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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 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.
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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.

Mentions: To examine the possible effects of Src on PYK2-mediated apoptosis, we expressed PYK2 in LA29 cells, a temperature-sensitive v-Src rat-1 cell line. When PYK2-WT was transiently expressed in LA29 cells grown at permissive temperature (35°C), the apoptotic index was significantly reduced from 0.97 to 0.25 (Fig. 7). No significant inhibition of apoptosis was observed when LA29 cells expressing PYK2 were grown at nonpermissive temperature (39°C) (data not shown), indicating that the tyrosine kinase activity of v-Src was required for this event. To examine whether suppression of apoptosis could be mimicked by c-Src, PYK2 was transiently expressed in mouse 10T1/2 cells stably over-expressing c-Src (5H), as well as its parental 10T1/2 cells. 10T1/2 cells expressing full length PYK2 underwent apoptosis (apoptotic index of 0.95). The apoptotic index was significantly reduced to 0.45 when PYK2 was expressed in the c-Src (5H) cell line (Fig. 7). To determine if the reduced apoptotic index of c-Src was dependent on kinase activity, PYK2 was transiently expressed in cells stably over-expressing either catalytically inactive c-Src (pm430 mutant) or an SH2-defective variant of c-Src (dl155; Wilson et al., 1989). The extent of cell death (apoptotic index of 0.98) mediated by PYK2 in cells expressing catalytically inactive Src, was not appreciably different from the parental 10T1/2 cells (Fig. 5). However, in the SH2-defective c-Src cell line (dl155), the apoptotic index was significantly reduced to 0.55 (Fig. 7). These results suggested that the catalytic activity, but not the SH2 domain of c-Src, appeared to be required for the suppression of PYK2-mediated apoptosis in fibroblasts.


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 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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2139789&req=5

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.
Mentions: To examine the possible effects of Src on PYK2-mediated apoptosis, we expressed PYK2 in LA29 cells, a temperature-sensitive v-Src rat-1 cell line. When PYK2-WT was transiently expressed in LA29 cells grown at permissive temperature (35°C), the apoptotic index was significantly reduced from 0.97 to 0.25 (Fig. 7). No significant inhibition of apoptosis was observed when LA29 cells expressing PYK2 were grown at nonpermissive temperature (39°C) (data not shown), indicating that the tyrosine kinase activity of v-Src was required for this event. To examine whether suppression of apoptosis could be mimicked by c-Src, PYK2 was transiently expressed in mouse 10T1/2 cells stably over-expressing c-Src (5H), as well as its parental 10T1/2 cells. 10T1/2 cells expressing full length PYK2 underwent apoptosis (apoptotic index of 0.95). The apoptotic index was significantly reduced to 0.45 when PYK2 was expressed in the c-Src (5H) cell line (Fig. 7). To determine if the reduced apoptotic index of c-Src was dependent on kinase activity, PYK2 was transiently expressed in cells stably over-expressing either catalytically inactive c-Src (pm430 mutant) or an SH2-defective variant of c-Src (dl155; Wilson et al., 1989). The extent of cell death (apoptotic index of 0.98) mediated by PYK2 in cells expressing catalytically inactive Src, was not appreciably different from the parental 10T1/2 cells (Fig. 5). However, in the SH2-defective c-Src cell line (dl155), the apoptotic index was significantly reduced to 0.55 (Fig. 7). These results suggested that the catalytic activity, but not the SH2 domain of c-Src, appeared to be required for the suppression of PYK2-mediated apoptosis in fibroblasts.

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