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

Mentions: In this paper, we examined the ability of the HCMV-encoded chemokine receptor pUS28 to activate Pyk2 and determined the role of Pyk2 in pUS28 mediated SMC migration (Figure 7). Pyk2 expression is limited to a subset of cell types in vivo including brain, hematopoetic cells, endothelial cells, SMC and fibroblasts. Interestingly, many of these cell types are capable of undergoing migration events in response to various external stimuli including integrin, growth factor, hormone and chemokine-mediated signals. There is very little consensus in the literature regarding the participation of Pyk2 in such migration events. Indeed, requirements for Pyk2 signaling, and downstream effectors of Pyk2 activation are highly cell type and signal type-specific [28].


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-mediated Pro-migratory Signaling in SMC. Schematic model for known components of US28-mediated pro-migratory signaling in response to CC-chemokine ligands.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3050435&req=5

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.
Mentions: In this paper, we examined the ability of the HCMV-encoded chemokine receptor pUS28 to activate Pyk2 and determined the role of Pyk2 in pUS28 mediated SMC migration (Figure 7). Pyk2 expression is limited to a subset of cell types in vivo including brain, hematopoetic cells, endothelial cells, SMC and fibroblasts. Interestingly, many of these cell types are capable of undergoing migration events in response to various external stimuli including integrin, growth factor, hormone and chemokine-mediated signals. There is very little consensus in the literature regarding the participation of Pyk2 in such migration events. Indeed, requirements for Pyk2 signaling, and downstream effectors of Pyk2 activation are highly cell type and signal type-specific [28].

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