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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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Axl cross-phosphorylates Tyro3 and both receptors co-immunoprecipitate.Wild-type and kinase dead forms of Tyro3 were tested for their ability to auto-phosphorylate (panel A) and phosphorylate Axl (panel B). Rat2 cells were transiently transfected kinase dead (kd)Tyro3 (kdT3, lane 2) or with wild-type (wt) Tyro3 (wtT3, lane 3). The cells were activated with 350 ng/ml of Gas6 for 20 min. After protein normalization the extracts were divided in two, for Tyro3 immunoprecipitation (IP) (panel A) and for Axl IP (panel B). After SDS-PAGE using 8% gels and Western blotting, 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 reblotted with α-Tyro3 serum 5424 (1∶3,500, α-Tyro3) (panel A bottom) or re-probed with α-Axl (1∶3,500) (panel B bottom). These blots (panels A and B) are representative of 4 experiments. To determine if Axl can induce Tyro3 phosphorylation (panel C) Rat2 cells were transiently transfected with vectors encoding lacz (lane 1), kdTyro3 (kdT3, lane 2), wtTyro3 (wtT3, lane 3) or doubly transfected with lacz/Axl (lane 4), kdTyro3/Axl (kdT3/Axl,lane 5) and wtTyro3/Axl (wtT3/Axl, lane 6).The cells were activated with 350 ng/ml of Gas6 for 20 min. After protein normalization the extracts were divided in two, for Tyro3 immunoprecipitation (IP) (top and center panels) and for Axl IP (bottom panel). After SDS-PAGE in 8% gels and transfer, the membranes were probed with anti-phosphotyrosine (α-pTyr) (PY20 and P99 mixture 1∶3,500) antibodies (top panel), and rabbit α-Axl (bottom panel). The membrane probed with α-pTyr (Tyro3 IPs) was re-probed with α-Tyro3 (center panel). These blots are representative of 5 experiments. To determine whether Tyro3 and Axl co-immunoprecipitate, (panel D) Rat2/T3V5 cells were activated with 350 ng/ml Gas6 for 10 min. Detergent extracts were normalized and divided in two, for Tyro3 immunoprecipitation (IP) using α-FN2 Tyro3 antibodies (IP Tyro3, lanes 1 and 3) and for Axl IP using mouse monocolonal α-Axl antibodies (IP Axl, lanes 2 and 4). The samples were separated by SDS-PAGE using 6% gels and blotted with α-Tyro3 serum 5424 (1∶3,500) (lanes 1 and 2) or rabbit α-Axl antibodies (1∶3,500) (lanes 3 and 4). These blots are representative of 4 experiments.
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pone-0036800-g005: Axl cross-phosphorylates Tyro3 and both receptors co-immunoprecipitate.Wild-type and kinase dead forms of Tyro3 were tested for their ability to auto-phosphorylate (panel A) and phosphorylate Axl (panel B). Rat2 cells were transiently transfected kinase dead (kd)Tyro3 (kdT3, lane 2) or with wild-type (wt) Tyro3 (wtT3, lane 3). The cells were activated with 350 ng/ml of Gas6 for 20 min. After protein normalization the extracts were divided in two, for Tyro3 immunoprecipitation (IP) (panel A) and for Axl IP (panel B). After SDS-PAGE using 8% gels and Western blotting, 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 reblotted with α-Tyro3 serum 5424 (1∶3,500, α-Tyro3) (panel A bottom) or re-probed with α-Axl (1∶3,500) (panel B bottom). These blots (panels A and B) are representative of 4 experiments. To determine if Axl can induce Tyro3 phosphorylation (panel C) Rat2 cells were transiently transfected with vectors encoding lacz (lane 1), kdTyro3 (kdT3, lane 2), wtTyro3 (wtT3, lane 3) or doubly transfected with lacz/Axl (lane 4), kdTyro3/Axl (kdT3/Axl,lane 5) and wtTyro3/Axl (wtT3/Axl, lane 6).The cells were activated with 350 ng/ml of Gas6 for 20 min. After protein normalization the extracts were divided in two, for Tyro3 immunoprecipitation (IP) (top and center panels) and for Axl IP (bottom panel). After SDS-PAGE in 8% gels and transfer, the membranes were probed with anti-phosphotyrosine (α-pTyr) (PY20 and P99 mixture 1∶3,500) antibodies (top panel), and rabbit α-Axl (bottom panel). The membrane probed with α-pTyr (Tyro3 IPs) was re-probed with α-Tyro3 (center panel). These blots are representative of 5 experiments. To determine whether Tyro3 and Axl co-immunoprecipitate, (panel D) Rat2/T3V5 cells were activated with 350 ng/ml Gas6 for 10 min. Detergent extracts were normalized and divided in two, for Tyro3 immunoprecipitation (IP) using α-FN2 Tyro3 antibodies (IP Tyro3, lanes 1 and 3) and for Axl IP using mouse monocolonal α-Axl antibodies (IP Axl, lanes 2 and 4). The samples were separated by SDS-PAGE using 6% gels and blotted with α-Tyro3 serum 5424 (1∶3,500) (lanes 1 and 2) or rabbit α-Axl antibodies (1∶3,500) (lanes 3 and 4). These blots are representative of 4 experiments.

Mentions: In order to determine if the kinase activity of Tyro3 is required to induce Axl phosphorylation, we generated a kinase inactive or “kinase dead” construct (kdTyro3) by mutating K535 to M (K535M). This results in a receptor that is unable to undergo autophosphorylation [44], but that can still be trans-phosphorylated. As shown in Fig. 5A lane 2, we failed to detect tyrosine phosphorylation of the kdTyro3 mutant when activated by Gas6 in contrast to the sharp increases in phosphotyrosine levels observed in the wild-type (wt) Tyro3 construct (compare kdT3, Fig. 5 lane 2 with wtT3, lane 3). We then compared Axl phosphorylation levels (Fig. 5B) in Rat2 cells transiently transfected with either kdTyro3 (lane 2) or wtTyro3 (lane 3). As shown in Fig. 5B, Axl phosphorylation levels in untransfected cells (that do not express Tyro3) (lane 1), are comparable to those observed in cells transfected with the kdTyro3 construct (lane 2). In contrast, in cells expressing wtTyro3 (lane 3) the levels of phosphorylation of both Tyro3 and Axl are significantly higher than in the untransfected cells (Fig. 5, compare lane 3 with lane 2 in panels A and B). These results indicated that the increase in Axl phosphorylation observed upon Tyro3 overexpression is due to Tyro3 activation.


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)

Axl cross-phosphorylates Tyro3 and both receptors co-immunoprecipitate.Wild-type and kinase dead forms of Tyro3 were tested for their ability to auto-phosphorylate (panel A) and phosphorylate Axl (panel B). Rat2 cells were transiently transfected kinase dead (kd)Tyro3 (kdT3, lane 2) or with wild-type (wt) Tyro3 (wtT3, lane 3). The cells were activated with 350 ng/ml of Gas6 for 20 min. After protein normalization the extracts were divided in two, for Tyro3 immunoprecipitation (IP) (panel A) and for Axl IP (panel B). After SDS-PAGE using 8% gels and Western blotting, 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 reblotted with α-Tyro3 serum 5424 (1∶3,500, α-Tyro3) (panel A bottom) or re-probed with α-Axl (1∶3,500) (panel B bottom). These blots (panels A and B) are representative of 4 experiments. To determine if Axl can induce Tyro3 phosphorylation (panel C) Rat2 cells were transiently transfected with vectors encoding lacz (lane 1), kdTyro3 (kdT3, lane 2), wtTyro3 (wtT3, lane 3) or doubly transfected with lacz/Axl (lane 4), kdTyro3/Axl (kdT3/Axl,lane 5) and wtTyro3/Axl (wtT3/Axl, lane 6).The cells were activated with 350 ng/ml of Gas6 for 20 min. After protein normalization the extracts were divided in two, for Tyro3 immunoprecipitation (IP) (top and center panels) and for Axl IP (bottom panel). After SDS-PAGE in 8% gels and transfer, the membranes were probed with anti-phosphotyrosine (α-pTyr) (PY20 and P99 mixture 1∶3,500) antibodies (top panel), and rabbit α-Axl (bottom panel). The membrane probed with α-pTyr (Tyro3 IPs) was re-probed with α-Tyro3 (center panel). These blots are representative of 5 experiments. To determine whether Tyro3 and Axl co-immunoprecipitate, (panel D) Rat2/T3V5 cells were activated with 350 ng/ml Gas6 for 10 min. Detergent extracts were normalized and divided in two, for Tyro3 immunoprecipitation (IP) using α-FN2 Tyro3 antibodies (IP Tyro3, lanes 1 and 3) and for Axl IP using mouse monocolonal α-Axl antibodies (IP Axl, lanes 2 and 4). The samples were separated by SDS-PAGE using 6% gels and blotted with α-Tyro3 serum 5424 (1∶3,500) (lanes 1 and 2) or rabbit α-Axl antibodies (1∶3,500) (lanes 3 and 4). These blots are representative of 4 experiments.
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pone-0036800-g005: Axl cross-phosphorylates Tyro3 and both receptors co-immunoprecipitate.Wild-type and kinase dead forms of Tyro3 were tested for their ability to auto-phosphorylate (panel A) and phosphorylate Axl (panel B). Rat2 cells were transiently transfected kinase dead (kd)Tyro3 (kdT3, lane 2) or with wild-type (wt) Tyro3 (wtT3, lane 3). The cells were activated with 350 ng/ml of Gas6 for 20 min. After protein normalization the extracts were divided in two, for Tyro3 immunoprecipitation (IP) (panel A) and for Axl IP (panel B). After SDS-PAGE using 8% gels and Western blotting, 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 reblotted with α-Tyro3 serum 5424 (1∶3,500, α-Tyro3) (panel A bottom) or re-probed with α-Axl (1∶3,500) (panel B bottom). These blots (panels A and B) are representative of 4 experiments. To determine if Axl can induce Tyro3 phosphorylation (panel C) Rat2 cells were transiently transfected with vectors encoding lacz (lane 1), kdTyro3 (kdT3, lane 2), wtTyro3 (wtT3, lane 3) or doubly transfected with lacz/Axl (lane 4), kdTyro3/Axl (kdT3/Axl,lane 5) and wtTyro3/Axl (wtT3/Axl, lane 6).The cells were activated with 350 ng/ml of Gas6 for 20 min. After protein normalization the extracts were divided in two, for Tyro3 immunoprecipitation (IP) (top and center panels) and for Axl IP (bottom panel). After SDS-PAGE in 8% gels and transfer, the membranes were probed with anti-phosphotyrosine (α-pTyr) (PY20 and P99 mixture 1∶3,500) antibodies (top panel), and rabbit α-Axl (bottom panel). The membrane probed with α-pTyr (Tyro3 IPs) was re-probed with α-Tyro3 (center panel). These blots are representative of 5 experiments. To determine whether Tyro3 and Axl co-immunoprecipitate, (panel D) Rat2/T3V5 cells were activated with 350 ng/ml Gas6 for 10 min. Detergent extracts were normalized and divided in two, for Tyro3 immunoprecipitation (IP) using α-FN2 Tyro3 antibodies (IP Tyro3, lanes 1 and 3) and for Axl IP using mouse monocolonal α-Axl antibodies (IP Axl, lanes 2 and 4). The samples were separated by SDS-PAGE using 6% gels and blotted with α-Tyro3 serum 5424 (1∶3,500) (lanes 1 and 2) or rabbit α-Axl antibodies (1∶3,500) (lanes 3 and 4). These blots are representative of 4 experiments.
Mentions: In order to determine if the kinase activity of Tyro3 is required to induce Axl phosphorylation, we generated a kinase inactive or “kinase dead” construct (kdTyro3) by mutating K535 to M (K535M). This results in a receptor that is unable to undergo autophosphorylation [44], but that can still be trans-phosphorylated. As shown in Fig. 5A lane 2, we failed to detect tyrosine phosphorylation of the kdTyro3 mutant when activated by Gas6 in contrast to the sharp increases in phosphotyrosine levels observed in the wild-type (wt) Tyro3 construct (compare kdT3, Fig. 5 lane 2 with wtT3, lane 3). We then compared Axl phosphorylation levels (Fig. 5B) in Rat2 cells transiently transfected with either kdTyro3 (lane 2) or wtTyro3 (lane 3). As shown in Fig. 5B, Axl phosphorylation levels in untransfected cells (that do not express Tyro3) (lane 1), are comparable to those observed in cells transfected with the kdTyro3 construct (lane 2). In contrast, in cells expressing wtTyro3 (lane 3) the levels of phosphorylation of both Tyro3 and Axl are significantly higher than in the untransfected cells (Fig. 5, compare lane 3 with lane 2 in panels A and B). These results indicated that the increase in Axl phosphorylation observed upon Tyro3 overexpression is due to Tyro3 activation.

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