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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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Schematic diagrams of PYK2, PYK2 mutants, FAK,  and FAK mutants. The numbers represent the amino acid residues deleted from wild-type PYK2. The shaded block represents  the kinase domain of PYK2. The putative FAT domain and proline-rich sequences are indicated. The constructs tagged with either c-Myc or GST are also indicated. 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, and listed in the right-hand column.
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Figure 3: Schematic diagrams of PYK2, PYK2 mutants, FAK, and FAK mutants. The numbers represent the amino acid residues deleted from wild-type PYK2. The shaded block represents the kinase domain of PYK2. The putative FAT domain and proline-rich sequences are indicated. The constructs tagged with either c-Myc or GST are also indicated. 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, and listed in the right-hand column.

Mentions: The morphological changes in rat-1 fibroblasts induced by PYK2 expression was indicative of apoptosis. We used both propidium iodide staining and TdT-mediated dUTP-fluorescence nick end labeling (TUNEL) to determine whether DNA condensation and fragmentation, a hallmark of apoptosis, occurred in the cells expressing PYK2 (Fisher, 1994; Muzio et al., 1996). In parallel, the transfected cells were also immunostained with either anti-PYK2 or anti-Myc (9E10) antibodies to monitor the protein expression. Propidium iodide staining of cells expressing wild-type PYK2 demonstrated condensed nuclei (Fig. 2, B and D). This effect was not observed in cells transfected with vector alone (data not shown) or constructs encoding COOH-terminal PYK2 protein (PYK2Δ1-680; Fig. 3, A and C). In addition, PYK2-expressing cells exhibited DNA fragmentation, detected using modified TUNEL (Fig. 2, F and H), whereas no fragmentation was observed in cells transfected with the COOH-terminal PYK2, PYK2Δ1-680 (Fig. 2, E and G). The concomitant induction of cell death with expression of wild-type PYK2 suggested that the expression of PYK2 may be directly responsible for the induction of cell death. Similar morphological changes and apoptosis were observed after transfection of PYK2 into mouse 10T1/2, swiss 3T3, quail QT6, and HEK 293 cells (data not shown). In PC12 cells that express significant levels of endogenous PYK2, no detectable cell death was observed when PYK2 was over-expressed (data not shown).


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

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

Schematic diagrams of PYK2, PYK2 mutants, FAK,  and FAK mutants. The numbers represent the amino acid residues deleted from wild-type PYK2. The shaded block represents  the kinase domain of PYK2. The putative FAT domain and proline-rich sequences are indicated. The constructs tagged with either c-Myc or GST are also indicated. 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, and listed in the right-hand column.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2139789&req=5

Figure 3: Schematic diagrams of PYK2, PYK2 mutants, FAK, and FAK mutants. The numbers represent the amino acid residues deleted from wild-type PYK2. The shaded block represents the kinase domain of PYK2. The putative FAT domain and proline-rich sequences are indicated. The constructs tagged with either c-Myc or GST are also indicated. 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, and listed in the right-hand column.
Mentions: The morphological changes in rat-1 fibroblasts induced by PYK2 expression was indicative of apoptosis. We used both propidium iodide staining and TdT-mediated dUTP-fluorescence nick end labeling (TUNEL) to determine whether DNA condensation and fragmentation, a hallmark of apoptosis, occurred in the cells expressing PYK2 (Fisher, 1994; Muzio et al., 1996). In parallel, the transfected cells were also immunostained with either anti-PYK2 or anti-Myc (9E10) antibodies to monitor the protein expression. Propidium iodide staining of cells expressing wild-type PYK2 demonstrated condensed nuclei (Fig. 2, B and D). This effect was not observed in cells transfected with vector alone (data not shown) or constructs encoding COOH-terminal PYK2 protein (PYK2Δ1-680; Fig. 3, A and C). In addition, PYK2-expressing cells exhibited DNA fragmentation, detected using modified TUNEL (Fig. 2, F and H), whereas no fragmentation was observed in cells transfected with the COOH-terminal PYK2, PYK2Δ1-680 (Fig. 2, E and G). The concomitant induction of cell death with expression of wild-type PYK2 suggested that the expression of PYK2 may be directly responsible for the induction of cell death. Similar morphological changes and apoptosis were observed after transfection of PYK2 into mouse 10T1/2, swiss 3T3, quail QT6, and HEK 293 cells (data not shown). In PC12 cells that express significant levels of endogenous PYK2, no detectable cell death was observed when PYK2 was over-expressed (data not shown).

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