Untersuchungen zur Beteiligung Aktin-assoziierter Proteine an intrazellulären Signalwegen

In dieser Arbeit wurden zwei Aktin-assoziierte Proteine, WIP und Par-4, durch proteinbiochemische und zellbiologische Methoden untersucht. Zunächst wurde ausgehend von einer unvollständigen cDNA-Sequenz die komplette cDNA eines neuen Gens aus einer Ratten-cDNA-Bibliothek isoliert und anhand von computerunterstützten Sequenzanalysen als das Ratten-Ortholog des humanen WASP-interagierenden Proteins (WIP) identifiziert. Durch Northern Blot-Analysen konnten sechs verschiedene Spleißformen von WIP detektiert werden. Die Expression des Ratten-WIP in Fibroblasten zeigte, daß das Protein an die Aktin-Filamente des Zytoskeletts bindet. Durch Koimmunpräzipitationsversuche wurde die Interaktion des Ratten-WIP mit N-WASP nachgewiesen. Die Koexpression von WIP und N-WASP in Ratten-Fibroblasten führte interessanterweise zu einer Relokalisation von N-WASP in das Zytoplasma und darüber hinaus zu einer Reorganisation des Aktin-Filamentsystems, wobei die Spannungsfasern zugunsten einer vermehrten Bildung von Filopodien aufgelöst wurden. Diese Ergebnisse zeigen, daß WIP möglicherweise die N-WASP-vermittelte Aktin-Polymerisation unterstützen kann. Das zweite Aktin-bindende Protein, das in dieser Arbeit untersucht wurde, ist das pro-apoptotische Protein Par-4. Die stabile Expression von Par-4 in CHO-Zellen führte zur Selektion von Zellinien mit partieller Apoptose-Resistenz. Außerdem zeigte sich, daß in 8 von 10 untersuchten Gehirntumorzellinien die Par-4-Expression herunterreguliert war, was ebenfalls zur Apoptose-Resistenz beitragen könnte. Die ektopische Expression von Par 4 führte in diesen Tumorzellinien zur Apoptose, jedoch konnte eine Par-4-vermittelte Inaktivierung der PKC ζ-Signalwege bzw. eine Herunterregulation von Bcl-2 ausgeschlossen werden. Durch Immunfluoreszenz-Analysen und In vitro-Filament-Bindungsversuche wurde gezeigt, daß der N-Terminus von Par-4 die direkte Aktin-Bindung vermittelt, der C-Terminus dagegen für die starke Bindung an das Aktin-Zytoskelett notwendig ist. Durch Deletionsmutanten sowie durch die Zerstörung des Aktin-Zytoskeletts mit Cytochalasin D konnte gezeigt werden, daß die Bindung an Aktin für die Par-4-vermittelte Apoptose wichtig zu sein scheint. Extraktionsversuche mit Triton X-100 haben gezeigt, daß die C-terminale Bindung an Aktin-Filamente von Par-4 pH-sensitiv ist, was möglicherweise auf Konformationsänderungen des C-Terminus zurückzuführen ist. Par-4 könnte daher ein Sensor-Protein darstellen, dessen Interaktion mit seinen Effektorproteinen sowie mit dem Aktin-Zytoskelett durch äußere Bedingungen beeinflußt werden kann

EFP1 is an ER stress-induced glycoprotein which interacts with the pro-apoptotic protein Par-4

We have isolated the rat ortholog of EFP1 (EF-hand binding protein 1) as a novel interaction partner of the pro-apoptotic protein Par-4 (prostate apoptosis response-4). Rat EFP1 contains two thioredoxin domains, the COOH-terminal one harboring a CGFC motif, and has a similar protein domain structure as members of the protein disulfide isomerase (PDI) family. In REF52.2 and CHO cells, EFP1 colocalized with the endoplasmic reticulum (ER) marker PDI. Furthermore, EFP1 possesses catalytic activity as demonstrated by an insulin disulfide reduction assay. Western blot analysis revealed two EFP1 protein bands of approximately 136 and 155 kDa, representing different glycosylation states of the protein. Complex formation between EFP1 and Par-4 was confirmed in vitro and in vivo by co-immunoprecipitation, dot blot overlay and pull-down experiments. In CHO cells, coexpression of EFP1 and Par-4 resulted in enhanced Par-4-mediated apoptosis, which required the catalytic activity of EFP1. Interestingly, EFP1 was specifically upregulated in NIH3T3 cells after induction of ER stress by thapsigargin, tunicamycin, and brefeldin A, but not by agents that induce oxidative stress or ER-independent apoptosis. Furthermore, we could show that the induction of apoptosis by Ca2+ stress-inducing agents was significantly decreased after siRNA oligonucleotide-mediated knockdown of Par-4. Our data suggest that EFP1 might represent a cell-protective enzyme that could play an important role in the decision between survival and initiation of Par-4-mediated apoptosis

Stimulus-Specific Activation and Actin Dependency of Distinct, Spatially Separated ERK1/2 Fractions in A7r5 Smooth Muscle Cells

A proliferative response of smooth muscle cells to activation of extracellular signal regulated kinases 1 and 2 (ERK1/2) has been linked to cardiovascular disease. In fully differentiated smooth muscle, however, ERK1/2 activation can also regulate contraction. Here, we use A7r5 smooth muscle cells, stimulated with 12-deoxyphorbol 13-isobutylate 20-acetate (DPBA) to induce cytoskeletal remodeling or fetal calf serum (FCS) to induce proliferation, to identify factors that determine the outcomes of ERK1/2 activation in smooth muscle. Knock down experiments, immunoprecipitation and proximity ligation assays show that the ERK1/2 scaffold caveolin-1 mediates ERK1/2 activation in response to DPBA, but not FCS, and that ERK1/2 is released from caveolin-1 upon DPBA, but not FCS, stimulation. Conversely, ERK1/2 associated with the actin cytoskeleton is significantly reduced after FCS, but not DPBA stimulation, as determined by Triton X fractionation. Furthermore, cytochalasin treatment inhibits DPBA, but not FCS-induced ERK1/2 phosphorylation, indicating that the actin cytoskeleton is not only a target but also is required for ERK1/2 activation. Our results show that (1) at least two ERK1/2 fractions are regulated separately by specific stimuli, and that (2) the association of ERK1/2 with the actin cytoskeleto

ERK1/2 activation has stimulus-specific effects on the A7r5 cytoskeleton.

<p> (A) ERK1/2 is phosphorylated in response to both, DPBA and FCS. A7r5 cells stimulated with either DPBA or FCS for the indicated time points were processed for western blot analysis of total ERK1/2 and phospo-ERK1/2. After densitometry analysis, no significant differences in ERK1/2 phosphorylation (normalized to total ERK1/2) were detected. (B) A7r5 cells were pre-treated with the MEK inhibitor U0126 for 60 minutes, then stimulated with DPBA or FCS, or left untreated, for additional 60 minutes before preparation of cell lysates. Lysates were analyzed by western blotting and densitometry. Please note that in the presence of the MEK inhibitor, caldesmon phosphorylation in response to DPBA is completely blocked, whereas caldesmon phosphorylation (normalized to tubulin) in response to FCS is reduced by only about 30%. Statistical significance was tested by a two-tailed Student's T-Test (**p<0.01 U0126 vs. control, ^##p<0.01 DPBA vs. FCS). (C) A7r5 cells grown on coverslips were treated as indicated for 60 minutes, then fixed and stained for fluorescence microscopy with phalloidin to visualize actin filaments. Please note the cytoskeletal rearrangements and the appearance of podosomes in the DPBA treated cells (arrows). Scale bar, 20 µm.</p

Stimulus-specific cytoskeletal localization of phospho-ERK1/2 depends on caveolin-1.

<p>A7r5 cells grown on coverslips were transfected with caveolin-1 siRNA and control siRNA. Five days after transfection, cells were stimulated with either DPBA or FCS for 60 minutes, or left untreated. After stimulation, cells were fixed and stained for caveolin-1 (a, e, I, m), phospho-ERK1/2 (b, f, j, n) and DAPI (c, g, k, o); merged images are shown in panels d, h, l, p. Please note that in the caveolin-1 knock down cells, the filamentous phospho-ERK1/2 staining seen after DPBA stimulation is absent in caveolin-1 knock down cells (arrows). Scale bar, 20 µm.</p

The cytoskeletal fraction of ERK1/2 is mobilized upon FCS, but not DPBA, stimulation.

<p>(A) A7r5 cells were treated with either DPBA or FCS and then subjected to subcellular fractionation into cytoskeletal and soluble fractions by Triton X-100 extraction. ERK1/2 subcellular distribution was analyzed by western blotting and densitometry. The graph shows the percentage of total ERK1/2 found in the caveolar and non-caveolar (cytoskeletal) TX-insoluble fractions at late time points of stimulation (30 minutes and 60 minutes). Statistical significance was tested by two-tailed Student's T-Tests (non-caveolar insoluble fraction: *p<0.05 DPBA vs. FCS, **p<0.01 unstimulated vs. FCS; caveolar insoluble fraction: ^#p<0.05 DPBA vs. FCS). (B) A7r5 cells were grown on coverslips were subjected to TX extraction before fixing and staining for phospho-ERK1/2. Please note that filamentous phospho-ERK1/2 staining after DPBA stimulation is TX resistant, while the nuclear and diffuse perinuclear phospho-ERK1/2 staining after FCS stimulation is not. Scale bar, 20 µm. (C) A7r5 cells, pretreated with Cytochalasin D for one hour or control treated (solvent only), were stimulated with either FCS or DPBA, or left unstimulated, for another hour. Lysates were subjected to western blotting with a total ERK1/2 antibody and a phospho-ERK1/2 antibody. The graph shows phospho-ERK1/2 relative to total ERK1/2. Statistical significance was tested by a paired two-tailed Student's T-Test (**p<0.01 Cyto D vs. control, #p<0.05 DPBA vs. FCS, n.s. = not significant).</p

Knock down of caveolin-1 interferes with DPBA induced ERK1/2 phosphorylation.

<p>A7r5 cells were transfected with caveolin-1 siRNA, caveolin-2 siRNA as control or left untransfected. Experiments were performed five days after transfection. (A) Western blot analysis and statistical analysis of densitometry analysis show efficient knock down of caveolin-1. Statistical significance was tested by two-tailed Student's T-Tests (***p<0.001 siRNA vs. control, ^###p<0.001 siRNA vs. untransfected). (B) Five days after siRNA transfection, cells were stimulated with either DPBA or FCS, or left untreated. Lysates were analyzed for ERK1/2 phosphorylation by western blotting and densitometry. The graph shows that after caveolin-1 knock down, the rate of ERK1/2 phosphorylation is significantly reduced in response to DPBA, but not FCS. Statistical significance was tested by two-tailed Student's T-Tests (**p<0.01 siRNA vs. control, ^##p<0.01 DPBA vs. FCS).</p

A caveolar fraction of ERK1/2 is phosphorylated and mobilized in a stimulus-specific manner.

<p>(A) A7r5 cells grown on coverslips were stimulated with DPBA or FCS, or left unstimulated, and then fixed and stained with a caveolin-1 antibody and either an ERK1/2 or phospho-ERK1/2 antibody. Cells were processed for proximity ligation assays (Olink) according to the manufacturer's protocol and then analyzed for signal dots indicative of close proximity. (B and C) A7r5 cells were treated with either DPBA or FCS for the indicated times. Lysates were then subjected to immunoprecipitation with an anti-caveolin-1 antibody. Immunoprecipitates were analyzed for co-immunoprecipitation of (B) phospho-ERK1/2 and (C) total ERK1/2 by western blotting and densitometry. Please note that interaction of caveolin-1 with phospho-ERK1/2 is increased at early time points after DPBA, but not FCS stimulation, whereas interaction with total ERK1/2 is reduced at later time points of DPBA, but not FCS stimulation. Statistical significance was tested by two-tailed Student's T-Tests (*p<0.05, ***p<0.001 DPBA vs. unstimulated; ^#p<0.05, ^##p<0.01, ^###p<0.001 DPBA vs. FCS; ⁺p<0.05 FCS vs. unstimulated).</p