Human Cytomegalovirus Chemokine Receptor US28-induced Smooth Muscle Cell Migration Is Mediated by Focal Adhesion Kinase and Src

Abstract

The human cytomegalovirus-encoded chemokine receptor US28 induces arterial smooth muscle cell (SMC) migration; however, the underlying mechanisms involved in this process are unclear. We have previously shown that US28-mediated SMC migration occurs by a ligand-dependent process that is sensitive to proteintyrosine kinase inhibitors. We demonstrate here that US28 signals through the non-receptor protein-tyrosine kinases Src and focal adhesion kinase (FAK) and that this activity is necessary for US28-mediated SMC migration. In the presence of RANTES (regulated on activation normal T cell expressed and secreted), US28 stimulates the production of a FAK⅐Src kinase complex. Interestingly, Src co-immunoprecipitates with US28 in a ligand-dependent manner. This association occurs earlier than the formation of the FAK⅐Src kinase complex, suggesting that US28 activates Src before FAK. US28 binding to RANTES also promotes the formation of a Grb2⅐FAK complex, which is sensitive to treatment with the Src inhibitor PP2, further highlighting the critical role of Src in US28 activation of FAK. Human cytomegalovirus US28-mediated SMC migration is inhibited by treatment with PP2 and through the expression of either of two dominant negative inhibitors of FAK (F397Y and NH 2 -terminal amino acids 1-401). These findings demonstrate that activation of FAK and Src plays a critical role in US28-mediated signaling and SMC migration. Human cytomegalovirus (HCMV)1 is a ubiquitous herpesvirus that establishes a life-long latent infection after the primary infection has been cleared. Although anti-viral therapy has appreciably reduced disease in transplant and AIDS patients, HCMV is still a significant problem in congenital disease and bone marrow transplant patients. In addition, HCMV has also been associated with long term diseases such as atherosclerosis, restenosis after angioplasty, chronic rejection after solid organ transplantation, and malignancies (1-4). The development of vascular disease involves a chronic inflammatory process with many contributing factors, and of these, chemokines and their receptors have been identified as key mediators. Interestingly, HCMV encodes a CXC chemokine (UL146), a potential CC chemokine (UL128), and four potential chemokine receptors (US27, US28, UL33, and UL78) with the most characterized being US28 (5-8). We have previously reported that US28 mediates arterial smooth muscle cell (SMC) migration and that this activity may contribute to viral dissemination and/or acceleration of vascular disease development (9).US28 contains homology to the CC-chemokine receptors (10) and binds to a broad spectrum of chemokines including the CC chemokines RANTES, MCP-1, MCP-3, and MIP-1␤ and the CX 3 C chemokine Fractalkine/CX3CL1 (11, 12). That CC chemokines fail to compete out Fractalkine binding suggests that Fractalkine binds to additional unique regions of US28 compared with the CC chemokines (11). In 293 cells, RANTES binding to US28 activates ERK1/2 pathways through the Gproteins G␣ i1 and G␣ 16 (13). We have...

Functional analysis of human cytomegalovirus pUS28 mutants in infected cells

“…While there are several reports of agonist independent signalling for cellular receptors, this signalling is usually far more conservative than that exhibited by pUS28 (Carroll et al, 2001;Quitterer et al, 1996;Seifert & Wenzel-Seifert, 2002). Although pUS28-dependent PLC-b signalling does not require chemokine-binding, several other pUS28-dependent signalling pathways, including RhoA and cSrc, do appear to require chemokine (Melnychuk et al, 2004;Minisini et al, 2003;Streblow et al, 2003). Our mutants will be particularly interesting in future studies aimed at understanding how pUS28 activates molecules such as RhoA and c-Src on a more mechanistic level.…”

“…Interaction of C-C chemokines with pUS28 requires the presence of a 6 aa segment between amino acids 11 and 16, as pUS28 mutants deleted for this region are unable to bind chemokine . Although these chemokines bind with high affinity to pUS28, their roles in signalling remain unclear as some pUS28 signalling pathways appear to be chemokinedependent, while others appear to be chemokine-independent (Billstrom et al, 1998;Casarosa et al, 2001;Melnychuk et al, 2004;Minisini et al, 2003;Streblow et al, 2003). HCMV-infected cells secrete CCL5/RANTES and CCL2/MCP-1, which further complicates issues regarding chemokines and pUS28 signalling activity, as the secreted chemokines could bind to pUS28 and activate it in an autocrine manner (Bodaghi et al, 1998;Michelson et al, 1997;Randolph-Habecker et al, 2002;Taylor & Bresnahan, 2006).…”

“…More detailed analyses of the biological and signalling activities of pUS28 are required to gain a better understanding regarding how this protein contributes to viral pathogenesis. pUS28 exhibits significant signalling activity, including the ability to activate the phospholipase C-b (PLC-b), the tyrosine kinase c-Src and the small G protein RhoA (Billstrom et al, 1998;Casarosa et al, 2001;Gao & Murphy, 1994;McLean et al, 2004;Melnychuk et al, 2004;Minisini et al, 2003;Streblow et al, 2003;Waldhoer et al, 2002). pUS28 shares significant homology to C-C chemokine receptors and, accordingly, can bind C-C chemokines, including CCL5/RANTES, CCL2/MCP-1 and CCL4/MIP1b (Bodaghi et al, 1998;Kledal et al, 1998;Kuhn et al, 1995;Neote et al, 1993).…”

Cell transformation mediated by the Epstein–Barr virus G protein-coupled receptor BILF1 is dependent on constitutive signaling

“…Furthermore, both US28 and ORF74 are constitutively active (Arvanitakis et al, 1997;Waldhoer et al, 2002). US28 signals through Ga q , Ga s and Ga 12/13 , further activating kinases and transcription factors, whereas ORF74 signals through Ga q , Ga s and Ga i , virtually activating the same downstream signal transduction pathways as US28 (Smit et al, 2002;Waldhoer et al, 2002;Streblow et al, 2003;McLean et al, 2004). Furthermore, ORF74 and US28 stimulate the secretion of cytokines and growth factors, including vascular endothelial growth factor (VEGF) (Bais et al, 1998;Sodhi et al, 2000;Pati et al, 2001;Maussang et al, 2006).…”

Desensitization of herpesvirus-encoded G protein-coupled receptors

“…US28 can stimulate smooth muscle cell migration through activation of G 12 proteins and Rho, (Melnychuk et al, 2004;Streblow et al, 1999;Streblow et al, 2003). This ability of US28 to promote cellular migration may serve as a means for enhancing HCMV spread throughout the host as well as underlie the link between HCMV and the development of vascular disease.…”