Proteomic-based analysis of components involved in Human cytomegalovirus US28-induced cellular migration
EMSL Project ID
30468
Abstract
Our goal is to identify the molecular mechanisms involved in Pyk2 signaling in response to activation by the Human cytomegalovirus (HCMV)-encoded chemokine receptor US28. We propose to utilize quantitative mass spectrometry analysis to study the effects of US28 signaling on Pyk2 phosphorylation and coupling to other signaling molecules. HCMV is a ubiquitous β-herpesvirus that establishes life-long persistent infections. While HCMV is generally asymptomatic in healthy individuals, the virus is associated with long-term vascular diseases such as atherosclerosis, restenosis, and chronic allograft rejection. However, the mechanism(s) involved in the acceleration of vascular disease by HCMV is unknown. HCMV encodes four chemokine receptors (US27, US28, UL33 and UL78). US28 is the best characterized of these and has been implicated in the development of long-term disease, including vascular disease and malignancies. Chemokine receptors play an important role in vasculopathy development and we hypothesize that CMV chemokine receptors are integral in the acceleration of this disease.We have shown that the HCMV-US28 induces vascular smooth muscle migration (SMC), which is implicated in the development of vascular disease. US28-induced SMC migration involves the activation of protein tyrosine kinases (PTKs) Src and Focal adhesion kinase. Currently, we are examining the involvement of the PTK Pyk2 in US28-induced SMC migration. We have established that Pyk2 is important for US28-mediated SMC migration and specifically the autophosphorylation of Pyk2. Over-expression of the autophosphorylation site mutant of Pyk2 (Tyr-402) blocks SMC migration, while the kinase-inactive mutant failed to cause the same effect. In addition, US28 stimulation with RANTES results in ligand-dependent Pyk2 Tyr-402-phosphorylation, and this phosphorylation event can be abrogated via chelation of intracellular calcium. Similarly, using Pyk2 co-immnoprecipitation/in vitro kinase coupled assay we show that RANTES stimulation of SMC expressing US28 induced the formation of an active kinase complex containing Pyk2. Indeed these findings indicate the importance of phosphorylation of Pyk2 in US28 signaling and migration, however, we still do not know which of the 4 potential Tyr-phosphorylation sites and unknown numbers of Ser/Thr sites on Pyk2 are activated during these processes. In addition, Pyk2 acts as a signaling scaffold but the proteins that interact with Pyk2 are still, for the most part, unknown. Thus, we propose to use the nanoLC-12T FTICR at EMSL to determine the specific protein modifications made to Pyk2 and proteins that complex with Pyk2 following US28 activation. Since we will employ both bottom-up and top-down approaches, we feel our proposed project is ideal for the EMSL Science Theme: "Mass-Spectrometry-Characterization of Combinatorial Posttranslational Modifications by an Integrated Top-down and Bottom-up Proteomics strategy. In addition, our US28/Pyk2 story has yet to be published and we predict that the combined results (current + proposed) should result in a high-impact journal article. Lastly, our findings would be highly valuable for the signaling and proteomics fields and the techniques and results could be applied to other areas of signaling research.
Project Details
Project type
Large-Scale EMSL Research
Start Date
2008-12-05
End Date
2011-09-30
Status
Closed
Released Data Link
Team
Principal Investigator
Team Members
Related Publications
Vomaske J, SM Varnum, R Melnychuk, P Smith, L Pasa-Tolic, JI Shutthanandan, and DN Streblow. 2010. "HCMV pUS28 initiates pro-migratory signaling via activation of Pyk2 kinase." Herpesviridae 1(1):Article No.: 2. doi:10.1186/2042-4280-1-2