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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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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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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).

Mentions: We next examined the effects of PI3 kinase on PYK2-mediated apoptosis. The plasmids encoding c-Myc–tagged constitutively active or catalytically inactive P110/PI3 kinases, in which a truncated p85 subunit was linked to the catalytic subunit (Hu et al., 1995), were cotransfected in rat-1 cells with wild-type PYK2. Co-expression of the constitutively active PI3 kinase (PI3K) significantly reduced the apoptotic effects of PYK2 (apoptotic index of 0.59; Fig. 8). The reduction in PYK2-induced apoptosis was not observed when the catalytically inactive PI3 kinase (PI3KΔ) was cotransfected into rat-1 cells (Fig. 8), indicating that the catalytic activity of PI3 kinase was required for suppression of apoptosis. The expression of catalytically active (PI3K) and inactive (PI3KΔ) PI3 kinases was examined by immunostaining using antibodies against the c-Myc epitope and p85 subunit of PI3 kinase.


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

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

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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Related In: Results  -  Collection

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

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).
Mentions: We next examined the effects of PI3 kinase on PYK2-mediated apoptosis. The plasmids encoding c-Myc–tagged constitutively active or catalytically inactive P110/PI3 kinases, in which a truncated p85 subunit was linked to the catalytic subunit (Hu et al., 1995), were cotransfected in rat-1 cells with wild-type PYK2. Co-expression of the constitutively active PI3 kinase (PI3K) significantly reduced the apoptotic effects of PYK2 (apoptotic index of 0.59; Fig. 8). The reduction in PYK2-induced apoptosis was not observed when the catalytically inactive PI3 kinase (PI3KΔ) was cotransfected into rat-1 cells (Fig. 8), indicating that the catalytic activity of PI3 kinase was required for suppression of apoptosis. The expression of catalytically active (PI3K) and inactive (PI3KΔ) PI3 kinases was examined by immunostaining using antibodies against the c-Myc epitope and p85 subunit of PI3 kinase.

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