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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 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.
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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.

Mentions: pUS28-expressing FAK-/- stimulated with CCL5 showed sustained phosphorylation of Pyk2 at Y402. Levels of Pyk2 phosphorlyation were ~2-fold higher in the presence of pUS28 than control Ad-Trans infected cells similarly stimulated. CCL5 stimulation had a small but quantifiable effect on Pyk2 phosphorylation in FAK-/- cells infected with Trans only, we believe this activation is attributable to the effects of calcium gradients produced by the addition of media upon stimulation (Figure 2A). Pyk2 kinase activity has been shown to be dependent on intracellular calcium levels [28-30]. Therefore, we treated pUS28-expressing FAK-/- with BAPTA-AM to chelate intracellular calcium prior to performing signaling assays. The cells were then stimulated with CCL5 and examined for Pyk2 Y402 phosphorylation via western blot. As expected, chelation of intracellular calcium abrogated phosphorylation of Pyk2 in response to pUS28 signaling (Figure 2B) and prevented Pyk2 activation in Ad-Trans controls (data not shown). We observed a low, but measurable, effect of US28 expression on Pyk2 activity in the absence of ligand (Figure 2A and 2B), which could be attributed to either low-level constitutive signaling or minor effects of endogenously expressed ligands.


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

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getmorefigures.php?uid=PMC3050435&req=5

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.
Mentions: pUS28-expressing FAK-/- stimulated with CCL5 showed sustained phosphorylation of Pyk2 at Y402. Levels of Pyk2 phosphorlyation were ~2-fold higher in the presence of pUS28 than control Ad-Trans infected cells similarly stimulated. CCL5 stimulation had a small but quantifiable effect on Pyk2 phosphorylation in FAK-/- cells infected with Trans only, we believe this activation is attributable to the effects of calcium gradients produced by the addition of media upon stimulation (Figure 2A). Pyk2 kinase activity has been shown to be dependent on intracellular calcium levels [28-30]. Therefore, we treated pUS28-expressing FAK-/- with BAPTA-AM to chelate intracellular calcium prior to performing signaling assays. The cells were then stimulated with CCL5 and examined for Pyk2 Y402 phosphorylation via western blot. As expected, chelation of intracellular calcium abrogated phosphorylation of Pyk2 in response to pUS28 signaling (Figure 2B) and prevented Pyk2 activation in Ad-Trans controls (data not shown). We observed a low, but measurable, effect of US28 expression on Pyk2 activity in the absence of ligand (Figure 2A and 2B), which could be attributed to either low-level constitutive signaling or minor effects of endogenously expressed ligands.

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