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Induction of apoptosis after expression of PYK2, a tyrosine kinase structurally related to focal adhesion kinase.

Xiong W, Parsons JT - J. Cell Biol. (1997)

Bottom Line: Disruption of the cell-ECM interactions mediated by the integrin receptors results in apoptosis.In addition, it could also be suppressed by overexpressing an ICE or ICE-like proteinase inhibitor, crmA, but not Bcl2.Collectively, our results suggest that PYK2 and FAK, albeit highly homologous in primary structure, appear to have different functions; FAK is required for cell survival, whereas PYK2 induces apoptosis in fibroblasts.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Health Science Center, University of Virginia, Charlottesville, Virginia 22908, USA.

ABSTRACT
Many cells (e.g., epithelial cells) require attachment to the extracellular matrix (ECM) to survive, a phenomenon known as anchorage-dependent cell survival. Disruption of the cell-ECM interactions mediated by the integrin receptors results in apoptosis. Focal adhesion kinase (FAK), a 125-kD protein tyrosine kinase activated by integrin engagement, appears to be involved in mediating cell attachment and survival. Proline-rich tyrosine kinase 2 (PYK2), also known as cellular adhesion kinase beta (CAKbeta) and related adhesion focal tyrosine kinase, is a second member of the FAK subfamily and is activated by an increase in intracellular calcium levels, or treatment with TNFalpha and UV light. However, the function of PYK2 remains largely unknown. In this study, we show that over-expression of PYK2, but not FAK, in rat and mouse fibroblasts leads to apoptotic cell death. Using a series of deletion mutants and chimeric fusion proteins of PYK2/FAK, we determined that the NH2-terminal domain and tyrosine kinase activity of PYK2 were required for the efficient induction of apoptosis. Furthermore, the apoptosis mediated by PYK2 could be suppressed by over-expressing catalytically active v-Src, c-Src, phosphatidylinositol-3-kinase, or Akt/protein kinase B. In addition, it could also be suppressed by overexpressing an ICE or ICE-like proteinase inhibitor, crmA, but not Bcl2. Collectively, our results suggest that PYK2 and FAK, albeit highly homologous in primary structure, appear to have different functions; FAK is required for cell survival, whereas PYK2 induces apoptosis in fibroblasts.

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Morphological changes of rat-1 cells expressing PYK2.  Rat-1 cells were transiently transfected with pCMV vector (A  and B), pCMV-PYK2-WT (C and D), and pCMV-FAK (E and  F) plasmids without (A, C, and E) and with pCMV-β-galactosidase (B, D, and F). 30 h after transfection, the cells were fixed  with 4% paraformaldehyde and stained with antibodies against  c-Myc (9E10) epitope (A, C, and E), or fixed by 0.5% glutaraldehyde and stained for the β-galactosidase activity of the cells  cotransfected with pCMV-β-galactosidase (B, D, and F).
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Figure 1: Morphological changes of rat-1 cells expressing PYK2. Rat-1 cells were transiently transfected with pCMV vector (A and B), pCMV-PYK2-WT (C and D), and pCMV-FAK (E and F) plasmids without (A, C, and E) and with pCMV-β-galactosidase (B, D, and F). 30 h after transfection, the cells were fixed with 4% paraformaldehyde and stained with antibodies against c-Myc (9E10) epitope (A, C, and E), or fixed by 0.5% glutaraldehyde and stained for the β-galactosidase activity of the cells cotransfected with pCMV-β-galactosidase (B, D, and F).

Mentions: To study the function of PYK2, we attempted to generate rat-1 fibroblastic cell lines stably expressing PYK2. After a number of failures, we examined the phenotype of rat-1 cells after transient transfection using a vector carrying cDNA encoding full length PYK2 with an NH2-terminal c-Myc epitope tag under the control of the CMV promoter (PYK2-WT). The expression of PYK2 as well as cell morphology were monitored by immunostaining of the cells using anti-PYK2 antibodies, anti-c-Myc (9E10) monoclonal antibodies, or by β-galactosidase staining of cells that were co-transfected with a reporter plasmid containing the cDNA encoding β-galactosidase (pCMV-β-gal). As shown in Fig. 1, the transfection with a control pCMV vector with or without pCMV-β-gal had no effect on cell morphology (Fig. 1, A and B). However, rat-1 cells expressing the PYK2 protein, identified by both PYK2 or Myc immunostaining or β-galactosidase staining, appeared to be round with condensed cytoplasm, blebbed membrane, and poorly attached to the dish (Fig. 1, C and D). These morphological changes were specific for PYK2- expressing rat-1 cells, since the rat-1 cells overexpressing FAK appeared similar to control cells (Fig. 1, E and F).


Induction of apoptosis after expression of PYK2, a tyrosine kinase structurally related to focal adhesion kinase.

Xiong W, Parsons JT - J. Cell Biol. (1997)

Morphological changes of rat-1 cells expressing PYK2.  Rat-1 cells were transiently transfected with pCMV vector (A  and B), pCMV-PYK2-WT (C and D), and pCMV-FAK (E and  F) plasmids without (A, C, and E) and with pCMV-β-galactosidase (B, D, and F). 30 h after transfection, the cells were fixed  with 4% paraformaldehyde and stained with antibodies against  c-Myc (9E10) epitope (A, C, and E), or fixed by 0.5% glutaraldehyde and stained for the β-galactosidase activity of the cells  cotransfected with pCMV-β-galactosidase (B, D, and F).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2139789&req=5

Figure 1: Morphological changes of rat-1 cells expressing PYK2. Rat-1 cells were transiently transfected with pCMV vector (A and B), pCMV-PYK2-WT (C and D), and pCMV-FAK (E and F) plasmids without (A, C, and E) and with pCMV-β-galactosidase (B, D, and F). 30 h after transfection, the cells were fixed with 4% paraformaldehyde and stained with antibodies against c-Myc (9E10) epitope (A, C, and E), or fixed by 0.5% glutaraldehyde and stained for the β-galactosidase activity of the cells cotransfected with pCMV-β-galactosidase (B, D, and F).
Mentions: To study the function of PYK2, we attempted to generate rat-1 fibroblastic cell lines stably expressing PYK2. After a number of failures, we examined the phenotype of rat-1 cells after transient transfection using a vector carrying cDNA encoding full length PYK2 with an NH2-terminal c-Myc epitope tag under the control of the CMV promoter (PYK2-WT). The expression of PYK2 as well as cell morphology were monitored by immunostaining of the cells using anti-PYK2 antibodies, anti-c-Myc (9E10) monoclonal antibodies, or by β-galactosidase staining of cells that were co-transfected with a reporter plasmid containing the cDNA encoding β-galactosidase (pCMV-β-gal). As shown in Fig. 1, the transfection with a control pCMV vector with or without pCMV-β-gal had no effect on cell morphology (Fig. 1, A and B). However, rat-1 cells expressing the PYK2 protein, identified by both PYK2 or Myc immunostaining or β-galactosidase staining, appeared to be round with condensed cytoplasm, blebbed membrane, and poorly attached to the dish (Fig. 1, C and D). These morphological changes were specific for PYK2- expressing rat-1 cells, since the rat-1 cells overexpressing FAK appeared similar to control cells (Fig. 1, E and F).

Bottom Line: Disruption of the cell-ECM interactions mediated by the integrin receptors results in apoptosis.In addition, it could also be suppressed by overexpressing an ICE or ICE-like proteinase inhibitor, crmA, but not Bcl2.Collectively, our results suggest that PYK2 and FAK, albeit highly homologous in primary structure, appear to have different functions; FAK is required for cell survival, whereas PYK2 induces apoptosis in fibroblasts.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Health Science Center, University of Virginia, Charlottesville, Virginia 22908, USA.

ABSTRACT
Many cells (e.g., epithelial cells) require attachment to the extracellular matrix (ECM) to survive, a phenomenon known as anchorage-dependent cell survival. Disruption of the cell-ECM interactions mediated by the integrin receptors results in apoptosis. Focal adhesion kinase (FAK), a 125-kD protein tyrosine kinase activated by integrin engagement, appears to be involved in mediating cell attachment and survival. Proline-rich tyrosine kinase 2 (PYK2), also known as cellular adhesion kinase beta (CAKbeta) and related adhesion focal tyrosine kinase, is a second member of the FAK subfamily and is activated by an increase in intracellular calcium levels, or treatment with TNFalpha and UV light. However, the function of PYK2 remains largely unknown. In this study, we show that over-expression of PYK2, but not FAK, in rat and mouse fibroblasts leads to apoptotic cell death. Using a series of deletion mutants and chimeric fusion proteins of PYK2/FAK, we determined that the NH2-terminal domain and tyrosine kinase activity of PYK2 were required for the efficient induction of apoptosis. Furthermore, the apoptosis mediated by PYK2 could be suppressed by over-expressing catalytically active v-Src, c-Src, phosphatidylinositol-3-kinase, or Akt/protein kinase B. In addition, it could also be suppressed by overexpressing an ICE or ICE-like proteinase inhibitor, crmA, but not Bcl2. Collectively, our results suggest that PYK2 and FAK, albeit highly homologous in primary structure, appear to have different functions; FAK is required for cell survival, whereas PYK2 induces apoptosis in fibroblasts.

Show MeSH
Related in: MedlinePlus