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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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Catalytic and apoptotic activity of wild-type and  mutant PYK2. (A) Schematic  representation of PYK2 and  its mutants. The numbers  represent the amino acid residues mutated 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 c-Myc  are also indicated. The tyrosine  phosphorylation and the apoptotic index of these mutants are listed in the right-hand column. (B) Tyrosine  phosphorylation of wild-type  PYK2 (PYK2-WT), kinase-inactive PYK2 (PYK2-KD),  the putative FAT domain deletion mutant (PYK2Δ936-1009),  the NH2-terminal domain  deletion mutants (PYK2Δ1-88  and PYK2Δ1-416), and the  autophosphorylation site mutant (PYK2-Y402F). Cell  lysates from 293 cells  overexpressing PYK2-WT,  PYK2-KD, PYK2Δ936-1009,  PYK2Δ1-88, PYK2Δ1-416,  and PYK2-Y402F proteins  were immunoprecipitated by  antibodies against PYK2. The  immunoprecipitatied proteins  were then subjected to immunoblotting with the antibodies against phosphotyrosine  (anti-P-tyr) or PYK2 (anti-PYK2). (C) Histograms of  apoptotic index of PYK2Δ936- 1009, PYK2Δ1-88, PYK2-WT,  PYK2-KD, PYK2Δ1-416, and  PYK2-Y402F.
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Figure 5: Catalytic and apoptotic activity of wild-type and mutant PYK2. (A) Schematic representation of PYK2 and its mutants. The numbers represent the amino acid residues mutated 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 c-Myc are also indicated. The tyrosine phosphorylation and the apoptotic index of these mutants are listed in the right-hand column. (B) Tyrosine phosphorylation of wild-type PYK2 (PYK2-WT), kinase-inactive PYK2 (PYK2-KD), the putative FAT domain deletion mutant (PYK2Δ936-1009), the NH2-terminal domain deletion mutants (PYK2Δ1-88 and PYK2Δ1-416), and the autophosphorylation site mutant (PYK2-Y402F). Cell lysates from 293 cells overexpressing PYK2-WT, PYK2-KD, PYK2Δ936-1009, PYK2Δ1-88, PYK2Δ1-416, and PYK2-Y402F proteins were immunoprecipitated by antibodies against PYK2. The immunoprecipitatied proteins were then subjected to immunoblotting with the antibodies against phosphotyrosine (anti-P-tyr) or PYK2 (anti-PYK2). (C) Histograms of apoptotic index of PYK2Δ936- 1009, PYK2Δ1-88, PYK2-WT, PYK2-KD, PYK2Δ1-416, and PYK2-Y402F.

Mentions: The PYK2-WT demonstrated the highest apoptotic activity or apoptotic index (0.97), while the basal apoptotic index in rat-1 cells transfected with control vector alone was 0.09–0.13. A significant reduction of the apoptotic index (0.3–0.5) was observed after transfection with the NH2-terminal domain deletion mutants (e.g., PYK2Δ1-88 and PYK2Δ1-416; Figs. 3 and 4, A and C), suggesting that the NH2-terminal domain of PYK2 was required for maximal apoptotic activity. This conclusion was further supported by the observation that transfection of cells with the NH2-terminal domain alone (PYK2Δ250-1009) was able to induce significant cell death (0.55; Figs. 3 and 4 C). We next determined if kinase activity was required for PYK2-induced apoptosis. The kinase-inactive PYK2 (K457A [PYK2-KD]), containing a lysine (K) to alanine (A) mutation in the ATP-binding site (Fig. 5 A), was transfected into rat-1 cells. The apoptotic index of rat-1 cells expressing the kinase-inactive mutant was 0.6, a significant decrease from that of the wild-type, suggesting that PYK2 catalytic activity was required for the maximal apoptotic activity (Fig. 5 B). This conclusion was further supported by the observation that the apoptotic index of rat-1 cells expressing the autophosphorylation mutant PYK2-Y402F, containing a tyrosine (Y) 402 to (F) mutant (Fig. 5 A), was also significantly reduced to 0.41 (Fig. 5 B). In contrast to the cells expressing PYK2, cells expressing wild-type FAK, an autophosphorylation mutant (Y397F), or the COOH-terminal domain of FAK (FRNK) exhibited a low level of apoptosis (apoptotic index of 0.1–0.3; Figs. 3 and 4 C). The expression levels of PYK2, FAK, FAK-Y397F, and FRNK were similar based on Western blot analysis of extracts from the transfected cells (Fig. 4 B). The transfection efficiencies using these constructs were similar (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)

Catalytic and apoptotic activity of wild-type and  mutant PYK2. (A) Schematic  representation of PYK2 and  its mutants. The numbers  represent the amino acid residues mutated 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 c-Myc  are also indicated. The tyrosine  phosphorylation and the apoptotic index of these mutants are listed in the right-hand column. (B) Tyrosine  phosphorylation of wild-type  PYK2 (PYK2-WT), kinase-inactive PYK2 (PYK2-KD),  the putative FAT domain deletion mutant (PYK2Δ936-1009),  the NH2-terminal domain  deletion mutants (PYK2Δ1-88  and PYK2Δ1-416), and the  autophosphorylation site mutant (PYK2-Y402F). Cell  lysates from 293 cells  overexpressing PYK2-WT,  PYK2-KD, PYK2Δ936-1009,  PYK2Δ1-88, PYK2Δ1-416,  and PYK2-Y402F proteins  were immunoprecipitated by  antibodies against PYK2. The  immunoprecipitatied proteins  were then subjected to immunoblotting with the antibodies against phosphotyrosine  (anti-P-tyr) or PYK2 (anti-PYK2). (C) Histograms of  apoptotic index of PYK2Δ936- 1009, PYK2Δ1-88, PYK2-WT,  PYK2-KD, PYK2Δ1-416, and  PYK2-Y402F.
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Figure 5: Catalytic and apoptotic activity of wild-type and mutant PYK2. (A) Schematic representation of PYK2 and its mutants. The numbers represent the amino acid residues mutated 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 c-Myc are also indicated. The tyrosine phosphorylation and the apoptotic index of these mutants are listed in the right-hand column. (B) Tyrosine phosphorylation of wild-type PYK2 (PYK2-WT), kinase-inactive PYK2 (PYK2-KD), the putative FAT domain deletion mutant (PYK2Δ936-1009), the NH2-terminal domain deletion mutants (PYK2Δ1-88 and PYK2Δ1-416), and the autophosphorylation site mutant (PYK2-Y402F). Cell lysates from 293 cells overexpressing PYK2-WT, PYK2-KD, PYK2Δ936-1009, PYK2Δ1-88, PYK2Δ1-416, and PYK2-Y402F proteins were immunoprecipitated by antibodies against PYK2. The immunoprecipitatied proteins were then subjected to immunoblotting with the antibodies against phosphotyrosine (anti-P-tyr) or PYK2 (anti-PYK2). (C) Histograms of apoptotic index of PYK2Δ936- 1009, PYK2Δ1-88, PYK2-WT, PYK2-KD, PYK2Δ1-416, and PYK2-Y402F.
Mentions: The PYK2-WT demonstrated the highest apoptotic activity or apoptotic index (0.97), while the basal apoptotic index in rat-1 cells transfected with control vector alone was 0.09–0.13. A significant reduction of the apoptotic index (0.3–0.5) was observed after transfection with the NH2-terminal domain deletion mutants (e.g., PYK2Δ1-88 and PYK2Δ1-416; Figs. 3 and 4, A and C), suggesting that the NH2-terminal domain of PYK2 was required for maximal apoptotic activity. This conclusion was further supported by the observation that transfection of cells with the NH2-terminal domain alone (PYK2Δ250-1009) was able to induce significant cell death (0.55; Figs. 3 and 4 C). We next determined if kinase activity was required for PYK2-induced apoptosis. The kinase-inactive PYK2 (K457A [PYK2-KD]), containing a lysine (K) to alanine (A) mutation in the ATP-binding site (Fig. 5 A), was transfected into rat-1 cells. The apoptotic index of rat-1 cells expressing the kinase-inactive mutant was 0.6, a significant decrease from that of the wild-type, suggesting that PYK2 catalytic activity was required for the maximal apoptotic activity (Fig. 5 B). This conclusion was further supported by the observation that the apoptotic index of rat-1 cells expressing the autophosphorylation mutant PYK2-Y402F, containing a tyrosine (Y) 402 to (F) mutant (Fig. 5 A), was also significantly reduced to 0.41 (Fig. 5 B). In contrast to the cells expressing PYK2, cells expressing wild-type FAK, an autophosphorylation mutant (Y397F), or the COOH-terminal domain of FAK (FRNK) exhibited a low level of apoptosis (apoptotic index of 0.1–0.3; Figs. 3 and 4 C). The expression levels of PYK2, FAK, FAK-Y397F, and FRNK were similar based on Western blot analysis of extracts from the transfected cells (Fig. 4 B). The transfection efficiencies using these constructs were similar (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.

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Related in: MedlinePlus