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

Vomaske J, Varnum S, Melnychuk R, Smith P, Pasa-Tolic L, Shutthanandan JI, Streblow DN - Herpesviridae (2010)

Bottom Line: 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 mutant Pyk2 F402Y did not abrogate the formation of an active Pyk2 kinase complex, but instead prevented pUS28-mediated activation of RhoA.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: The Vaccine & Gene Therapy Institute, Oregon Health & Science University, Beaverton OR 97006 USA. streblow@ohsu.edu.

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 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.

No MeSH data available.


Related in: MedlinePlus

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.
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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.

Mentions: Having established a functional link between pUS28, Pyk2 and migration in SMC, we hypothesized that pUS28 mediates pro-migratory signaling to Pyk2 in other CMV-susceptible cell types. Although HCMV infection has been associated with poor clinical outcome in glioblastoma multiforme (GBM) patients, a clear mechanistic link between HCMV and GBM tumorigenesis has not been established [5,46,57]. However, many studies have associated aberrant activation of Pyk2 with increased invasiveness in GBM [41,58-60]. To test whether pUS28 can signal to Pyk2 in a glioma model, we examined phosphorylation of Pyk2 at the Y402 site in pUS28 adenovirus infected U373. Interestingly, we observed pUS28-specific phosphorylation of Pyk2 in U373 in response to both CCL5 and CX3CL1 stimulation (Figure 6A). Interestingly, Pyk2 phosphorylation in response to CX3CL1 stimulation is biphasic, with two separate peaks of phosphorlyation, the first at 2.5 and second at 10 min post-ligand addition. This biphasic pattern of phosphorylation at the Y402 site has been observed in other systems [37].


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

Vomaske J, Varnum S, Melnychuk R, Smith P, Pasa-Tolic L, Shutthanandan JI, Streblow DN - Herpesviridae (2010)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

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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.
Mentions: Having established a functional link between pUS28, Pyk2 and migration in SMC, we hypothesized that pUS28 mediates pro-migratory signaling to Pyk2 in other CMV-susceptible cell types. Although HCMV infection has been associated with poor clinical outcome in glioblastoma multiforme (GBM) patients, a clear mechanistic link between HCMV and GBM tumorigenesis has not been established [5,46,57]. However, many studies have associated aberrant activation of Pyk2 with increased invasiveness in GBM [41,58-60]. To test whether pUS28 can signal to Pyk2 in a glioma model, we examined phosphorylation of Pyk2 at the Y402 site in pUS28 adenovirus infected U373. Interestingly, we observed pUS28-specific phosphorylation of Pyk2 in U373 in response to both CCL5 and CX3CL1 stimulation (Figure 6A). Interestingly, Pyk2 phosphorylation in response to CX3CL1 stimulation is biphasic, with two separate peaks of phosphorlyation, the first at 2.5 and second at 10 min post-ligand addition. This biphasic pattern of phosphorylation at the Y402 site has been observed in other systems [37].

Bottom Line: 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 mutant Pyk2 F402Y did not abrogate the formation of an active Pyk2 kinase complex, but instead prevented pUS28-mediated activation of RhoA.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: The Vaccine & Gene Therapy Institute, Oregon Health & Science University, Beaverton OR 97006 USA. streblow@ohsu.edu.

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 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.

No MeSH data available.


Related in: MedlinePlus