HCMV pUS28 initiates pro-migratory signaling via activation of Pyk2 kinase

Abstract

Background: Human Cytomegalovirus (HCMV) has been implicated in the acceleration of vascular disease and chronic allograft rejection. Recently, the virus has been associated with glioblastoma and other tumors. We have previously shown that the HCMV-encoded chemokine receptor pUS28 mediates smooth muscle cell (SMC) and macrophage motility and this activity has been implicated in the acceleration of vascular disease. pUS28 induced SMC migration involves the activation of the protein tyrosine kinases (PTKs) Src and Focal adhesion kinase as well as the small GTPase RhoA. The PTK Pyk2 has been shown to play a role in cellular migration and formation of cancer, especially glioblastoma. The role of Pyk2 in pUS28 signaling and migration are unknown.

Methods: In the current study, we examined the involvement of the PTK Pyk2 in pUS28-induced cellular motility. We utilized in vitro migration of SMC to determine the requirements for Pyk2 in pUS28 pro-migratory signaling. We performed biochemical analysis of Pyk2 signaling in response to pUS28 activation to determine the mechanisms involved in pUS28 migration. We performed mass spectrometric analysis of Pyk2 complexes to identify novel Pyk2 binding partners.

Results: Expression of a mutant form of Pyk2 lacking the autophosphorylation site (Tyr-402) blocks pUS28-mediated SMC migration in response to CCL5, while the kinase-inactive Pyk2 mutant failed to elicit the same negative effect on migration. pUS28 stimulation with CCL5 results in ligand-dependent and calcium-dependent phosphorylation of Pyk2 Tyr-402 and induced the formation of an active Pyk2 kinase complex containing several novel Pyk2 binding proteins. Expression of the autophosphorylation null mutant Pyk2 F402Y did not abrogate the formation of an active Pyk2 kinase complex, but instead prevented pUS28-mediated activation of RhoA. Additionally, pUS28 activated RhoA via Pyk2 in the U373 glioblastoma cells. Interestingly, the Pyk2 kinase complex in U373 contained several proteins known to participate in glioma tumorigenesis.

Conclusions: These findings represent the first demonstration that pUS28 signals through Pyk2 and that this PTK participates in pUS28-mediated cellular motility via activation of RhoA. Furthermore, these results provide a potential mechanistic link between HCMV-pUS28 and glioblastoma cell activation.

Figure 1

Pyk2 Autophosphorylation Mutant Inhibits HCMV-mediated SMC. (A) Schematic of Pyk2 domain structure with A457K and F402Y mutations annotated. (B) Sub-cellular localization of myc-tagged adenovirus constructs was determined via indirect immunofluorescence staining for the myc epitope. From left Ad-Pyk2 WT, Ad-Pyk2-F402Y and Ad-Pyk2-A457K in lamellipodia of adenovirus-infected PAT1 SMC. Gene expression was driven my co-infection with an adenovirus expressing the Tet-off transactivator (Ad-Trans) (C) SMC migration assays were performed on PAT1 SMC co-infected with HCMV at MOI 10 and adenoviruses expressing control Ad-Trans only or Ad-Trans+Pyk2-WT, Pyk2-A457K, or Pyk2-F402Y at MOI 50 in transwell chambers. At 2 hrs post-infection fresh medium containing 50 ng/ml CCL5 was added to the bottom chamber and cells were allowed to migrate for 48 hrs. Migrated cells in the lower chamber were counted via light microscopy. For each condition, n = 6 from two independent experiments.

Figure 2

pUS28 Signaling Causes Calcium-Dependent Phosphorylation of Pyk2 at Y402. (A) FAK-/- fibroblasts were infected with Ad-Trans only or Ad-Trans+Ad-pUS28 for 18 hrs. Cells were stimulated with 40 ng/ml CCL5 for the indicated times and analyzed via western blot with a phospho-specific Pyk2-Y402 antibody or total Pyk2 antibody. (B) Adenovirus-infected FAK-/- were pre-treated for 30 min with BAPTA-AM to chelate intracellular calcium and then stimulated with 40 ng/ml CCL5 for 5 min. Total cell lysates were analyzed via western blot for phospho-Y402 Pyk2 and total Pyk2. For both experiments, blots were stripped and reprobed to verify pUS28-HA expression. Phospho-specific blots were quantified via densitometry using ImageJ software and are expressed as fold change compared to unstimulated, Ad-Trans infected control.

Figure 3

pUS28 Signaling Causes Formation of an Active Kinase Complex Involving Pyk2. SMC were infected with Ad-Trans and Ad-Pyk2 +/- Ad-pUS28 and stimulated with 20% serum or 40 ng/ml CCL5 for indicated times. Pyk2 was immunoprecipitated using myc antibodies. In vitro kinase reactions were performed on immunoprecipitated material and reactions were loaded on SDS-PAGE, transferred to immobilon-P membranes and visualized via autoradiography. (A) Autoradiogram of in vitro kinase reactions. Total Pyk2 and pUS28 expression was determined by western blot for myc and HA tags, respectively. (B) Densitometric lane plots of results shown in panel A, generated using ImageJ software. The darker curve (overlay) is unstimulated and the lighter curve (background) is 5 min post-stimulation for Pyk2 only stimulated with serum (top) or Pyk2+pUS28 stimulated with CCL5 (bottom). Black arrows indicate bands present only in stimulated samples. Molecular weight markers are shown on the density plot to facilitate comparison to the autoradiogram in panel A. (C) Overall optical density quantification of each lane in Pyk2 in vitro kinase reactions shown in panel A. Values are displayed as percentages compared to unstimulated samples infected with Ad-Trans+Ad-Pyk2.

Figure 4

Pyk2-Associated Proteins are Cell-Type and Signal-Specific. SMC or U373 were infected with Ad-Trans and Ad-Pyk2 +/- Ad-pUS28 and stimulated with 20% serum or 40 ng/ml CCL5 for 0, 5, 10, 15, 30 or 60 min. Pyk2 was immunoprecipitated using myc antibodies. Pyk2 complexes were analyzed via tandem mass spectrometry. Results shown are proteins identified with >3 spectral hits for all peptides. (A) Total hits for Pyk2-associated proteins in RSMC and U373. (B) Proteins for which Pyk2-association was induced over the timecourse of stimulation in RSMC and U373. For data analysis, a protein was considered induced if the average spectral hits for all peptides for timepoints 5, 10, 15, 30 and 60 min was greater than twice the spectral hits for all peptides in unstimulated cells.

Figure 5

Pyk2-F402Y Blocks pUS28 Signaling to RhoA. FAK-/- fibroblasts were infected with Ad-Trans and Ad-pUS28 +/- Pyk2-WT or -F402Y for 18 hrs. Cells were stimulated with 40 ng/ml CCL5 for the indicated times. Lysates were immunoprecipitated with Rhotekin-RBD-GST Agarose and analyzed by western blot for RhoA. Input lysates were analyzed for total RhoA and to confirm adenovirus infection efficiency. The percent active RhoA was quantified via ImageJ densitometry of both IP and total lysate western blots.

Figure 6

pUS28 Activates Pyk2 and RhoA in U373 Glioblastoma Cells. (A) U373 cells were infected with Ad-Trans or Ad-pUS28 for 18 hrs. Cells were stimulated with 40 ng/ml CCL5 (left) or 40 ng/ml CX3CL1 (right) for the indicated times and analyzed via western blot with a phospho-specific Pyk2-Y402 antibody or total Pyk2 antibody. (B) U373 were infected with Ad-Trans, Ad-pUS28 or Ad-pUS28 + Pyk2 WT or F402Y for 18 hrs. Cells were stimulated with 40 ng/ml CCL5 for the indicated times. Lysates were immunoprecipitated with Rhotekin-RBD-GST Agarose and analyzed by western blot for RhoA. Input lysates were analyzed for total RhoA and to confirm adenovirus infection efficiency. The percent active RhoA was quantified via ImageJ densitometry of both IP and total lysate western blots.

Figure 7

pUS28-mediated Pro-migratory Signaling in SMC. Schematic model for known components of US28-mediated pro-migratory signaling in response to CC-chemokine ligands.