Limits...
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.

Show MeSH

Related in: MedlinePlus

Characterization of anti-TAM specific antibodies and TAM expression in Rat2 cells.To determine whether the antibodies used to immunoprecipitate (IP) and detect Tyro3, Axl, and Mer “TAMS” by Western blotting were specific for each receptor, we used tissues derived from knockout mice for each of the TAMS. Whole brain and spleen detergent extracts were prepared from wild-type (wt) (+/+) C57BL/6 mice and from tyro3−/−, axl−/−, and mer−/− knockout mice. As shown in panel A, After normalization for protein concentration, Tyro3 and Mer were IPed from brain detergent extracts (lanes 1–2 and 5–6 respectively) and Axl was IPed from spleen extracts (lanes 3–4). Tyro3 was IPed using α-FN2, Axl using #AF154 and Mer #AF591. SDS-PAGE was performed using 8% gels followed by Western blot analysis. The membranes were probed with α-Tyro3 (5424 serum 1∶3,000, top panel), affinity purified rabbit α-Axl (serum 1∶3,500, center panel), and rabbit α-Mer (1∶5,000 bottom panel). These antibodies were used to characterize Tyro3, Axl, and Mer expression in Rat2 and Rat2/T3V5 cells as shown in panel B. Detergent cell extracts were prepared from untransfected Rat2 cells (Rat, lane 1), stably Tyro3 transfected Rat2/T3V5 cells (Rat/T3 lane 2), brain tissue extract (lane 3, top and bottom panels) or spleen tissue extract (lane3, center panel). After normalization of tissue extracts for protein concentration, Tyro3, Axl and Mer were immunoprecipitated (IPed) (see Fig. 1A for antibodies). SDS-PAGE was performed using 8% gels followed by Western blot analysis. The membranes were probed against Tyro3 (top panel), Axl (center panel), and Mer (bottom panel), as described in Fig. 1A. The observed differences in Tyro3 molecular weight depending on the source are due in part by N-linked glycosylation as shown in panel C. Detergent extracts were prepared from Rat2/T3V5 cells (Rat/T3, lanes 1 and 2), and adult rat brain tissue (brain, lanes 3 and 4). Cell and tissue extracts were incubated overnight without (−) or with (+) PNGase F. SDS-PAGE was performed using 8% gels followed by Western blot analysis using α-Tyro3 antibodies.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3351477&req=5

pone-0036800-g001: Characterization of anti-TAM specific antibodies and TAM expression in Rat2 cells.To determine whether the antibodies used to immunoprecipitate (IP) and detect Tyro3, Axl, and Mer “TAMS” by Western blotting were specific for each receptor, we used tissues derived from knockout mice for each of the TAMS. Whole brain and spleen detergent extracts were prepared from wild-type (wt) (+/+) C57BL/6 mice and from tyro3−/−, axl−/−, and mer−/− knockout mice. As shown in panel A, After normalization for protein concentration, Tyro3 and Mer were IPed from brain detergent extracts (lanes 1–2 and 5–6 respectively) and Axl was IPed from spleen extracts (lanes 3–4). Tyro3 was IPed using α-FN2, Axl using #AF154 and Mer #AF591. SDS-PAGE was performed using 8% gels followed by Western blot analysis. The membranes were probed with α-Tyro3 (5424 serum 1∶3,000, top panel), affinity purified rabbit α-Axl (serum 1∶3,500, center panel), and rabbit α-Mer (1∶5,000 bottom panel). These antibodies were used to characterize Tyro3, Axl, and Mer expression in Rat2 and Rat2/T3V5 cells as shown in panel B. Detergent cell extracts were prepared from untransfected Rat2 cells (Rat, lane 1), stably Tyro3 transfected Rat2/T3V5 cells (Rat/T3 lane 2), brain tissue extract (lane 3, top and bottom panels) or spleen tissue extract (lane3, center panel). After normalization of tissue extracts for protein concentration, Tyro3, Axl and Mer were immunoprecipitated (IPed) (see Fig. 1A for antibodies). SDS-PAGE was performed using 8% gels followed by Western blot analysis. The membranes were probed against Tyro3 (top panel), Axl (center panel), and Mer (bottom panel), as described in Fig. 1A. The observed differences in Tyro3 molecular weight depending on the source are due in part by N-linked glycosylation as shown in panel C. Detergent extracts were prepared from Rat2/T3V5 cells (Rat/T3, lanes 1 and 2), and adult rat brain tissue (brain, lanes 3 and 4). Cell and tissue extracts were incubated overnight without (−) or with (+) PNGase F. SDS-PAGE was performed using 8% gels followed by Western blot analysis using α-Tyro3 antibodies.

Mentions: We first characterized antibodies against individual TAM receptors to determine their specificity. Tyro3 and Mer immunoprecipitates were prepared from total brain extracts obtained from wild-type (wt), tyro3−/− and mer−/− knockout mice, while immunoprecipitates for Axl were prepared from wt and axl−/− knockout mouse spleens. Western blotting was performed with immunoprecipitates of Tyro3, Axl and Mer, using antibodies directed against each of these receptors. As shown in Fig. 1A (top panel), anti-Tyro3 antibodies only recognize Tyro3 in wt (+/+) extracts (lane 1) but not in those derived from the tyro3−/− mice (lane 2). Furthermore this antibody did not recognize proteins immunoprecipitated with the anti-Axl (lanes 3–4) and anti-Mer antibodies (lanes 5–6). Similarly, anti-Axl and anti-Mer antibodies only recognized bands corresponding to their cognate receptors (middle and bottom panels respectively). These results indicated that the antibodies utilized for immunoprecipitation and Western blotting are specific for each receptor of the TAM family.


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)

Characterization of anti-TAM specific antibodies and TAM expression in Rat2 cells.To determine whether the antibodies used to immunoprecipitate (IP) and detect Tyro3, Axl, and Mer “TAMS” by Western blotting were specific for each receptor, we used tissues derived from knockout mice for each of the TAMS. Whole brain and spleen detergent extracts were prepared from wild-type (wt) (+/+) C57BL/6 mice and from tyro3−/−, axl−/−, and mer−/− knockout mice. As shown in panel A, After normalization for protein concentration, Tyro3 and Mer were IPed from brain detergent extracts (lanes 1–2 and 5–6 respectively) and Axl was IPed from spleen extracts (lanes 3–4). Tyro3 was IPed using α-FN2, Axl using #AF154 and Mer #AF591. SDS-PAGE was performed using 8% gels followed by Western blot analysis. The membranes were probed with α-Tyro3 (5424 serum 1∶3,000, top panel), affinity purified rabbit α-Axl (serum 1∶3,500, center panel), and rabbit α-Mer (1∶5,000 bottom panel). These antibodies were used to characterize Tyro3, Axl, and Mer expression in Rat2 and Rat2/T3V5 cells as shown in panel B. Detergent cell extracts were prepared from untransfected Rat2 cells (Rat, lane 1), stably Tyro3 transfected Rat2/T3V5 cells (Rat/T3 lane 2), brain tissue extract (lane 3, top and bottom panels) or spleen tissue extract (lane3, center panel). After normalization of tissue extracts for protein concentration, Tyro3, Axl and Mer were immunoprecipitated (IPed) (see Fig. 1A for antibodies). SDS-PAGE was performed using 8% gels followed by Western blot analysis. The membranes were probed against Tyro3 (top panel), Axl (center panel), and Mer (bottom panel), as described in Fig. 1A. The observed differences in Tyro3 molecular weight depending on the source are due in part by N-linked glycosylation as shown in panel C. Detergent extracts were prepared from Rat2/T3V5 cells (Rat/T3, lanes 1 and 2), and adult rat brain tissue (brain, lanes 3 and 4). Cell and tissue extracts were incubated overnight without (−) or with (+) PNGase F. SDS-PAGE was performed using 8% gels followed by Western blot analysis using α-Tyro3 antibodies.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3351477&req=5

pone-0036800-g001: Characterization of anti-TAM specific antibodies and TAM expression in Rat2 cells.To determine whether the antibodies used to immunoprecipitate (IP) and detect Tyro3, Axl, and Mer “TAMS” by Western blotting were specific for each receptor, we used tissues derived from knockout mice for each of the TAMS. Whole brain and spleen detergent extracts were prepared from wild-type (wt) (+/+) C57BL/6 mice and from tyro3−/−, axl−/−, and mer−/− knockout mice. As shown in panel A, After normalization for protein concentration, Tyro3 and Mer were IPed from brain detergent extracts (lanes 1–2 and 5–6 respectively) and Axl was IPed from spleen extracts (lanes 3–4). Tyro3 was IPed using α-FN2, Axl using #AF154 and Mer #AF591. SDS-PAGE was performed using 8% gels followed by Western blot analysis. The membranes were probed with α-Tyro3 (5424 serum 1∶3,000, top panel), affinity purified rabbit α-Axl (serum 1∶3,500, center panel), and rabbit α-Mer (1∶5,000 bottom panel). These antibodies were used to characterize Tyro3, Axl, and Mer expression in Rat2 and Rat2/T3V5 cells as shown in panel B. Detergent cell extracts were prepared from untransfected Rat2 cells (Rat, lane 1), stably Tyro3 transfected Rat2/T3V5 cells (Rat/T3 lane 2), brain tissue extract (lane 3, top and bottom panels) or spleen tissue extract (lane3, center panel). After normalization of tissue extracts for protein concentration, Tyro3, Axl and Mer were immunoprecipitated (IPed) (see Fig. 1A for antibodies). SDS-PAGE was performed using 8% gels followed by Western blot analysis. The membranes were probed against Tyro3 (top panel), Axl (center panel), and Mer (bottom panel), as described in Fig. 1A. The observed differences in Tyro3 molecular weight depending on the source are due in part by N-linked glycosylation as shown in panel C. Detergent extracts were prepared from Rat2/T3V5 cells (Rat/T3, lanes 1 and 2), and adult rat brain tissue (brain, lanes 3 and 4). Cell and tissue extracts were incubated overnight without (−) or with (+) PNGase F. SDS-PAGE was performed using 8% gels followed by Western blot analysis using α-Tyro3 antibodies.
Mentions: We first characterized antibodies against individual TAM receptors to determine their specificity. Tyro3 and Mer immunoprecipitates were prepared from total brain extracts obtained from wild-type (wt), tyro3−/− and mer−/− knockout mice, while immunoprecipitates for Axl were prepared from wt and axl−/− knockout mouse spleens. Western blotting was performed with immunoprecipitates of Tyro3, Axl and Mer, using antibodies directed against each of these receptors. As shown in Fig. 1A (top panel), anti-Tyro3 antibodies only recognize Tyro3 in wt (+/+) extracts (lane 1) but not in those derived from the tyro3−/− mice (lane 2). Furthermore this antibody did not recognize proteins immunoprecipitated with the anti-Axl (lanes 3–4) and anti-Mer antibodies (lanes 5–6). Similarly, anti-Axl and anti-Mer antibodies only recognized bands corresponding to their cognate receptors (middle and bottom panels respectively). These results indicated that the antibodies utilized for immunoprecipitation and Western blotting are specific for each receptor of the TAM family.

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