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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 increases Gas6-induced Axl phosphorylation.Rat2 (Rat lanes 1–3) and Rat2/T3V5 cells (Rat/T3, lanes 4–6) were treated with 0, 50, 350 ng/ml Gas6 for 20 min. Detergent cell lysates were prepared and normalized for protein concentration. The samples were divided in two for Tyro3 (panel A) and Axl (panel B) immunoprecipitations (IP). This was followed by SDS-PAGE using 8% gels followed and Western blot analysis. The membranes were probed with anti-phosphotyrosine (α-pTyr) antibodies (PY20 and P99 mixture 1∶3,500) (top, panels A and B). The membranes were stripped and reprobed with α-Tyro3 serum 5424 (1∶3,500) (A, bottom panel) or affinity purified rabbit α-Axl (1∶3,500) (B, bottom panel). These blots are representative of 4 experiments. To determine whether Tyro3 expression affects Axl levels in Rat2 cells (panel C), detergent cell extracts were prepared from Rat2 cells (lane 1), and independently derived stably transfected Rat2/T3V5 cell lines (clone (cl) 25, lane 2; clone 11, lane 3; clone 30, lane 4). SDS-PAGE using 4–20% gels followed by Western blot analysis was performed on these extracts. The membranes were cut at the level of the 66 kDa marker and the top portion was probed with rabbit α-Tyro3 (5424 serum 1∶3,500) or rabbit α-Axl (1∶3,500). The bottom portion of the membranes were blotted with α-GAPDH (1∶500). These blots are representative of 5 experiments. To determine if the levels of Axl phosphorylation depended on the levels of Tyro3 expressed (panel D) Rat2 cells (lanes 1 and 2) and Rat2/T3V5 cell lines cl25 (lanes 3 and 4) and cl30 (lanes 5 and 6) were activated with media only (0) or 350 ng/ml of Gas6 for 10 min. Detergent cell lysates were prepared and normalized for protein concentration. The samples were divided in two for Tyro3 and Axl immunoprecipitations (IP). SDS-PAGE using 8% gels followed by Western blot analysis was performed. The membranes were probed with anti-phosphotyrosine (α-pTyr) antibodies (PY20 and P99 mixture 1∶3,500) (top and third panels). An aliquot of the remaining Axl IPs were reloaded and probed with affinity purified rabbit α-Axl (1∶3,500) (second panel from the top). The membrane corresponding to Tyro3 IP’s was stripped and reprobed with α-Tyro3 serum 5424 (1∶3,500) (bottom panel). These blots are representative of 6 experiments.
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pone-0036800-g004: Tyro3 increases Gas6-induced Axl phosphorylation.Rat2 (Rat lanes 1–3) and Rat2/T3V5 cells (Rat/T3, lanes 4–6) were treated with 0, 50, 350 ng/ml Gas6 for 20 min. Detergent cell lysates were prepared and normalized for protein concentration. The samples were divided in two for Tyro3 (panel A) and Axl (panel B) immunoprecipitations (IP). This was followed by SDS-PAGE using 8% gels followed and Western blot analysis. The membranes were probed with anti-phosphotyrosine (α-pTyr) antibodies (PY20 and P99 mixture 1∶3,500) (top, panels A and B). The membranes were stripped and reprobed with α-Tyro3 serum 5424 (1∶3,500) (A, bottom panel) or affinity purified rabbit α-Axl (1∶3,500) (B, bottom panel). These blots are representative of 4 experiments. To determine whether Tyro3 expression affects Axl levels in Rat2 cells (panel C), detergent cell extracts were prepared from Rat2 cells (lane 1), and independently derived stably transfected Rat2/T3V5 cell lines (clone (cl) 25, lane 2; clone 11, lane 3; clone 30, lane 4). SDS-PAGE using 4–20% gels followed by Western blot analysis was performed on these extracts. The membranes were cut at the level of the 66 kDa marker and the top portion was probed with rabbit α-Tyro3 (5424 serum 1∶3,500) or rabbit α-Axl (1∶3,500). The bottom portion of the membranes were blotted with α-GAPDH (1∶500). These blots are representative of 5 experiments. To determine if the levels of Axl phosphorylation depended on the levels of Tyro3 expressed (panel D) Rat2 cells (lanes 1 and 2) and Rat2/T3V5 cell lines cl25 (lanes 3 and 4) and cl30 (lanes 5 and 6) were activated with media only (0) or 350 ng/ml of Gas6 for 10 min. Detergent cell lysates were prepared and normalized for protein concentration. The samples were divided in two for Tyro3 and Axl immunoprecipitations (IP). SDS-PAGE using 8% gels followed by Western blot analysis was performed. The membranes were probed with anti-phosphotyrosine (α-pTyr) antibodies (PY20 and P99 mixture 1∶3,500) (top and third panels). An aliquot of the remaining Axl IPs were reloaded and probed with affinity purified rabbit α-Axl (1∶3,500) (second panel from the top). The membrane corresponding to Tyro3 IP’s was stripped and reprobed with α-Tyro3 serum 5424 (1∶3,500) (bottom panel). These blots are representative of 6 experiments.

Mentions: We compared the ability of Gas6 to induce phosphorylation of Tyro3 (Fig. 4A) and Axl (Fig. 4B) in cells expressing Axl (Rat2) (lanes 1–3 both panels) and cells expressing both receptors (Rat2/T3V5) (lanes 4–6 both panels). Gas6 induced only a modest increase in phosphorylation of Axl in the untransfected cells (see Fig. 4B lanes 1–3 and Fig. 4D lanes 1 and 2), when compared to the higher Axl phosphorylation levels detected in the presence of Tyro3 (Fig. 4B lanes 4–6 and Fig. 4D lanes 3–6). To control for the possibility that higher levels of Axl phosphorylation were due to higher levels of its expression, we compared Axl levels across 3 different stably transfected Tyro3 clonal (cl) cell lines exhibiting different levels of Tyro3 expression (Fig. 4C, cl25 lane 2, cl11 lane 3, and cl30 lane 4 ). As shown in Fig. 4C no difference in total Axl levels were detected across the cell lines. Therefore changes in Axl phosphorylation in the presence of Tyro3 cannot be accounted for by changes in Axl expression as also indicated by the data in Fig. 3A. This suggests that increased levels of Axl phosphorylation are caused by differences in the levels of Tyro3 expression. To further test this possibility we used two of the clonal cell lines expressing different levels of Tyro3 described in Fig. 4C and tested whether increasing levels of Tyro3 would result in increased levels of Axl phosphorylation upon Gas6 stimulation. As shown in Fig. 4D, a small but detectable increase in Axl phosphorylation was observed in the absence of Tyro3 (Fig. 4D lanes 1 and 2), but the levels of Axl phosphorylation were much higher when Tyro3 was present (lanes 3–6). Furthermore they were proportional to the amount of Tyro3 present in the cells as observed when comparing the levels of Axl phosphorylation in the lower Tyro3 expressing clone (cl25 lanes 3 and 4) to the high Tyro3 expressing clone (cl30, lanes 5 and 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 increases Gas6-induced Axl phosphorylation.Rat2 (Rat lanes 1–3) and Rat2/T3V5 cells (Rat/T3, lanes 4–6) were treated with 0, 50, 350 ng/ml Gas6 for 20 min. Detergent cell lysates were prepared and normalized for protein concentration. The samples were divided in two for Tyro3 (panel A) and Axl (panel B) immunoprecipitations (IP). This was followed by SDS-PAGE using 8% gels followed and Western blot analysis. The membranes were probed with anti-phosphotyrosine (α-pTyr) antibodies (PY20 and P99 mixture 1∶3,500) (top, panels A and B). The membranes were stripped and reprobed with α-Tyro3 serum 5424 (1∶3,500) (A, bottom panel) or affinity purified rabbit α-Axl (1∶3,500) (B, bottom panel). These blots are representative of 4 experiments. To determine whether Tyro3 expression affects Axl levels in Rat2 cells (panel C), detergent cell extracts were prepared from Rat2 cells (lane 1), and independently derived stably transfected Rat2/T3V5 cell lines (clone (cl) 25, lane 2; clone 11, lane 3; clone 30, lane 4). SDS-PAGE using 4–20% gels followed by Western blot analysis was performed on these extracts. The membranes were cut at the level of the 66 kDa marker and the top portion was probed with rabbit α-Tyro3 (5424 serum 1∶3,500) or rabbit α-Axl (1∶3,500). The bottom portion of the membranes were blotted with α-GAPDH (1∶500). These blots are representative of 5 experiments. To determine if the levels of Axl phosphorylation depended on the levels of Tyro3 expressed (panel D) Rat2 cells (lanes 1 and 2) and Rat2/T3V5 cell lines cl25 (lanes 3 and 4) and cl30 (lanes 5 and 6) were activated with media only (0) or 350 ng/ml of Gas6 for 10 min. Detergent cell lysates were prepared and normalized for protein concentration. The samples were divided in two for Tyro3 and Axl immunoprecipitations (IP). SDS-PAGE using 8% gels followed by Western blot analysis was performed. The membranes were probed with anti-phosphotyrosine (α-pTyr) antibodies (PY20 and P99 mixture 1∶3,500) (top and third panels). An aliquot of the remaining Axl IPs were reloaded and probed with affinity purified rabbit α-Axl (1∶3,500) (second panel from the top). The membrane corresponding to Tyro3 IP’s was stripped and reprobed with α-Tyro3 serum 5424 (1∶3,500) (bottom panel). These blots are representative of 6 experiments.
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pone-0036800-g004: Tyro3 increases Gas6-induced Axl phosphorylation.Rat2 (Rat lanes 1–3) and Rat2/T3V5 cells (Rat/T3, lanes 4–6) were treated with 0, 50, 350 ng/ml Gas6 for 20 min. Detergent cell lysates were prepared and normalized for protein concentration. The samples were divided in two for Tyro3 (panel A) and Axl (panel B) immunoprecipitations (IP). This was followed by SDS-PAGE using 8% gels followed and Western blot analysis. The membranes were probed with anti-phosphotyrosine (α-pTyr) antibodies (PY20 and P99 mixture 1∶3,500) (top, panels A and B). The membranes were stripped and reprobed with α-Tyro3 serum 5424 (1∶3,500) (A, bottom panel) or affinity purified rabbit α-Axl (1∶3,500) (B, bottom panel). These blots are representative of 4 experiments. To determine whether Tyro3 expression affects Axl levels in Rat2 cells (panel C), detergent cell extracts were prepared from Rat2 cells (lane 1), and independently derived stably transfected Rat2/T3V5 cell lines (clone (cl) 25, lane 2; clone 11, lane 3; clone 30, lane 4). SDS-PAGE using 4–20% gels followed by Western blot analysis was performed on these extracts. The membranes were cut at the level of the 66 kDa marker and the top portion was probed with rabbit α-Tyro3 (5424 serum 1∶3,500) or rabbit α-Axl (1∶3,500). The bottom portion of the membranes were blotted with α-GAPDH (1∶500). These blots are representative of 5 experiments. To determine if the levels of Axl phosphorylation depended on the levels of Tyro3 expressed (panel D) Rat2 cells (lanes 1 and 2) and Rat2/T3V5 cell lines cl25 (lanes 3 and 4) and cl30 (lanes 5 and 6) were activated with media only (0) or 350 ng/ml of Gas6 for 10 min. Detergent cell lysates were prepared and normalized for protein concentration. The samples were divided in two for Tyro3 and Axl immunoprecipitations (IP). SDS-PAGE using 8% gels followed by Western blot analysis was performed. The membranes were probed with anti-phosphotyrosine (α-pTyr) antibodies (PY20 and P99 mixture 1∶3,500) (top and third panels). An aliquot of the remaining Axl IPs were reloaded and probed with affinity purified rabbit α-Axl (1∶3,500) (second panel from the top). The membrane corresponding to Tyro3 IP’s was stripped and reprobed with α-Tyro3 serum 5424 (1∶3,500) (bottom panel). These blots are representative of 6 experiments.
Mentions: We compared the ability of Gas6 to induce phosphorylation of Tyro3 (Fig. 4A) and Axl (Fig. 4B) in cells expressing Axl (Rat2) (lanes 1–3 both panels) and cells expressing both receptors (Rat2/T3V5) (lanes 4–6 both panels). Gas6 induced only a modest increase in phosphorylation of Axl in the untransfected cells (see Fig. 4B lanes 1–3 and Fig. 4D lanes 1 and 2), when compared to the higher Axl phosphorylation levels detected in the presence of Tyro3 (Fig. 4B lanes 4–6 and Fig. 4D lanes 3–6). To control for the possibility that higher levels of Axl phosphorylation were due to higher levels of its expression, we compared Axl levels across 3 different stably transfected Tyro3 clonal (cl) cell lines exhibiting different levels of Tyro3 expression (Fig. 4C, cl25 lane 2, cl11 lane 3, and cl30 lane 4 ). As shown in Fig. 4C no difference in total Axl levels were detected across the cell lines. Therefore changes in Axl phosphorylation in the presence of Tyro3 cannot be accounted for by changes in Axl expression as also indicated by the data in Fig. 3A. This suggests that increased levels of Axl phosphorylation are caused by differences in the levels of Tyro3 expression. To further test this possibility we used two of the clonal cell lines expressing different levels of Tyro3 described in Fig. 4C and tested whether increasing levels of Tyro3 would result in increased levels of Axl phosphorylation upon Gas6 stimulation. As shown in Fig. 4D, a small but detectable increase in Axl phosphorylation was observed in the absence of Tyro3 (Fig. 4D lanes 1 and 2), but the levels of Axl phosphorylation were much higher when Tyro3 was present (lanes 3–6). Furthermore they were proportional to the amount of Tyro3 present in the cells as observed when comparing the levels of Axl phosphorylation in the lower Tyro3 expressing clone (cl25 lanes 3 and 4) to the high Tyro3 expressing clone (cl30, lanes 5 and 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