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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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Tyro3 modulates MAPK and PI(3)K signaling pathways.As shown in panel A, Rat/T3V5 (Rat/T3, lanes 1–3) and Rat2 cells (Rat, lanes 4–6) were treated with 0, 50, and 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 antibodies directed against α-Tyro3, α-Axl, α-pAKT, αpp70S6K, α-pmTOR, and α-pERK1/2. The membranes were stripped or cut and reprobed with α-ERK1/2 or α-GAPDH and shown beneath each panel as protein loading control. These blots are representative of 5 experiments. As shown in panel B, transiently transfected Rat2/Axl cells (Rat/Axl, lanes 1–3) and Rat2 cells (Rat, lanes 4–6) were treated and processed as above. Western blotting was performed with α-Axl, α-pAKT, and α-pERK1/2. The membranes were cut and reprobed with α-GAPDH shown beneath each panel as protein loading control. These blots are representative of 4 experiments. The total levels of MAPK and PI(3)K signaling pathway molecules was compared in Rat2 cells overexpressing Tyro3 (panel C) and Axl (panel D). For Tyro3 overexpressing cells (panel C), Rat/T3V5 cells (Rat/T3, lanes 1–3), and Rat2 untransfected cells (Rat, lanes 4–6), were treated with 0, 50 and 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 antibodies directed against α-AKT, αp70S6K, α-mTOR. The membranes were cut and reprobed with α-GAPDH shown beneath each panel as protein loading control. For total levels of Tyro3, Axl, and ERK1/2 in Rat2 and Rat2/T3V5 cells, see Fig. 3 A and B. These blots are representative of 4 experiments. For Axl overexpressing cells (panel D), transiently transfected Rat2/Axl cells (Rat/Axl, lanes 1–3) and Rat2 untransfected cells (Rat, lanes 4–6) were treated and processed as above. Western blotting was performed with α-Axl, α-AKT, and α-ERK1/2. The membranes were cut and reprobed with α-GAPDH shown beneath each panel as protein loading control. These blots are representative of 4 experiments.
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pone-0036800-g003: Tyro3 modulates MAPK and PI(3)K signaling pathways.As shown in panel A, Rat/T3V5 (Rat/T3, lanes 1–3) and Rat2 cells (Rat, lanes 4–6) were treated with 0, 50, and 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 antibodies directed against α-Tyro3, α-Axl, α-pAKT, αpp70S6K, α-pmTOR, and α-pERK1/2. The membranes were stripped or cut and reprobed with α-ERK1/2 or α-GAPDH and shown beneath each panel as protein loading control. These blots are representative of 5 experiments. As shown in panel B, transiently transfected Rat2/Axl cells (Rat/Axl, lanes 1–3) and Rat2 cells (Rat, lanes 4–6) were treated and processed as above. Western blotting was performed with α-Axl, α-pAKT, and α-pERK1/2. The membranes were cut and reprobed with α-GAPDH shown beneath each panel as protein loading control. These blots are representative of 4 experiments. The total levels of MAPK and PI(3)K signaling pathway molecules was compared in Rat2 cells overexpressing Tyro3 (panel C) and Axl (panel D). For Tyro3 overexpressing cells (panel C), Rat/T3V5 cells (Rat/T3, lanes 1–3), and Rat2 untransfected cells (Rat, lanes 4–6), were treated with 0, 50 and 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 antibodies directed against α-AKT, αp70S6K, α-mTOR. The membranes were cut and reprobed with α-GAPDH shown beneath each panel as protein loading control. For total levels of Tyro3, Axl, and ERK1/2 in Rat2 and Rat2/T3V5 cells, see Fig. 3 A and B. These blots are representative of 4 experiments. For Axl overexpressing cells (panel D), transiently transfected Rat2/Axl cells (Rat/Axl, lanes 1–3) and Rat2 untransfected cells (Rat, lanes 4–6) were treated and processed as above. Western blotting was performed with α-Axl, α-AKT, and α-ERK1/2. The membranes were cut and reprobed with α-GAPDH shown beneath each panel as protein loading control. These blots are representative of 4 experiments.

Mentions: In order to further identify the signaling requirements for Tyro3-enhanced cell proliferation, we compared the activation of the PI(3)K and MAPK signaling pathways in Rat2/T3V5 cells expressing both Axl and Tyro3 (Fig. 3A, lanes 1–3) with the untransfected Rat2 cells that only express Axl (Fig. 3A, lanes 4–6).


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)

Tyro3 modulates MAPK and PI(3)K signaling pathways.As shown in panel A, Rat/T3V5 (Rat/T3, lanes 1–3) and Rat2 cells (Rat, lanes 4–6) were treated with 0, 50, and 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 antibodies directed against α-Tyro3, α-Axl, α-pAKT, αpp70S6K, α-pmTOR, and α-pERK1/2. The membranes were stripped or cut and reprobed with α-ERK1/2 or α-GAPDH and shown beneath each panel as protein loading control. These blots are representative of 5 experiments. As shown in panel B, transiently transfected Rat2/Axl cells (Rat/Axl, lanes 1–3) and Rat2 cells (Rat, lanes 4–6) were treated and processed as above. Western blotting was performed with α-Axl, α-pAKT, and α-pERK1/2. The membranes were cut and reprobed with α-GAPDH shown beneath each panel as protein loading control. These blots are representative of 4 experiments. The total levels of MAPK and PI(3)K signaling pathway molecules was compared in Rat2 cells overexpressing Tyro3 (panel C) and Axl (panel D). For Tyro3 overexpressing cells (panel C), Rat/T3V5 cells (Rat/T3, lanes 1–3), and Rat2 untransfected cells (Rat, lanes 4–6), were treated with 0, 50 and 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 antibodies directed against α-AKT, αp70S6K, α-mTOR. The membranes were cut and reprobed with α-GAPDH shown beneath each panel as protein loading control. For total levels of Tyro3, Axl, and ERK1/2 in Rat2 and Rat2/T3V5 cells, see Fig. 3 A and B. These blots are representative of 4 experiments. For Axl overexpressing cells (panel D), transiently transfected Rat2/Axl cells (Rat/Axl, lanes 1–3) and Rat2 untransfected cells (Rat, lanes 4–6) were treated and processed as above. Western blotting was performed with α-Axl, α-AKT, and α-ERK1/2. The membranes were cut and reprobed with α-GAPDH shown beneath each panel as protein loading control. These blots are representative of 4 experiments.
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pone-0036800-g003: Tyro3 modulates MAPK and PI(3)K signaling pathways.As shown in panel A, Rat/T3V5 (Rat/T3, lanes 1–3) and Rat2 cells (Rat, lanes 4–6) were treated with 0, 50, and 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 antibodies directed against α-Tyro3, α-Axl, α-pAKT, αpp70S6K, α-pmTOR, and α-pERK1/2. The membranes were stripped or cut and reprobed with α-ERK1/2 or α-GAPDH and shown beneath each panel as protein loading control. These blots are representative of 5 experiments. As shown in panel B, transiently transfected Rat2/Axl cells (Rat/Axl, lanes 1–3) and Rat2 cells (Rat, lanes 4–6) were treated and processed as above. Western blotting was performed with α-Axl, α-pAKT, and α-pERK1/2. The membranes were cut and reprobed with α-GAPDH shown beneath each panel as protein loading control. These blots are representative of 4 experiments. The total levels of MAPK and PI(3)K signaling pathway molecules was compared in Rat2 cells overexpressing Tyro3 (panel C) and Axl (panel D). For Tyro3 overexpressing cells (panel C), Rat/T3V5 cells (Rat/T3, lanes 1–3), and Rat2 untransfected cells (Rat, lanes 4–6), were treated with 0, 50 and 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 antibodies directed against α-AKT, αp70S6K, α-mTOR. The membranes were cut and reprobed with α-GAPDH shown beneath each panel as protein loading control. For total levels of Tyro3, Axl, and ERK1/2 in Rat2 and Rat2/T3V5 cells, see Fig. 3 A and B. These blots are representative of 4 experiments. For Axl overexpressing cells (panel D), transiently transfected Rat2/Axl cells (Rat/Axl, lanes 1–3) and Rat2 untransfected cells (Rat, lanes 4–6) were treated and processed as above. Western blotting was performed with α-Axl, α-AKT, and α-ERK1/2. The membranes were cut and reprobed with α-GAPDH shown beneath each panel as protein loading control. These blots are representative of 4 experiments.
Mentions: In order to further identify the signaling requirements for Tyro3-enhanced cell proliferation, we compared the activation of the PI(3)K and MAPK signaling pathways in Rat2/T3V5 cells expressing both Axl and Tyro3 (Fig. 3A, lanes 1–3) with the untransfected Rat2 cells that only express Axl (Fig. 3A, lanes 4–6).

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