus (for a recent review see [45]).
Overexpression of PKI prevented forskolin-induced nuclear export of MK5 in PC12 cells. Of the EGFP-MK5 expressing cells, in 98% of the cells (+/- 2%; 50 cells were counted in several independent experiments) MK5 was mainly in the nucleus (Figure 5A). This is in contrast with the transient nuclear exclusion of MK5 in forskolin-treated PC12 cells (Figure 1). To further ascertain the necessity of nuclear entry of Cα in MK5 cytoplasmic relocation, we compared the residence of EGFP-MK5 in cells co-transfected with an expression plasmid for EGFP-MK5 plus an expression vector for Cα tagged with either a nuclear localisation signal (NLS) or with a nuclear export signal (NES). In order to visualise Cα and MK5 simultaneously, we used red fluorescence protein-Calpha fusion proteins (AsRed-PKA-NLS and AsRedPKA-NES, respectively). The NES-tagged Cα subunit resided in the cytoplasm and was unable to induce nuclear export of MK5, while in cells expressing a Cα-NLS fusion protein both cytoplasmic and nuclear MK5 location was observed (in 90% +/- 2% of the analysed cells; Figures 5B and 5C). The levels of cytoplasmic MK5 in Cα-NLS expressing cells were increased compared to cells not expressing Calpha -NLS (compare untreated cells in Figures 1A and 5C).

PKA-induced nuclear export of MK5 requires protein kinase activity of PKA as well as MK5

Next, we investigated whether the kinase activity of Calpha was required to provoke nuclear export of MK5. Thereto, expression plasmids for the kinase dead Cα mutant (CL72H) and EGFP-MK5 were co-transfected in PC12 cells and subcellular localisation of MK5 was monitored. In contrast to an active Cα enzyme, no nuclear export was observed with the mutant catalytic PKA subunit. In fact, in 98% (+/- 2%; 50 cells were counted in several
independent experiments) of the cells, MK5 resided predominantly in the nucleus (Figure 6A). Western blot analysis of cell extracts transfected with the kinase dead Cα L72H mutant confirmed that this mutant had no enzymatic activity because phosphorylation of the transcription factor CREB, which is a genuine substrate for PKA, did not increase in the presence of this mutant, while increased phospho-CREB was detected in
extracts prepared from cells transfected with the active Cα expression plasmid. Moreover, the kinase dead Cα mutant was unable to stimulate CRE-dependent transcription (bottom panel Figure 6A). To decide whether MK5 enzyme activity is required for PKA-regulated nuclear export, we monitored the subcellular distribution of the kinase dead MK5 K51E mutant and the T182A mutation in the phosphorylation loop. Both mutants also failed to relocate to the cytoplasm in the presence of activated PKA (Figure 6B). In agreement with our observations in PC12 cells, forskolin treatment of HeLa cells also induced transient nuclear export of EGFP-MK5 protein, but not of the kinase dead T182A mutant, indicating a non cell-specific mechanism (results not shown). These findings indicate that both an active Cα and active MK5 are required for PKA-induced cytoplasmic translocation in PC12 cells.

MK5 mediates forskolin-induced F-actin rearrangements

Others and we noticed that forskolin treatment of PC12 cells resulted in altered cytoskeleton morphology [11,30]. Therefore, we assessed the amount of F-actin in response to forskolin. A transient increase in F-actin was observed 30 min after forskolin treatment (Figure 7A). Interestingly, the kinetics of F-actin rearrangement and increase coincided with the nuclear exclusion of MK5, presuming the involvement of the MK5 in PKA-induced F-actin remodelling. To address the possible implication of MK5 in cAMP/PKA-induced F-actin rearrangement, several different experimental approaches were conducted. We reasoned that the coupled event nuclear import of Cα/cytoplasmic export of MK5 would be required for F-actin reorganisation. Therefore, we compared the microfilament structure in untreated and forskolin-treated non-transfected cells or cells transfected with a PKI expression plasmid. F-actin remodelling was inhibited in cells
overexpressing PKI (>75% of the PKI transfected cells did not display F-actin rearrangement after forskolin exposure; Figure 7B). This underscores that nuclear import of PKA is necessary and suggests that nuclear export of MK5 may be required for cAMP/PKA-induced F-actin remodelling. Next, we examined forskolin-induced F-actin rearrangement in control cells and in cells in which the levels of MK5 protein had been depleted by validated siRNA against MK5. In agreement with previous reports, we observed that forskolin treatment of PC12 cells, which had been transfected with scrambled siRNA, resulted in F-actin remodelling (Figure 7C). However, the majority of cells (>75%; 50 cells counted in two independent experiments)
treated with MK5-directed siRNA did not show F-actin rearrangements after forskolin treatment (Figure 7D). While siRNA directed against MK5 transcripts strongly reduced MK5, but not ERK2 protein levels, scrambled siRNA did not reduce the levels of either of these proteins (Figure 7E). Taken together, these results indicate that MK5 mediates forskolin-induced F-actin remodelling. To elaborate the role of activated, cytoplasmic MK5 in F-actin rearrangement, we generated a MK5 mutant that is constitutive active and that locates to the cytoplasm. Our previous study had shown that a single amino-acid substitution in the functional NES motif of MK5 (L337A) converted MK5 in a constitutive active kinase. However, this mutant has lost its ability to shuttle to the cytoplasm [37]. To conduct this active MK5 variant to the cytoplasm, we fused the NES sequence of the human immunodeficiency virus (HIV) Rev protein to EGFP-MK5 to generate the fusion protein EGFP-NES-MK5 L337A. Studies by others had shown that this NES motif
could direct nuclear exclusion of EGFP [40]. Concordantly, the EGFP-NES-MK5 L337A protein resides exclusively in the cytoplasm (Figure 7F, top panel). All cells expressing this cytoplasmic, activated MK5 mutant (50 cells counted in three independent experiments) could induce some F-actin rearrangement in the absence of forskolin (Figure 7F, top panel). Overlay of the confocal microscopy images confirmed F-actin rearrangements in cells transfected with this MK5 mutant (results not shown), while no such changes were observed in non-transfected cells. Cytoplasmic expression of EGFP-NES-MK5 T182A (Figure 7D bottom panel) or EGFP-NES-MK5 (results not shown) did not affect the microfilament structure in these cells, despite the cytoplasmic location of MK5. These results proof that an activated, cytoplasmic MK5 can partially mimic forskolin-induced F-actin rearrangement, and suggest that PKA-dependent nuclear export and activation of MK5 is implicated in PKA-triggered reorganisation of F-actin.


Discussion

The MAPKAPK MK5 was first described almost 10 years ago, but as to date little is known about its activators, in vivo substrates, and biological functions. The atypical MAPKs ERK3 and ERK4 were the first identified genuine substrates for MK5, but the functional importance of these interactions remains unclear [23-26]. The original study with MK5-/- mice revealed no obvious phenotypical defects [22], but a recent study with MK5 deficient mice led to the recognition of the implication of MK5 in ras-induced senescence and tumour suppression in a p38 MAPK-dependent manner through activation of p53 [27].

Results from our studies assign

  1. a novel mode of regulating the subcellular localisation of MK5,

  2. PKA as a new activator for MK5, and

  3. a not previously recognised biological function of MK5 in F-actin rearrangements.


MK5, which contains functional, but overlapping NLS and NES motifs, accumulates in the nucleus of resting cells. The NLS signal seems to be dominant over NES, probably due to the conformation of the MK5 protein in which the
NES is less accessible to exportin. However, in response to cellular stress, the NES motif may be unmasked and MK5 can be exported to the cytoplasm [37,43]. In this study, we observed that activated PKA triggered nuclear export of MK5. Time studies revealed that at least 15 min were required for MK5 to exit the nucleus in response to forskolin (Figure 1), while nuclear translocation of the catalytic subunit of PKA in response to forskolin was reported to plateau after 15 minutes [46]. This similar kinetics may indicate that nuclear import of activated PKA is necessary for nuclear export of MK5. Cytoplasmic redistribution of MK5 is also observed when ERK3 and ERK4 are overexpressed. The mechanisms of ERK3/ERK4-mediated nuclear export of MK5 differ, however, from Calpha -mediated export. Co-expression of a kinase dead Calpha did not lead to accumulation of MK5 in the cytoplasm, and similarly, the kinase dead MK5 variants K51E and T182A were not excluded from the nucleus by an active Cα or forskolin.
On the contrary, kinase dead ERK3 and ERK4 mutants were able to induce nuclear export of wild-type MK5, but also of catalytic dead MK5 mutant proteins [23-26]. Further studies that identify the interaction regions on MK5 will provide additional information on the mechanistic insight how Calpha regulates the nucleocytoplasmic relocation of MK5. Our preliminary results show that a C-terminal truncated MK5 consisting of residues 1-423 is not transported out of the nucleus upon forskolin treatment. On the other hand, ERK3 still relocates this truncated MK5 mutant to the cytoplasm [24], indicating that nuclear exclusion of MK5 by PKA is not mediated by ERK3. ERK4, however, does not translocate this MK5 variant to the cytoplasm. Whether ERK4 is implicated in PKA-induced nuclear export of MK5 remains to be established. The C-terminal part of MK5, which is absent in MK2 and MK3, may be critical for PKA-induced MK5 nuclear export. Since the carboxy-terminal part of MK5 is absent in MK2 and MK3, it may explain the unique role of MK5 in PKA-triggered F-actin remodelling.

Next, we have provided experimental evidence that, similar to ERK3 and ERK4, co-expression of Calpha with MK5
stimulated the kinase activity of MK5 in vivo. We also found enhanced phosphorylation of MK5 by in vitro kinase assay. Whether this is due to increased phosphorylation of Thr-182 in the activation loop of MK5 remains unsolved. We have tried to inspect whether forskolin or Calpha trigger phosphorylation of MK5 at Thr-182 by using two different commercially available antibodies (anti-phospho-PRAK (Thr182) antibodies from Upstate, Charlottesville, VA, USA, and anti-phospho-Threonine-Proline antibodies from Cell signalling, Beverly, MA, USA). Unfortunately, none of these antibodies worked in our hands, as they did not recognise in vitro phosphorylated MK5 or immunoprecipitated MK5 from forskolin-treated or Calpha -transfected cells. Moreover, we were unable to detect PKA-induced phosphorylation of MK5 in vitro and in cell extracts with a phospho-Ser/Thr PKA substrate antibody (Cell signalling #9624).

The Calpha -MK5 interaction described here represents a new example of crosstalk between the cAMP/PKA and MAPK signalling pathways. Various branches of interaction between the cAMP/PKA pathway and the different MAPK cascades have been demonstrated, e.g. PKA-mediated regulation of Raf and p38 MAPK, and recently also of another MAPKAPK, RSK1 [32,34,36,47]. Unphosphorylated RSK1 interacted with the regulatory RI subunits, while activated RSK1 bound the catalytic subunits of PKA. The interaction with PKA also regulated the subcellular localisation of RSK1. The authors demonstrated that disruption of the interaction of RI subunits with A-kinase PKA anchoring protein (AKAP) resulted in an increase in cytosolic active RSK1 levels. The mechanism by which PKA-induces nuclear export of MK5 appears to be different from RSK1 because we did not detect an in vivo interaction between MK5 and RI. Chaturvedi and co-workers did not investigate whether Calpha phosphorylated RSK1, nor was the effect of this interaction on the intrinsic kinase activity of RSK1 tested [36]. We found that Calpha increased the phosphorylation levels of MK5, but not vice versa.

Finally, we solved the biological relevance for PKA-induced subcellular redistribution and activation of MK5 by demonstrating that MK5 acted as a mediator for PKA-induced F-actin remodelling. Cytoplasm-directed expression of a constitutive active MK5 variant mimicked forskolin-induced F-actin rearrangement, while ablation of MK5 expression by siRNA prevented forskolin-provoked F-actin rearrangements. MK5 is not the only protein that is involved in cytoskeletal architecture and whose subcellular location is regulated by PKA. The RNA-binding protein,
polypyrimidine tract-binding protein (PTB), was exported from the nucleus during PKA-induced F-actin rearrangement/neurite outgrowth in PC12 cells. Nuclear export of PTB required PKA-mediated phosphorylation of Ser-16. PTB preferentially associated with beta -actin mRNA and thwarting PTB expression by RNA interference disrupted PKA-, but not NGF-induced neurite outgrowth in PC12 cells. Similar to MK5, PTB represents a protein involved in cytoskeletal dynamics whose subcellular distribution is regulated by PKA [48].

Several experimental observations indicate that p38 MAPK, MK2 and Hsp27 play a role in mediating alternations in filamentous actin in response to stimuli. First, MK2, p38 MAPK, Hsp27 and Akt can form a multiprotein complex [49,50]. In addition, stress-inducing stimuli activated p38 MAPK, and this coincided with phosphorylation of MK2 and Hsp27, and changes in F-actin dynamics in different cell lines [51-55] . Furthermore, a regulatory role for MK2 in F-actin dynamics during embryonic development was also proposed by the work of Paliga and colleagues [56]. However, none of the above mentioned studies directly addressed the role of MK5 in F-actin remodelling, nor was the participation of MK2 unequivocally proven by e.g. RNA interference studies for overexpression of dominant negative variants of these kinases. Studies in rat pulmonary artery microvascular endothelial cells revealed a more direct causal involvement of MK2 in hypoxia-induced actin reorganisation. Indeed, overexpression of a constitutive active MK2 resembled, while a dominant negative MK2 mutant prevented the effect of hypoxia on F-actin structure [57]. Stimuli-induced alterations in microfilament structure may require different pathways that signal through distinct MKs. For example, stimuli that activate p38 MAPK (e.g. oxidative stress) may engage MK2, while stimuli signalling through the cAMP/PKA (e.g. secretin) pathway may utilise MK5 to
provoke changes in F-actin structures [51,55,58]. The precise molecular mechanism by which MK5 participates in F-actin remodelling remains elusive, but may in concordance with MK2 imply phosphorylation of Hsp27. We could detect MK5-Hsp27 interaction by co-immunoprecipitation and found that MK5 phosphorylated Hsp27 in vitro (our unpublished results). On the other hand, studies by the group of Gaestel with MK5 deficient mice jeopardise the role for MK5 in Hsp27 phosphorylation. The authors showed that Hsp27 did not interact with MK5 in vivo, at least in primary murine cells isolated from a strain with a mixed 129xC57/B6 genetic background [22]. It cannot be ruled out that Hsp27 is a bona fide substrate for MK5 in rat PC12 cells or that PKA-provoked phosphorylation/activation or conformational changes of MK5 are required to enable in vivo interaction with Hsp27. Observations in mk5-/- mice further support a putative role for MK5 in regulation of the cytoskeleton structure and cell migration. Cell motility requires reorganisation of the cytoskeleton and directed cell migration is important for embryonic development. Depending on the genetic background, MK5 deficient animals, but not mk2-/- mice, displayed embryonic lethality with incomplete penetrance around day E11.5. This developmental failure may be the result of impaired cell migration [23].

Activation of the nuclear transcription factor CREB by phosphorylation at Ser-133 has been documented to be involved in cAMP-provoked cytoskeletal changes in PC12 cells [59]. CREB can be phosphorylated by numerous protein kinases, including MAPKAPK [60] . It is unlikely that MK5-mediated F-actin rearrangements in forskolin-treated PC12 cells requires the action of CREB because forskolin induced redistribution of MK5 to another subcellular compartment than CREB, and MK5 did not interact with CREB in vivo, nor did MK5 phosphorylate CREB or activate CREB-mediated transcription [61].

In conclusion, our studies have revealed a novel function of MK5 in microfilament rearrangements in response to increased cAMP levels and disclosed a new interaction between the cAMP/PKA and the MAPK signalling pathways. These findings may increase our molecular understanding of stimulus-induced changes in microfilament and crosstalk between signal transduction pathways. Whether MK5 is involved in other cytoskeletal rearrangements and/or cell motility events awaits to be proven, but phosphorylation of the putative MK5 substrate, Hsp25/27 also contributes to cell migration. In addition, the F-actin interacting myosin light chain protein was shown to be an in vitro substrate for MK5 [10,62-64].

Where, when, and under which circumstances MK5 participates in cytoskeletal dynamics in an organism are questions that need to be answered to increase our understanding of MK5its role in normal and possible pathological processes in order to apply therapeutic strategies in diseases associated with perturbed MK5 activity.



Acknowledgements

We thank following colleagues for kindly providing us with important reagents in this study: Dr. M. Gaestel (pEGFP-MK2), Dr. G. Thiel (Cα-NLS expression plasmid), Dr. B. Skålhegg (RIα subunit antibodies), Dr. J. Saraste (PC12 cells), Dr R. A. Maurer (PKI expression plasmid), Dr. S.H. Green (GPKAnes and GPKAnls plasmids), Dr. J. Yao (PKA deficient PC12 cells), Division of Signal Transduction Therapy, University of Dundee (ERK3 protein and antibodies). The help of Linda Helander with the PC12 cells is greatly appreciated. This work was supported by grants from the Norwegian Research Council (NFR, project S5228), The Norwegian Cancer Society (Kreftforeningen, project A01037), the Erna and Olav Aakres Foundation, and the Family Blix Foundation.


Figures

Figure 1 : Activation of the cAMP/PKA signalling pathway induces transient nuclear exclusion of MK5


Figure 1A : PC12 cells were transfected with an expression plasmid for EGFP-MK5, and then stimulated with either 10 μM forskolin (FSK) or vehicle (DMSO) for the time points indicated. The subcellular localisation of the EGFP-MK5 fusion protein over time was visualised by confocal microscopy. Cell nuclei were visualised by DRAQ5 staining. The subcellular distribution of EGFP-MK5 was monitored by confocal microscopy. Fifty cells were counted and more than 80% displayed both nuclear and cytoplasmic presence of EGFP-MK5 after forskolin exposure.


Figure 1B : PC12 cells were treated with 10 μM forskolin for the indicated time points and endogenous MK5 was visualised with anti-PRAK antibody (Santa Cruz) as primary antibody and Alexa 488 as secondary antibody. Nuclei were stained with DRAQ5.

Figure 1C – First panel : PC12 cells were transfected with EGFP-MK5. Nuclear and cytoplasmic extracts were prepared from forskolin-treated cells (30 min) or vehicle- (DSMO) treated cells. The presence of EGFP-MK5 was assayed by western blotting. To ensure equal loading and to test for contamination between cytoplasmic and nuclear fractions, the membrane was re-probed with anti-Ras antibodies.










Figure 1C – Second panel : The intensity of the signals was determined by densitometry and the values are shown as relative densitometric units (RDU). Bottom panel: the ratio of RDUMK5:RDURAS in nuclear (respectively cytoplasmic) fraction of DMSO-treated cells was arbitrary set as 100% and the ratios of RDUMK5:RDURAS in the fractions of forskolin-treated cells was related to this.


Figure 2 : MK5, but not MK2, interacts with Cα in vivo





















Figure 2A : HeLa cells were co-transfected with expression plasmids for Cα and EGFP-MK5 or corresponding empty vectors (pCMV and pEGFP, respectively). After 24 h, cells were lysed and Cα was immunoprecipitated with anti-Cα antibody (left panel) or EGFP-MK5 was immunoprecipitated with anti-EGFP antibodies (right panel). The immunoprecipitated proteins were separated by SDS-PAGE and western blotting was performed using either anti-MK5 antibody (left panel) or anti-Cα antibody (right panel). The coimmunoprecipitates with anti-Cα were also assayed with anti-EGFP antibodies (middle part of left panel). Lanes 1-3: input control of the transfected cells; lanes 4-6: immunoprecipitates. Lanes 1 and 4: cells transfected with expression vectors for EGFP-MK5 and Cα; lanes 2 and 5: cells transfected with pEGFP vector and Cα expression vector; lanes 3 and 6: cells transfected with EGFP-MK5 and empty vector for Cα (pCMV). The positions of Cα, MK5, the EGFP-MK5 fusion proteins, and the heavy (H) and light (L) IgG chains are indicated. To ascertain equal protein loading, the membranes were re-probed with an antibody against the immunoprecipitated protein (bottom panel).

Figure 2.B: HeLa cells were co-transfected with expression plasmids for EGFP-MK2 and Cα or with the corresponding empty vectors (EGFP and pCMV, respectively). After 24 h, cells were lysed and Cα was immunoprecipitated with anti-Cα antibody (left panel) or EGFP-MK2 was immunoprecipitated with anti-EGFP antibodies (right panel). Lanes 1-3: input control of the transfected cells; lanes 4-6: western blotting of the immunoprecipitates with anti-Cα antibody. Lanes 1 and 4: cells transfected with expression vectors for EGFP-MK2 and Cα; lanes 2 and 5: cells transfected with empty vector for MK2 (EGFP) and Cα expression vector; lanes 3 and 6: cells transfected with MK2 expression plasmid and empty vector for Cα (pCMV). The molecular mass marker and the positions of MK2, MK2-EGFP fusion protein, and the heavy (H) and light (L) IgG chains are indicated. To ascertain equal protein loading, the membranes were re-probed with an antibody against the immunoprecipitated protein (bottom panel in the figure).




Figure 3 : The catalytic subunit Cα of PKA increased the phosphorylation level of MK5, but not MK2 in vitro


























Figure 3A : Top panel (IVK) shows in vitro phosphorylation of MK5 by Cα. Left lane: purified Cα; middle lane: purified MK5; right lane: Cα plus MK5. The positions of phosphorylated Cα (pCα) and phosphorylated MK5 (pMK5) are indicated. Middle panel: the samples used for in vitro kinase assay were tested for equal amounts of MK5 protein by western blot with anti-MK5 antibodies. The values under the top and middle panel represent relative densitometric units (RDU) of the scanned phosphoMK5 (pMK5) and MK5 signals, respectively. The ratio of RDU of total MK5 protein (western blot signals) and of phosphorylated MK5 (in vitro kinase signals) is shown in the bottom part.

Figure 3B Cα does not phosphorylate MK2 in vitro. Lane 1: MK2 protein; lane 2: purified Cα protein; lane 3: MK2 protein plus Cα; lane 4: myelin basic protein (MBP) plus Cα. The lower panels represent western blot with anti-MK2 and anti-Cα antibodies, respectively. Bovine serum albumin (BSA), which is present in the kinase assay buffer, is also phosphorylated by Cα. The positions of the phosphorylated proteins (pBSA, pCα, and pMBP) are shown. The amount of MK2 protein (middle panel) and Cα (bottom panel) in each sample was assayed by western blot.



Figure 4 : The PKA catalytic subunit Cα stimulates the kinase activity of MK5 in vitro and in vivo























Figure 4A : An in vitro kinase (IVK) assay (top panel) was performed with purified Cα or/and MK5 with recombinant ERK3 as substrate. Lane 1: purified Cα; lane 2: purified inactive MK5; lane 3: purified ERK3; lane 4: Cα plus ERK3; lane 5: inactive MK5 plus ERK3; lane 6: Cα plus inactive MK5 plus ERK3. The total amount of ERK3 protein in the samples was monitored by western blot (lower panel).

Figure 4B : Cells were transfected with expression plasmids for the EGFP-MK5 fusion protein and Cα or empty vector. As a positive control for the assay, cells were either transfected with plasmids encoding the constitutive active MK5 L337A mutant (EGFP-MK5 L337A) or with EGFP-MK5 and constitutive active MKK6E/E plus p38 MAPK. Cell lysates were immunoprecipitated with anti-EGFP antibody and the MK5 kinase activity was determined by an immune complex kinase assay with PRAKtide as a substrate. The relative kinase activity in of wild-type MK5 in the absence of Cα was arbitrary set as 100% and the activity of the other samples was related to this. The results represent the average of 5 to 6 independent experiments.




Figure 5 : Forskolin- or Cα-induced nuclear export of MK5 requires nuclear entrance of Cα


























Figure 5A : PC12 cell that were co-transfected with an expression plasmid encoding PKI and EGFP-MK5 were treated with forskolin and the subcellular localization of MK5 was monitored over time. The nuclei of the cells were visualized with DRAQ5 staining (blue channel).





























Figure 5B : Forskolin does not induce transient nuclear exclusion of the kinase death MK5 K51E and MK5 T182A mutants. Cells were transfected with expression plasmids for the EGFP-MK5 K51E (top panel) or EGFP-MK5 T182 (bottom panel) fusion proteins. Green fluorescence was monitored at different time points after stimulation with 10 μM forskolin.




















Figure 5C : Nucleus-directed localization of Cα results in nuclear export of MK5. PC12 cells were co-transfected with expression vectors for EGFP-MK5 and for red fluorescent-Cα fusion protein tagged with a functional NLS signal (As-Red-PKA-NLS). Panels A and D: NLS-tagged Cα was visualized directly (red channel); panels B and E: EGFP-MK5 was visualized directly (green channel); panels C and F: merged images of the red and green channels.




Figure 6 : The kinase activities of both the PKA catalytic subunit Cα and MK5 are required to induce nuclear export of MK5
































Figure 6A : PC12 cells were co-transfected with expression plasmids encoding EGFP-MK5 fusion protein and with nucleus-directed kinase dead Cα L72H mutant. The subcellular distribution of MK5 was monitored by confocal microscopy (left panel on the top). Middle panel on the top: phosphospecific CREB antibodies failed to detect increased phosphorylation of CREB at Ser-133 in cells expressing kinase dead Cα L72H, while phosphoCREB was monitored in cells expressing kinase active Cα. Lane 1: extracts of cells transfected with empty vector pRcCMV; lane 2: extracts of cells transfected with wild-type Cα expression vector; lane 3: extracts of cells transfected with expression plasmid for Cα L72H. The cells were serum-starved overnight after transfection before they were harvested for western blotting analysis. The phosphoCREB antibodies cross-react with phospho-ATF1, a member of the CREB superfamily. The membrane was stripped and blotted with anti-CREB antibodies to ensure equal loading. The molecular mass (in kDa) of the protein marker is indicated. Right panel on the top: HEK293 cells were transfected using Lipofectamine 2000 with 100 ng pCRE-LUC reporter plasmid and 100 ng of expression vector encoding wild-type Cα or kinase dead Cα L72H. Four hours after transfection, the medium was replaced with fresh medium containing 0.2% serum. Luciferase activity (expressed as relative luciferase units; RLU) was measured 20 hrs later. The results represent the average +/- SD of three independent experiments.



Figure 6B : Forskolin does not induce transient nuclear exclusion of the kinase death MK5 K51E and MK5 T182A mutants. Cells were transfected with expression plasmids for the EGFP-MK5 K51E (top panel) or EGFP-MK5 T182 (bottom panel) fusion proteins. Green fluorescence was monitored at different time points after stimulation with 10 μM forskolin.





Figure 7 : Forskolin alters the amount of F-actin and PKA-induced F-actin remodeling is mediated by MK5











Figure 7A : PC12 cells were left untreated (lanes 1-4) or treated with forskolin for the time points indicated (lanes 5-12). After the cells were lysed, G-actin and F-actin were separated by ultracentrifugation, with G-actin found in the supernatant and F-actin present in the pellet. Both fractions were run on a denaturating polyacrylamide gel and G-actin (G) and F-actin (F) were detected by western blotting using an anti-actin antibody. F-actin enhancing solution was added to the lysate of cells of which the fractions after ultracentrifugation are shown in lanes 1 and 2. F-actin depolymerization solution was added was to the lysate of cells of which the fractions after ultracentrifugation are shown in lanes 3 and 4.





















Figure 7B : Nuclear exclusion of activated PKA by PKI prevents forskolin-induced F-actin remodeling. Cells transfected with a PKI expression plasmid were stimulated with forskolin 24 hrs after transfection and were then monitored for microfilament structure by staining F-actin.

Figure 7C : PC12 cells, transfected with scrambled siRNA, were treated with forskolin (10 μM) for the indicated time points and the architecture of the microfilaments was examined by staining F-actin with Alexa Fluor 594 phalloidin.

Figure 7D : Depletion of MK5 protein ablates forskolin-induced F-actin rearrangements in PC12 cells. Cells were transfected with 100 μM siRNA/106 cells and were stained for F-actin 48 hrs after transfection.









Figure 7E : Treatment of PC12 cells with siRNA against MK5, but not with scrambled siRNA, reduced the MK5 protein levels, while the protein amounts of ERK2 remained unaffected. Cells were transfected with siRNA or left untreated and lysates were prepared 24 h after transfection. The amount of MK5 was visualised by western blotting.
































Figure 7F : Cytoplasmic expression of activated MK5 is sufficient to induce F-actin rearrangement. PC12 cells were transfected with either expression plasmids encoding EGFP fusion proteins with activated MK5L337A or MK5T182A mutants tagged with a functional NES. Twenty-four hours after transfection, F-actin was stained and the nuclear localisation of MK5 was monitored.





Supplementary Data



Figure 1




Figure 2









Bibliography

1. Huang, C., Jacobson, K., and Schaller, M.D. (2004) J. Cell Sci. 117, 4619-4628

2. Roux, P.P., and Blenis, J. (2004) Microbiol. Mol. Biol. Rev. 68, 320-344

3. Imajo, M., Tsuchiya, Y., and Nishida, E. (2006). IUBMB Life 58, 312-317

4. Krens, S.F., Spaink, H.P., and Snaar-Jagalska B.E. (2006) FEBS Lett. 580, 4984-4990

5. Whitmarsh, A.J. (2007) Biochim Biophys Acta. 1773, 1285-1298

6. Manning, G., Whyte, D.B., Martinez, R., Hunter, T., and Sudarsanam, S. (2002) Science 298, 1912-1934

7. Stokoe, D., Campbell, D.G., Nakielny, S., Hidaka, H., Leevers, S.J., Marshall, C., and Cohen, P. (1992) EMBO J. 11, 3985-3994

8. Sithanandam, G., Latif, F., Duh, F.M., Bernal, R., Smola, U., Li, H., Kuzmin, I., Wixler, V., Geil, L. and Shrestha, S. (1996) Mol. Cell. Biol. 16, 868-876

9. New, L., Jiang, Y., Zhao, M., Liu, K., Zhu, W., Flood, L.J., Kato, Y., Parry, G.C.N., and Han, J. (1998) EMBO J. 17, 3372-3384

10. Ni, H., Wang, X.S., Diener, K., and Yao, Z. (1998) Biochem. Biophys. Res. Commun. 243, 492-496

11. Gaestel, M. (2006) Nature Rev. Mol. Cell Biol. 7, 120-130

12. Gerits, N., Kostenko, S., and Moens, U. (2007) Transgenic Res. 16, 281-314

13. Han, J., and Ulevitch, R.J. (1999) Nature Cell Biol. 1, E39-40

14. Kotlyarov, A., Neininger, A., Schubert, C., Eckert, R., Birchmeier, C., Volk, H.D., and Gaestel, M. (1999) Nature Cell Biol. 1, 94-97

15. Kotlyarov, A., Yannoni, Y., Fritz, S., Laas, K., Telliez, J.B., Pitman, D., Lin, L.L., and Gaestel, M. (2002) Mol. Cell. Biol. 22, 4827-4835

16. Manke I.A., Nguyen, A., Lim, D., Stewart, M.Q., Elia, A.E.H., and Yaffe, M.B. (2005) Mol. Cell, 17, 37-48

17. Culbert, A.A., Skaper, S.D., Howlett, D.R., Evans, N.A., Facci, L., Soden, P.E., Seymour, Z.M., Guillot, F., Gaestel, M. , and Richardson, J.C. (2006) J. Biol. Chem. 281, 23658-23667

18. Kobayashi, M., Nishita, M., Mishima, T., Ohashi, K., and Mizuno, K. (2006) EMBO J. 25,713-726

19. Neufeld, B., Grosse-Wilde, A., Hoffmeyer, A., Jordan B.W., Chen, P., Dinev, D., Ludwig, S. and Rapp, U.R. (2000) J. Biol. Chem. 275, 20239-20242

20. Voncken, J.W., Niessen, H., Neufeld, B., Rennefahrt, U., Dahlmans, V., Kubben, N., Holzer, B., Ludwig, S., and Rapp, U.R. (2005) J. Biol. Chem. 280, 5178-5187

21. Houben, R., Becker, J.C., and Rapp, U.R. (2005) J. Carcinogen. 20, 23

22. Shi, Y., Kotlyarov, A., Laas, K., Gruber, A.D., Butt, E., Marcus, K., Meyer, H.E., Friedrich, A., Volk, H.D., and Gaestel, M. (2003) Mol. Cell. Biol. 23, 7732-7741

23. Schumacher, S., Laass, K., Kant, S., Shi, Y., Visel, A., Gruber, A.D., Kotlyarov, A., and Gaestel, M. (2004) EMBO J. 23, 4770-4779

24. Seternes, O.M., Mikalsen, T., Johansen, B., Michaelsen, E., Armstrong, C.G., Morrice, N.A., Turgeon, B., Meloche, S., Moens, U., and Keyse, S.M. (2004) EMBO J. 23, 4780-4791

25. Kant, S., Schumacher, S., Singh, M.K., Kisper, A., Kotlyarov, A., and Gaestel, M. (2006) J. Biol. Chem. 281, 35511-35519

26. Åberg, E., Perander, M., Johansen, B., Julien, C., Meloche, S., Keyse, S.M., and Seternes, O.M. (2006) J. Biol. Chem. 281, 35499-35510

27. Sun, P., Yoshizuka, N., New, L., Moser, B.A., Li, Y., Liao, R., Xie, C., Chen, J., Deng, Q., Yamout, M., Dong, M.Q., Frangou, C.G., Yates III, J.R., Wright, P.E., and Han, J. (2007) Cell 128, 295-308

28. Skålhegg, B., and Taskén, K. (2000) Front. Biosci. 5, D678-693

29. Stork, P.J., and Schmitt, J.M. (2002) Trends Cell Biol. 12, 258-266

30. Howe, A.K. (2004) Biochim. Biophys. Acta 1692, 159-174

31. Johannessen, M., Moens, U. (2005) Trends in Cellular Signalling, 41-78. Nova Science Publishers, NY

32. Saxena, M., Williams, S., Taskén, K., and Mustelin, T. (1999) Nature Cell Biol. 1, 305-311

33. Delghandi, M.P., Johannessen, M., and Moens, U. (2005) Cell Signal. 17, 1343-1351

34. Houslay, M.D. (2006) Sci STKE. 349, pe32

35. Pearson, G.W., Earnest, S., and Cobb, M.H. (2006) Mol. Cell. Biol. 26, 3039-3047

36. Chaturvedi, D., Poppleton, H.M., Stringfield, T., Barbier, A., and Patel, T.B. (2006) Mol Cell. Biol. 26, 4586-4600

37. Seternes, O.M., Johansen, B., Hegge, B., Johannessen, M., Keyse, S.M. and Moens, U. (2002) Mol. Cell. Biol. 22, 6931-6945

38. Day, R.N., Walder, J.A., and Maurer, R.A. (1989) J. Biol. Chem. 264, 431-436

39. Thiel, G., Al Sarraj, V., Vinson, C., Stefano, L., and Bach, K. (2005) J. Neurochem. 92, 321-336

40. Thyssen, G., Li, T.H., Lehmann, L., Zhuo, M., Sharma, M., and Sun, Z. (2006) Mol. Cell. Biol. 26, 8857-8867

41. Bok, J., Zha, X.M., Cho, Y.S., and Green, S.H. (2003) J. Neurosci. 23, 777-787

42. Johannessen, M., Delghandi, M.P., Seternes, O.M., Johansen, B. and Moens, U. (2004) Cell Signal. 16, 1187-1199

43. New, L., Jiang, Y., and Han, J. (2003) Mol. Biol. Cell 14, 2603-2616

44. Yao, J., Erickson, J.D., and Hersch, L.B. (2004) Traffic 5, 1006-1016

45. Dalton, G.D. and Dewey, W.L. (2006) Neuropeptides 40, 23-34

46. Montminy, M. (1997) Annu. Rev. Biochem. 66, 807-822

47. Dumaz, N., and Marais R. (2005) FEBS J. 272, 3491-504

48. Ma, S., Liu, G., Sun, Y., Xie, J. (2007) Biochim Biophys. Acta 1773, 912-923

49. Zheng, C., Lin, Z., Zhao, Z.J., Yang, Y., Niu, H. and Shen, X. (2006) J. Biol. Chem. 281, 37215-37226

50. Wu, R., Kausar, H., Johnson, P., Montoya-Durango, D.E., Merchant, M. and Rane, M.J. (2007) J. Biol. Chem. 282, 21598-21608

51. Huot J., Houle, F., Marceau, F., and Landry, J. (1997) Circ. Res. 80, 383-392

52. Larsen, J.K., Yamboliev, I.A., Weber, L.A., and Gerthoffer, W.T. (1997) Am. J. Physiol. 273, L930-L940

53. Huot, J., Houle, F., Rousseau, S., Deschesnes, R.G., Shah, G.M., and Landry, J. (1998) J. Cell Biol. 143, 1361-1373

54. Schäfer, C., Ross, S.E., Bragado, M.J., Groblewski, G.E., Ernst, S.A., and Williams, J.A. (1998) J. Biol. Chem. 273, 24173-24180

55. Azuma, N., Akasaka, N., Kito, H., Ikeda, M., Gahtan, V., Sasajima, T. and Sumpio, B.E. (2001) Am. J. Physiol. Heart Circ. Physiol. 280, H189-197

56. Paliga, A.J.M., Natale, D.R., and Watson, A.J. (2005) Biol. Cell 97, 629-640

57. Kayyali, U.S., Pennella, C.M., Trujillo, C., Villa, O., Gaestel, M., and Hassoun, P.M. (2002) J. Biol. Chem. 277, 42596-42602

58. Kim, H.S., Yumkham, S., Kim, S.H., Yea, K., Shin, Y.C., Ryu, S.H., and Suh, P.G. (2006) Exp. Mol. Med. 38, 85-93

59. Shimomura, A., Okamoto, Y., Hirata, Y., Kobayashi, M., Kawakami, K., Kiuchi, K., Wakabayashi, T., and Hagiwara, M. (1998) J. Neurochem. 70, 1029-1034

60. Johannessen, M., and Moens, U. (2007) Front Biosci. 12, 1814-1832

61. Johannessen, M., Delghandi, M.P., Rykx, A., Dragset, M., Vandenheede, J.R., Van Lint, J., and Moens, U. (2007) J. Biol. Chem. 282, 14777-14787

62. Hedges, J.C., Dechert, M.A:, Yamboliev, I.A., Martin, J.L., Hickey, E., Weber, L.A., and Gerthoffer, W.T. (1999) J. Biol. Chem. 274, 24211-24219

63. Pichon, S., Bryckaert, M., and Berrou, E. (2004) J. Cell Sci. 117, 2569-2577

64. Rousseau, S., Dolado, I., Beardmore, V., Shpiro, N., Marquez, R., Nebrada, A.R., Arthur, J.S., Case, L.M., Tessier-Lavigne, M., Gaestel, M., Cuenda, A., and Cohen, P. (2006) Cell Signal. 18, 1897-1905


Footnotes

1
Abbreviations: Cα, catalytic subunit PKA isoform α; CRE, cAMP-response element; DMSO, dimethyl sulfoxide; EGFP, enhanced green fluorescence protein; MAPK, mitogen-activated protein kinase; MAPKAPK, MAPK-activated protein kinase; MBP, myelin basic protein; MK, MAPKAPK; NES, nuclear export signal; NLS, nuclear localisation signal; PKA, cAMP-dependent protein kinase; PTB, polypyrimidine tract-binding protein; RI, regulatory subunit PKA type I.


About this document ...

Modulation of F-actin rearrangements by the cyclic AMP/PKA pathway is mediated by MAPKAP Kinase 5 and requires PKA-induced nuclear export of MK5

This document was generated using the LaTeX2HTML translator Version 2002-2-1 (1.71)

Copyright © 1993, 1994, 1995, 1996, Nikos Drakos, Computer Based Learning Unit, University of Leeds.
Copyright © 1997, 1998, 1999, Ross Moore, Mathematics Department, Macquarie University, Sydney.

The translation was initiated on 2007-12-21