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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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Apoptotic activities and Western blotting of PYK2,  PYK2 mutants, FAK, and FAK mutants. (A) Western blotting of  HEK 293 cells expressing proteins of PYK2 and PYK2 mutants  using antibodies against PYK2 and c-Myc epitope. The position  of protein molecular weight markers is indicated at the right. (B)  Western blotting of HEK 293 cells expressing proteins of PYK2,  FAK, and FAK mutants using antibodies against c-Myc epitope.  The position of protein molecular weight markers is also indicated at the right. (C) Histograms of the apoptotic index of cells  transfected with constructs encoding PYK2, PYK2 mutants,  FAK, and FAK mutants. Apoptotic index (mean ± SEM) was  determined by counting the apoptotic PYK2 or FAK-expressing  cells (or PYK2-positive) divided by total number of PYK2 or  FAK expression cells.
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Figure 4: Apoptotic activities and Western blotting of PYK2, PYK2 mutants, FAK, and FAK mutants. (A) Western blotting of HEK 293 cells expressing proteins of PYK2 and PYK2 mutants using antibodies against PYK2 and c-Myc epitope. The position of protein molecular weight markers is indicated at the right. (B) Western blotting of HEK 293 cells expressing proteins of PYK2, FAK, and FAK mutants using antibodies against c-Myc epitope. The position of protein molecular weight markers is also indicated at the right. (C) Histograms of the apoptotic index of cells transfected with constructs encoding PYK2, PYK2 mutants, FAK, and FAK mutants. Apoptotic index (mean ± SEM) was determined by counting the apoptotic PYK2 or FAK-expressing cells (or PYK2-positive) divided by total number of PYK2 or FAK expression cells.

Mentions: PYK2 contains a central catalytic kinase domain (from amino acid 419 to 679), flanked by noncatalytic NH2-terminal (amino acids 1 to 418) and COOH-terminal (amino acids 680 to 1009) domains (Fig. 3). Within the COOH-terminal region, there are two proline-rich sequences and a putative FAT domain (Fig. 3). To determine which domains of PYK2 were required for its apoptotic activity, we generated a series of PYK2 deletion mutants, whose structures are summarized in Fig. 3. Mutant proteins were tagged either with GST or a c-Myc epitope at the NH2 terminus. Transfection of individual mutant constructs into HEK 293 cells followed by Western blotting of cell extracts using anti-PYK2, antibodies to the c-Myc epitope or GST, confirmed the expression of mutant proteins of the appropriate molecular sizes (Fig. 4 A). The apoptotic activity of individual mutant proteins was tested by transient transfection of rat-1 cells with each mutant construct in the presence or absence of pCMV β-galactosidase reporter. The “apoptotic index” for each mutant was determined by counting the apoptotic blue cells or apoptotic PYK2-expressing cells and dividing by the total number of transfected cells (total number of blue or PYK2-expressing cells).


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

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

Apoptotic activities and Western blotting of PYK2,  PYK2 mutants, FAK, and FAK mutants. (A) Western blotting of  HEK 293 cells expressing proteins of PYK2 and PYK2 mutants  using antibodies against PYK2 and c-Myc epitope. The position  of protein molecular weight markers is indicated at the right. (B)  Western blotting of HEK 293 cells expressing proteins of PYK2,  FAK, and FAK mutants using antibodies against c-Myc epitope.  The position of protein molecular weight markers is also indicated at the right. (C) Histograms of the apoptotic index of cells  transfected with constructs encoding PYK2, PYK2 mutants,  FAK, and FAK mutants. Apoptotic index (mean ± SEM) was  determined by counting the apoptotic PYK2 or FAK-expressing  cells (or PYK2-positive) divided by total number of PYK2 or  FAK expression cells.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2139789&req=5

Figure 4: Apoptotic activities and Western blotting of PYK2, PYK2 mutants, FAK, and FAK mutants. (A) Western blotting of HEK 293 cells expressing proteins of PYK2 and PYK2 mutants using antibodies against PYK2 and c-Myc epitope. The position of protein molecular weight markers is indicated at the right. (B) Western blotting of HEK 293 cells expressing proteins of PYK2, FAK, and FAK mutants using antibodies against c-Myc epitope. The position of protein molecular weight markers is also indicated at the right. (C) Histograms of the apoptotic index of cells transfected with constructs encoding PYK2, PYK2 mutants, FAK, and FAK mutants. Apoptotic index (mean ± SEM) was determined by counting the apoptotic PYK2 or FAK-expressing cells (or PYK2-positive) divided by total number of PYK2 or FAK expression cells.
Mentions: PYK2 contains a central catalytic kinase domain (from amino acid 419 to 679), flanked by noncatalytic NH2-terminal (amino acids 1 to 418) and COOH-terminal (amino acids 680 to 1009) domains (Fig. 3). Within the COOH-terminal region, there are two proline-rich sequences and a putative FAT domain (Fig. 3). To determine which domains of PYK2 were required for its apoptotic activity, we generated a series of PYK2 deletion mutants, whose structures are summarized in Fig. 3. Mutant proteins were tagged either with GST or a c-Myc epitope at the NH2 terminus. Transfection of individual mutant constructs into HEK 293 cells followed by Western blotting of cell extracts using anti-PYK2, antibodies to the c-Myc epitope or GST, confirmed the expression of mutant proteins of the appropriate molecular sizes (Fig. 4 A). The apoptotic activity of individual mutant proteins was tested by transient transfection of rat-1 cells with each mutant construct in the presence or absence of pCMV β-galactosidase reporter. The “apoptotic index” for each mutant was determined by counting the apoptotic blue cells or apoptotic PYK2-expressing cells and dividing by the total number of transfected cells (total number of blue or PYK2-expressing cells).

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