Induction of apoptosis after expression of PYK2, a tyrosine kinase structurally related to focal adhesion kinase - PubMed (original) (raw)

W Xiong et al. J Cell Biol. 1997.

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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Figures

Figure 1

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).

Figure 2

Figure 2

Apoptosis of rat-1 cells expressing PYK2. Rat-1 cells were transiently transfected with PYK2 COOH-terminal (PYK2Δ1-680; A, C, E, and G) and wild-type PYK2 (PYK2-WT; B, D, F, and H) plasmids. 24–30 h after transfection, the cells were fixed and immunostained with anti-PYK2 antibodies and propidium iodide (A–D). In a separate experiment, the transfected cells were immunostained using antibodies against PYK2 and labeled with fluorescence-conjugated dUTP using terminal transferase (E–H). Nuclei of rat-1 cells overexpressing PYK2Δ1-680 with normal morphology stained uniformly with propidium iodide, indicating intact nuclei (C, arrows). Propidium iodide staining of rat-1 cells overexpressing PYK2-WT showed apoptotic nuclei (condensed, indicated by arrows) in a fraction of cells (D). While there was no labeling of fluorescence-conjugated dUTP in cells expressing PYK2Δ1-680 with normal morphology (G), there was labeling of dUTP in cells expressing PYK2-WT (H).

Figure 3

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.

Figure 4

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.

Figure 4

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.

Figure 5

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.

Figure 6

Figure 6

Apoptotic activity of PYK2/FAK chimeric fusion proteins. (A) Schematic representation of PYK2, PYK2/FAK1, PYK2/FAK2, and FAK. The shaded block represents the regions of PYK2. The open block represents the regions of FAK. The numbers represent the number of amino acids in FAK or PYK2. The kinase domain sequence is indicated. The constructs tagged with c-Myc are also indicated. NH2-terminal domain of PYK2 (amino acid 2 to 385) was fused with the kinase and COOH-terminal domains of FAK (amino acids 380 to 1,052) in construct PYK2/FAK1. In construct PYK2/FAK2, PYK2 (amino acids 2 to 695) was fused with FAK (amino acids 692 to 1,052). The apoptotic index of these PYK2/FAK chimeric proteins was listed in the right-hand column. (B) Western blotting of HEK 293 cells expressing PYK2, PYK2/FAK1, PYK2/FAK2, and FAK proteins using antibodies against cMyc epitope, goat anti-PYK2 (from Santa Cruz Biotechnology; recognizing epitope in PYK2 NH2-terminal domain), or FAK (2A7, recognizing epitope in FAK COOH-terminal domain). (C) Histograms of the apoptotic index of rat-1 cells transfected with FAK-WT, PYK2/FAK1, PYK2/FAK2, and PYK2-WT.

Figure 7

Figure 7

Suppression of the PYK2-induced apoptosis by overexpression of catalytically active Src. Wild-type PYK2 was transiently transfected into different cell lines stably expressing wild-type Src and various Src mutants. (A) Immunostaining of PYK2 with anti–c-Myc antibodies (9E10 mAB) in rat-1, temperature-sensitive v-Src (ts v-Src, LA29), 10T1/2, c-Src (5HD47), kinase-dead c-Src (430), and SH2-defective c-Src (c-Src dSH2, dl155) cell lines expressing wild-type c-Myc–tagged PYK2. (B) Histograms of apoptotic index mediated by wild-type PYK2 in different Src cell lines.

Figure 7

Figure 7

Suppression of the PYK2-induced apoptosis by overexpression of catalytically active Src. Wild-type PYK2 was transiently transfected into different cell lines stably expressing wild-type Src and various Src mutants. (A) Immunostaining of PYK2 with anti–c-Myc antibodies (9E10 mAB) in rat-1, temperature-sensitive v-Src (ts v-Src, LA29), 10T1/2, c-Src (5HD47), kinase-dead c-Src (430), and SH2-defective c-Src (c-Src dSH2, dl155) cell lines expressing wild-type c-Myc–tagged PYK2. (B) Histograms of apoptotic index mediated by wild-type PYK2 in different Src cell lines.

Figure 8

Figure 8

Suppression of PYK2-induced apoptosis by overexpression of active PI3 kinase and Akt. Full length PYK2 was cotransfected into rat-1 cells with constitutively active and inactive PI3 kinase (PI3K, PI3KΔ) and active and inactive Akt (Akt, AktΔ; at 1:1 molar ratio of DNA). (A) Cotransfected cells were doubly immunostained using antibodies against PYK2 (a, c, e, and g), p85 subunit for PI3 kinase (b and d), and HA-epitope for Akt proteins (f and h). (B) Histograms of the apoptotic index mediated by cotransfection of PYK2-WT with vector alone (1), active PI3 kinase (2), inactive PI3 kinase (3), active Akt (4), inactive Akt (5), crmA (6), and Bcl2 (7).

Figure 8

Figure 8

Suppression of PYK2-induced apoptosis by overexpression of active PI3 kinase and Akt. Full length PYK2 was cotransfected into rat-1 cells with constitutively active and inactive PI3 kinase (PI3K, PI3KΔ) and active and inactive Akt (Akt, AktΔ; at 1:1 molar ratio of DNA). (A) Cotransfected cells were doubly immunostained using antibodies against PYK2 (a, c, e, and g), p85 subunit for PI3 kinase (b and d), and HA-epitope for Akt proteins (f and h). (B) Histograms of the apoptotic index mediated by cotransfection of PYK2-WT with vector alone (1), active PI3 kinase (2), inactive PI3 kinase (3), active Akt (4), inactive Akt (5), crmA (6), and Bcl2 (7).

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