Characterization of a Novel Interaction Between Vasodilator-Stimulated Phosphoprotein and Abelson Interactor 1 in Human Platelets: A Concerted Computational and Experimental Approach

Abstract

Objective-The goal of this study was systematic profiling of vasodilator-stimulated phosphoprotein (VASP)-Ena/VASP homology 1 (EVH1) interactors in human platelets using a combined in silico and in vitro approach. Methods and Results-Exploiting the information of the comprehensive proteome catalogue in the PlateletWeb database (http://plateletweb.bioapps.biozentrum.uni-wuerzburg.de/PlateletWeb.php), we performed a motif search of all sequences and identified potential target sites of class I EVH1 domains in human platelet proteins. Performing affinity purification with VASP-EVH1 domain and the lysates of platelets, we examined complex partners by mass spectrometry. Combining the results of both analyses, we identified Abelson interactor 1 (Abi-1) as a novel EVH1 domain-specific interaction partner of VASP in human platelets and investigated this interaction by yeast 2-hybrid mutational studies and immunoprecipitation. Immunofluorescence microscopy indicated colocalization of both proteins at the lamellipodia of spread human platelets, suggesting a role in reorganizing the cytoskeleton during spreading. Conclusion-The combination of experimental and computational interactome research has emerged as a valuable tool for the analysis of protein-protein interaction networks and facilitates the discovery and characterization of novel interactions as detailed here for Abi-1 and VASP in human platelets. System biological approaches can be expected to play an important role in basic and clinical platelet research, Key Words: Abi-1 Ⅲ bioinformatics Ⅲ EVH1 domain Ⅲ interactome Ⅲ platelet T he focus of biological research is currently shifting from the investigation of individual genes and proteins toward the analysis of entire biological systems. In connection with this, high-throughput analyses accumulate growing amounts of proteome, transcriptome, and interactome data. Particularly in the field of platelet research, a considerable number of mass spectrometric studies have set out to identify the complete repertoire of proteins expressed in human platelets. 1,2 To make this wealth of information biologically exploitable, we recently established a platelet database integrating proteomic and interaction data sources and assembled a first model of the platelet proteome and interactome from these data. 3 The entire network can be browsed online.Apart from their central physiological role in hemostasis, platelets are involved in inflammatory processes of the immune system and play a role in the pathophysiology of several cardiovascular diseases. Aggregation and spreading of platelets are accompanied by major changes in platelet morphology and in reorganization of the actin cytoskeleton, 4 up to the appearance of actin nodules. It has been proposed that these nodules play a major role in actin dynamics. 5 Defects in key proteins of the cytoskeleton organization, such as Wiskott-Aldrich syndrome protein, result in severe effects associated with impaired platelet function. 6 The importance of actin organization and intracellul...

Abi-1-bridged tyrosine phosphorylation of VASP by Abelson kinase impairs association of VASP to focal adhesions and regulates leukaemic cell adhesion

“…Gel-filtration analysis showed co-fractionation of VASP, c-Abl and Abi-1 at approximately 600 kDa. In addition to the previously identified EVH1 domain [48], we identified the CC domain of VASP as another binding site for Abi-1. The PP region of Abi-1 is critical for this interaction, which plays a crucial role in promoting phosphorylation of VASP by Abl.…”

“…VASP localizes to focal adhesions and to the tips of lamellipodia and filopodia [21]. Others have also reported that VASP and Abi-1 partially co-localize at the tip of lamellipodia in human platelets when cells are stimulated with adenosine diphosphate or thrombin [48]. Therefore c-Abl translocates to the tip of lamellipodia, where VASP is enriched, with the help of Abi-1.…”

“…These results indicate that the CC domain of VASP is the major interaction site with Abi-1, whereas the EVH1 domain also contributes to the efficient interaction. Abi-1 was recently identified as the binding partner of the EVH1 domain of VASP [48]. This previous study concluded that Abi-1 does not interact with the CC domain of VASP, on the basis of negative results in the yeast two-hybrid assay.…”

Time-resolved in silico modeling of fine-tuned cAMP signaling in platelets: feedback loops, titrated phosphorylations and pharmacological modulation

“…Therefore, we focused on VASP, a prominent PKA and PKG substrate and highly connected hub protein. Signal integration by VASP protein relies on the activation of the downstream cytoskeletal regulating signaling cascade [35,47]. Taking into account the two major known PKA/PKG phosphorylation sites, Ser157 and Ser239, we accurately predict and experimentally validate resulting differential activation in quantitative terms of specific PKA mediated phosphorylations on VASP.…”

Systems Biology of Platelet–Vessel Wall Interactions

“…For example, cPlateletWeb (http://plateletweb.bioapps.biozentrum.uni-wuerzburg.de) is an Internet-based platform organizing signaling network [59, 60]. A study based on PlateletWeb found a novel interaction between vasodilator-stimulated phosphoprotein and Abelson interactor 1 in human platelets [61]. Another database is Reactome (http://www.reactome.org), where extensive data have been collected, analyzed, and grouped in different pathways [62].…”