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Cross-phosphorylation, signaling and proliferative functions of the Tyro3 and Axl receptors in Rat2 cells.

Brown JE, Krodel M, Pazos M, Lai C, Prieto AL - PLoS ONE (2012)

Bottom Line: Overexpression of Tyro3 in the Rat2 cell line that expresses Axl, but not Mer or Tyro3, resulted in a 5 fold increase in cell proliferation.Co-immunoprecipitation experiments confirmed that the Axl and Tyro3 receptors are closely associated.These findings show that overexpression of Tyro3 in the presence of Axl promotes cell proliferation, and that co-expression of Axl and Tyro3 can affect the outcome of Gas6-initiated signaling.

View Article: PubMed Central - PubMed

Affiliation: Department of Psychological and Brain Sciences, Indiana University, Bloomington, Indiana, United States of America.

ABSTRACT
The dysregulation of receptor protein tyrosine kinase (RPTK) function can result in changes in cell proliferation, cell growth and metastasis leading to malignant transformation. Among RPTKs, the TAM receptor family composed of three members Tyro3, Axl, and Mer has been recognized to have a prominent role in cell transformation. In this study we analyzed the consequences of Tyro3 overexpression on cell proliferation, activation of signaling pathways and its functional interactions with Axl. Overexpression of Tyro3 in the Rat2 cell line that expresses Axl, but not Mer or Tyro3, resulted in a 5 fold increase in cell proliferation. This increase was partially blocked by inhibitors of the mitogen-activated protein kinase (MAPK) signaling pathway but not by inhibitors of the phosphatidylinositol 3-kinase (PI(3)K) signaling pathway. Consistent with these findings, an increase in ERK1/2 phosphorylation was detected with Tyro3 but not with Axl overexpression. In contrast, activation of Axl stimulated the PI(3)K pathway, which was mitigated by co-expression of Tyro3. The overexpression of Tyro3 enhanced Gas6-mediated Axl phosphorylation, which was not detected upon overexpression of a "kinase dead" form of Tyro3 (kdTyro3). In addition, the overexpression of Axl induced kdTyro3 phosphorylation. Co-immunoprecipitation experiments confirmed that the Axl and Tyro3 receptors are closely associated. These findings show that overexpression of Tyro3 in the presence of Axl promotes cell proliferation, and that co-expression of Axl and Tyro3 can affect the outcome of Gas6-initiated signaling. Furthermore, they demonstrate a functional interaction between the members of the TAM receptor family which can shed light on the molecular mechanisms underlying the functional consequences of TAM receptor activation in cell transformation, neural function, immune function, and reproductive function among others.

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Gas6 induced cell proliferation of Rat2 and Rat2/T3V5 cells.Serum starved Rat2 and Rat2/T3V5 cells were stimulated with 250 ng/ml of Gas6 for 0–72 hrs (panel A). Proliferative activity is expressed as % increase over the optical density (OD) obtained at 0 hrs which was considered 0%. * = a significant increase in OD was observed in the Rat2/T3V5 cells when compared to Rat2 cells at 24, 48 and 72 hrs. p<0.01, two-sample t-test. Each experiment consisted of 4 wells for each Rat2 and Rat2/T3V5. All comparisons for n = 3 experiments. To determine the effectiveness of the signaling pathway inhibitors (panel B), Rat/T3V5 cells were incubated 45 min prior to activation with vehicle (DMSO), 1.5 µM LY294002, or 5.5 µM wortmannin (top panel) or with 1 µM or 10 µM U0126 (bottom panel). The cells were activated with DMEM (-) or 350 ng/ml of Gas6 (+) for 20 min. Detergent cell extracts normalized for protein concentration were separated by SDS-PAGE using 4–20% gels. Western blotting was performed with α-pAKT (top panel), and α-pERK1/2 (bottom panel). The membranes were stripped and the blots were reprobed with α-GAPDH shown beneath each panel as protein loading control. These blots are representative of 3 experiments. To determine the effects of the signaling-pathway inhibitors on Gas6 mediated cell proliferation (panel C), serum starved cells were stimulated with DMEM only (control) or 250 ng/ml of Gas6 for 72 hrs (Gas6) in the absence or presence of the indicated inhibitors. Proliferative activity is expressed as % increase of the optical density (OD). The OD obtained in the absence of addition of Gas6 (control) or inhibitors was considered 100%. Asterisks * denote  =  a significant increase in proliferation in the Gas6 treated cells relative to the untreated controls (*: p<0.05; **: p<0.01) two-sample t-test. The differences between Gas6 treated cells and their respective controls for vehicle only, LY294002 and wortmannin (left panel) were found to be the same at p>0.05. The differences between Gas6 treated cells and their controls for the two conditions, vehicle only, and U0126 (right panels) were different at p<0.01. Comparisons are based on n = 3 for the PI(3)K inhibitor panel (left) and n = 4 for the MAPK inhibitor panel (right).
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pone-0036800-g002: Gas6 induced cell proliferation of Rat2 and Rat2/T3V5 cells.Serum starved Rat2 and Rat2/T3V5 cells were stimulated with 250 ng/ml of Gas6 for 0–72 hrs (panel A). Proliferative activity is expressed as % increase over the optical density (OD) obtained at 0 hrs which was considered 0%. * = a significant increase in OD was observed in the Rat2/T3V5 cells when compared to Rat2 cells at 24, 48 and 72 hrs. p<0.01, two-sample t-test. Each experiment consisted of 4 wells for each Rat2 and Rat2/T3V5. All comparisons for n = 3 experiments. To determine the effectiveness of the signaling pathway inhibitors (panel B), Rat/T3V5 cells were incubated 45 min prior to activation with vehicle (DMSO), 1.5 µM LY294002, or 5.5 µM wortmannin (top panel) or with 1 µM or 10 µM U0126 (bottom panel). The cells were activated with DMEM (-) or 350 ng/ml of Gas6 (+) for 20 min. Detergent cell extracts normalized for protein concentration were separated by SDS-PAGE using 4–20% gels. Western blotting was performed with α-pAKT (top panel), and α-pERK1/2 (bottom panel). The membranes were stripped and the blots were reprobed with α-GAPDH shown beneath each panel as protein loading control. These blots are representative of 3 experiments. To determine the effects of the signaling-pathway inhibitors on Gas6 mediated cell proliferation (panel C), serum starved cells were stimulated with DMEM only (control) or 250 ng/ml of Gas6 for 72 hrs (Gas6) in the absence or presence of the indicated inhibitors. Proliferative activity is expressed as % increase of the optical density (OD). The OD obtained in the absence of addition of Gas6 (control) or inhibitors was considered 100%. Asterisks * denote  =  a significant increase in proliferation in the Gas6 treated cells relative to the untreated controls (*: p<0.05; **: p<0.01) two-sample t-test. The differences between Gas6 treated cells and their respective controls for vehicle only, LY294002 and wortmannin (left panel) were found to be the same at p>0.05. The differences between Gas6 treated cells and their controls for the two conditions, vehicle only, and U0126 (right panels) were different at p<0.01. Comparisons are based on n = 3 for the PI(3)K inhibitor panel (left) and n = 4 for the MAPK inhibitor panel (right).

Mentions: We tested whether the introduction of Tyro3 would promote Gas6-mediated proliferation in Rat2 cells. Concentrations of Gas6 ranging from 0 to 500 ng/ml were evaluated, with a maximal effect achieved between 100 and 250 ng/ml (not shown). These concentrations are within those previously reported to induce Gas6-mediated proliferation [36]. As shown in Fig. 2A, treatment with 250 ng/ml of Gas6 induced a significant increase in cell proliferation of the Rat2/T3V5 cells compared to the Rat2 cells at all times points tested (24, 48 and 72 hrs). At 72 hrs, the increase in optical density was 5 times greater than that observed for the Rat2 cells.


Cross-phosphorylation, signaling and proliferative functions of the Tyro3 and Axl receptors in Rat2 cells.

Brown JE, Krodel M, Pazos M, Lai C, Prieto AL - PLoS ONE (2012)

Gas6 induced cell proliferation of Rat2 and Rat2/T3V5 cells.Serum starved Rat2 and Rat2/T3V5 cells were stimulated with 250 ng/ml of Gas6 for 0–72 hrs (panel A). Proliferative activity is expressed as % increase over the optical density (OD) obtained at 0 hrs which was considered 0%. * = a significant increase in OD was observed in the Rat2/T3V5 cells when compared to Rat2 cells at 24, 48 and 72 hrs. p<0.01, two-sample t-test. Each experiment consisted of 4 wells for each Rat2 and Rat2/T3V5. All comparisons for n = 3 experiments. To determine the effectiveness of the signaling pathway inhibitors (panel B), Rat/T3V5 cells were incubated 45 min prior to activation with vehicle (DMSO), 1.5 µM LY294002, or 5.5 µM wortmannin (top panel) or with 1 µM or 10 µM U0126 (bottom panel). The cells were activated with DMEM (-) or 350 ng/ml of Gas6 (+) for 20 min. Detergent cell extracts normalized for protein concentration were separated by SDS-PAGE using 4–20% gels. Western blotting was performed with α-pAKT (top panel), and α-pERK1/2 (bottom panel). The membranes were stripped and the blots were reprobed with α-GAPDH shown beneath each panel as protein loading control. These blots are representative of 3 experiments. To determine the effects of the signaling-pathway inhibitors on Gas6 mediated cell proliferation (panel C), serum starved cells were stimulated with DMEM only (control) or 250 ng/ml of Gas6 for 72 hrs (Gas6) in the absence or presence of the indicated inhibitors. Proliferative activity is expressed as % increase of the optical density (OD). The OD obtained in the absence of addition of Gas6 (control) or inhibitors was considered 100%. Asterisks * denote  =  a significant increase in proliferation in the Gas6 treated cells relative to the untreated controls (*: p<0.05; **: p<0.01) two-sample t-test. The differences between Gas6 treated cells and their respective controls for vehicle only, LY294002 and wortmannin (left panel) were found to be the same at p>0.05. The differences between Gas6 treated cells and their controls for the two conditions, vehicle only, and U0126 (right panels) were different at p<0.01. Comparisons are based on n = 3 for the PI(3)K inhibitor panel (left) and n = 4 for the MAPK inhibitor panel (right).
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pone-0036800-g002: Gas6 induced cell proliferation of Rat2 and Rat2/T3V5 cells.Serum starved Rat2 and Rat2/T3V5 cells were stimulated with 250 ng/ml of Gas6 for 0–72 hrs (panel A). Proliferative activity is expressed as % increase over the optical density (OD) obtained at 0 hrs which was considered 0%. * = a significant increase in OD was observed in the Rat2/T3V5 cells when compared to Rat2 cells at 24, 48 and 72 hrs. p<0.01, two-sample t-test. Each experiment consisted of 4 wells for each Rat2 and Rat2/T3V5. All comparisons for n = 3 experiments. To determine the effectiveness of the signaling pathway inhibitors (panel B), Rat/T3V5 cells were incubated 45 min prior to activation with vehicle (DMSO), 1.5 µM LY294002, or 5.5 µM wortmannin (top panel) or with 1 µM or 10 µM U0126 (bottom panel). The cells were activated with DMEM (-) or 350 ng/ml of Gas6 (+) for 20 min. Detergent cell extracts normalized for protein concentration were separated by SDS-PAGE using 4–20% gels. Western blotting was performed with α-pAKT (top panel), and α-pERK1/2 (bottom panel). The membranes were stripped and the blots were reprobed with α-GAPDH shown beneath each panel as protein loading control. These blots are representative of 3 experiments. To determine the effects of the signaling-pathway inhibitors on Gas6 mediated cell proliferation (panel C), serum starved cells were stimulated with DMEM only (control) or 250 ng/ml of Gas6 for 72 hrs (Gas6) in the absence or presence of the indicated inhibitors. Proliferative activity is expressed as % increase of the optical density (OD). The OD obtained in the absence of addition of Gas6 (control) or inhibitors was considered 100%. Asterisks * denote  =  a significant increase in proliferation in the Gas6 treated cells relative to the untreated controls (*: p<0.05; **: p<0.01) two-sample t-test. The differences between Gas6 treated cells and their respective controls for vehicle only, LY294002 and wortmannin (left panel) were found to be the same at p>0.05. The differences between Gas6 treated cells and their controls for the two conditions, vehicle only, and U0126 (right panels) were different at p<0.01. Comparisons are based on n = 3 for the PI(3)K inhibitor panel (left) and n = 4 for the MAPK inhibitor panel (right).
Mentions: We tested whether the introduction of Tyro3 would promote Gas6-mediated proliferation in Rat2 cells. Concentrations of Gas6 ranging from 0 to 500 ng/ml were evaluated, with a maximal effect achieved between 100 and 250 ng/ml (not shown). These concentrations are within those previously reported to induce Gas6-mediated proliferation [36]. As shown in Fig. 2A, treatment with 250 ng/ml of Gas6 induced a significant increase in cell proliferation of the Rat2/T3V5 cells compared to the Rat2 cells at all times points tested (24, 48 and 72 hrs). At 72 hrs, the increase in optical density was 5 times greater than that observed for the Rat2 cells.

Bottom Line: Overexpression of Tyro3 in the Rat2 cell line that expresses Axl, but not Mer or Tyro3, resulted in a 5 fold increase in cell proliferation.Co-immunoprecipitation experiments confirmed that the Axl and Tyro3 receptors are closely associated.These findings show that overexpression of Tyro3 in the presence of Axl promotes cell proliferation, and that co-expression of Axl and Tyro3 can affect the outcome of Gas6-initiated signaling.

View Article: PubMed Central - PubMed

Affiliation: Department of Psychological and Brain Sciences, Indiana University, Bloomington, Indiana, United States of America.

ABSTRACT
The dysregulation of receptor protein tyrosine kinase (RPTK) function can result in changes in cell proliferation, cell growth and metastasis leading to malignant transformation. Among RPTKs, the TAM receptor family composed of three members Tyro3, Axl, and Mer has been recognized to have a prominent role in cell transformation. In this study we analyzed the consequences of Tyro3 overexpression on cell proliferation, activation of signaling pathways and its functional interactions with Axl. Overexpression of Tyro3 in the Rat2 cell line that expresses Axl, but not Mer or Tyro3, resulted in a 5 fold increase in cell proliferation. This increase was partially blocked by inhibitors of the mitogen-activated protein kinase (MAPK) signaling pathway but not by inhibitors of the phosphatidylinositol 3-kinase (PI(3)K) signaling pathway. Consistent with these findings, an increase in ERK1/2 phosphorylation was detected with Tyro3 but not with Axl overexpression. In contrast, activation of Axl stimulated the PI(3)K pathway, which was mitigated by co-expression of Tyro3. The overexpression of Tyro3 enhanced Gas6-mediated Axl phosphorylation, which was not detected upon overexpression of a "kinase dead" form of Tyro3 (kdTyro3). In addition, the overexpression of Axl induced kdTyro3 phosphorylation. Co-immunoprecipitation experiments confirmed that the Axl and Tyro3 receptors are closely associated. These findings show that overexpression of Tyro3 in the presence of Axl promotes cell proliferation, and that co-expression of Axl and Tyro3 can affect the outcome of Gas6-initiated signaling. Furthermore, they demonstrate a functional interaction between the members of the TAM receptor family which can shed light on the molecular mechanisms underlying the functional consequences of TAM receptor activation in cell transformation, neural function, immune function, and reproductive function among others.

Show MeSH
Related in: MedlinePlus