Susanne Illenberger

Phosphorylation of the Vasodilator-stimulated Phosphoprotein Regulates Its Interaction with Actin

The vasodilator-stimulated phosphoprotein (VASP) is a major substrate for cyclic nucleotide-dependent kinases in platelets and other cardiovascular cells. It promotes actin nucleation and binds to actin filaments in vitro and associates with stress fibers in cells. The VASP-actin interaction is salt-sensitive, arguing for electrostatic interactions. Hence, phosphorylation may significantly alter the actin binding properties of VASP. This hypothesis was investigated by analyzing complex formation of recombinant murine VASP with actin after phosphorylation with cAMP-dependent kinase in different assays. cAMP-dependent kinase phosphorylation had a negative effect on both actin nucleation and VASP interaction with actin filaments, with the actin nucleating capacity being more affected than actin filament binding and bundling. Replacing VASP residues known to be phosphorylated in vivo by acidic residues to mimic phosphorylation had similar although less dramatic effects on VASP-actin interactions. In contrast, phosphorylation had no significant effect on VASP oligomerization or its interaction with its known ligands profilin, vinculin, and zyxin. When overexpressing VASP mutants in eukaryotic cells, they all showed targeting to focal contacts and stress fibers. Our results imply that VASP phosphorylation may act as an immediate negative regulator of actin dynamics.

Characterization of the actin binding properties of the vasodilator-stimulated phosphoprotein VASP

The vasodilator‐stimulated phosphoprotein (VASP) colocalizes with the ends of stress fibers in cell‐matrix and cell‐cell contacts. We report here that bacterially expressed murine VASP directly interacts with skeletal muscle actin in several test systems including cosedimentation, viscometry and polymerization assays. It nucleates actin polymerization and tightly bundles actin filaments. The interaction with actin is salt‐sensitive, indicating that the complex formation is primarily based on electrostatic interactions. Actin binding is confined to the C‐terminal domain of VASP (EVH2). This domain, when expressed as a fusion protein with EGFP, associates with stress fibers in transiently transfected cells.

Raver1, a dual compartment protein, is a ligand for PTB/hnRNPI and microfilament attachment proteins

By screening a yeast two-hybrid library with COOH-terminal fragments of vinculin/metavinculin as the bait, we identified a new protein termed raver1. Raver1 is an 80-kD multidomain protein and widely expressed but to varying amounts in different cell lines. In situ and in vitro, raver1 forms complexes with the microfilament-associated proteins vinculin, metavinculin, and α-actinin and colocalizes with vinculin/metavinculin and α-actinin at microfilament attachment sites, such as cell–cell and cell matrix contacts of epithelial cells and fibroblasts, respectively, and in costameres of skeletal muscle. The NH2-terminal part of raver1 contains three RNA recognition motifs with homology to members of the heterogeneous nuclear RNP (hnRNP) family. Raver1 colocalizes with polypyrimidine tract binding protein (PTB)/hnRNPI, a protein involved in RNA splicing of microfilament proteins, in the perinucleolar compartment and forms complexes with PTB/hnRNPI. Hence, raver1 is a dual compartment protein, which is consistent with the presence of nuclear location signal and nuclear export sequence motifs in its sequence. During muscle differentiation, raver1 migrates from the nucleus to the costamere. We propose that raver1 may coordinate RNA processing and targeting as required for microfilament anchoring in specific adhesion sites.

The vasodilator-stimulated phosphoprotein promotes actin polymerisation through direct binding to monomeric actin

The vasodilator‐stimulated phosphoprotein (VASP) functions as a cellular regulator of actin dynamics. VASP may initialise actin polymerisation, suggesting a direct interaction with monomeric actin. The present study demonstrates that VASP directly binds to actin monomers and that complex formation depends on a conserved four amino acid motif in the EVH2 domain. Point mutations within this motif drastically weaken VASP/G‐actin interactions, thereby abolishing any actin‐nucleating activity of VASP. Additionally, actin nucleation was found to depend on VASP oligomerisation since VASP monomers fail to induce the formation of actin filaments. Phosphorylation negatively affects VASP/G‐actin interactions preventing VASP‐induced actin filament formation.

Raver2, a new member of the hnRNP family

Raver2 was identified as a novel member of the hnRNP family based on sequence homology within three RNA recognition motifs and its general domain organization reminiscent of the previously described raver1 protein. Like raver1, raver2 contains two putative nuclear localization signals and a potential nuclear export sequence, and also displays nucleo‐cytoplasmic shuttling in a heterokaryon assay. In glia cells and neurons, raver2 localizes to the nucleus. Moreover, the protein interacts with polypyrimidine tract binding protein (PTB) suggesting that it may participate in PTB‐mediated nuclear functions. In contrast to ubiquitously expressed raver1, raver2 exerts a distinct spatio‐temporal expression pattern during embryogenesis and is essentially restricted to brain, lung, and kidney in the adult mouse.

Raver1 is an integral component of muscle contractile elements

Raver1, a ubiquitously expressed protein, was originally identified as a ligand for metavinculin, the muscle-specific isoform of the microfilament-associated protein vinculin. The protein resides primarily in the nucleus, where it colocalises and may interact with polypyrimidine-tract-binding protein, which is involved in alternative splicing processes. During skeletal muscle differentiation, raver1 translocates to the cytoplasm and eventually targets the Z-line of sarcomeres. Here, it colocalises with metavinculin, vinculin and alpha-actinin, all of which have biochemically been identified as raver1 ligands. To obtain more information about the potential role of raver1 in muscle structure and function, we have investigated its distribution and fine localisation in mouse striated and smooth muscle, by using three monoclonal antibodies that recognise epitopes in different regions of the raver1 protein. Our immunofluorescence and immunoelectron-microscopic results indicate that the cytoplasmic accumulation of raver1 is not confined to skeletal muscle but also occurs in heart and smooth muscle. Unlike vinculin and metavinculin, cytoplasmic raver1 is not restricted to costameres but additionally represents an integral part of the sarcomere. In isolated myofibrils and in ultrathin sections of skeletal muscle, raver1 has been found concentrated at the I-Z-I band. A minor fraction of raver1 is present in the nuclei of all three types of muscle. These data indicate that, during muscle differentiation, raver1 might link gene expression with structural functions of the contractile machinery of muscle.

A conserved peptide motif in Raver2 mediates its interaction with the polypyrimidine tract-binding protein

Raver2 was originally identified as a member of the hnRNP family through database searches revealing three N-terminal RNA recognition motifs (RRMs) bearing highest sequence identity in the RNP sequences to the related hnRNP Raver1. Outside the RRM region, both Raver proteins are quite divergent in sequence except for conserved peptide motifs of the [S/G][I/L]LGxxP consensus sequence. The latter have been implicated in Raver1 binding to the polypyrimidine tract-binding protein (PTB) a regulatory splicing repressor and common ligand of both Raver proteins. In the present study we investigated the association of Raver2 with RNA and PTB in more detail. The isolated RRM domain of Raver2 weakly interacted with ribonucleotides, but the full-length protein failed to directly bind to RNA in vitro. However, trimeric complexes with RNA were formed via binding to PTB. Raver2 harbors two putative PTB binding sequences in the C-terminal half of the protein, whose influence on Raver2-PTB complex formation was analyzed in a mutational approach, replacing critical leucine residues with alanines. While mutation of either sequence motif alone negatively affected Raver2 binding to PTB in vitro, only mutation of the more C-terminally located SLLGEPP motif significantly reduced the recruitment of Raver2 into perinucleolar compartments (PNCs) in HeLa cells. The latter observation was also confirmed for Raver1: out of four sequence motifs matching the PTB binding consensus, mutations in the SLLGEPP motif were the only ones attenuating the recruitment of Raver1 into PNCs. The conserved mode of PTB binding suggests that Raver2, like Raver1, may function as a modulator of PTB activity.