Cross-Phosphorylation, Signaling and Proliferative Functions of the Tyro3 and Axl Receptors in Rat2 Cells

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

Tyro3 modulates MAPK and PI(3)K signaling pathways.

<p>As shown in <b>panel A</b>, Rat/T3V5 <b>(Rat/T3, lanes 1–3)</b> and Rat2 cells <b>(Rat, lanes 4–6)</b> 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 <b>panel B</b>, transiently transfected Rat2/Axl cells <b>(Rat/Axl, lanes 1–3)</b> and Rat2 cells <b>(Rat, lanes 4–6)</b> 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 <b>(panel C)</b> and Axl <b>(panel D).</b> For Tyro3 overexpressing cells <b>(panel C),</b> Rat/T3V5 cells <b>(Rat/T3, lanes 1–3),</b> and Rat2 untransfected cells <b>(Rat, lanes 4–6),</b> 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 <b>Fig. 3 A and B</b>. These blots are representative of 4 experiments. For Axl overexpressing cells <b>(panel D), t</b>ransiently transfected Rat2/Axl cells <b>(Rat/Axl, lanes 1–3)</b> and Rat2 untransfected cells <b>(Rat, lanes 4–6)</b> 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.</p

Gas6 induced cell proliferation of Rat2 and Rat2/T3V5 cells.

<p>Serum starved Rat2 and Rat2/T3V5 cells were stimulated with 250 ng/ml of Gas6 for 0–72 hrs (<b>panel A</b>). 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 (<b>panel B</b>), Rat/T3V5 cells were incubated 45 min prior to activation with vehicle (DMSO), 1.5 µM LY294002, or 5.5 µM wortmannin <b>(top panel)</b> or with 1 µM or 10 µM U0126 <b>(bottom panel)</b>. 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 <b>(top panel)</b>, and α-pERK1/2 <b>(bottom panel)</b>. 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 <b>(panel C)</b>, 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).</p

Characterization of anti-TAM specific antibodies and TAM expression in Rat2 cells.

<p>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 <i>tyro3</i>^−/−, <i>axl^−/−</i>, and <i>mer</i>^−/− knockout mice. As shown in <b>panel A</b>, 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, <b>top panel),</b> affinity purified rabbit α-Axl (serum 1∶3,500, <b>center panel),</b> and rabbit α-Mer (1∶5,000 <b>bottom panel)</b>. These antibodies were used to characterize Tyro3, Axl, and Mer expression in Rat2 and Rat2/T3V5 cells as shown in <b>panel B.</b> Detergent cell extracts were prepared from untransfected Rat2 cells (<b>Rat, lane 1</b>), stably Tyro3 transfected Rat2/T3V5 cells (<b>Rat/T3 lane 2</b>), brain tissue extract (<b>lane 3, top and bottom panels</b>) or spleen tissue extract <b>(lane3, center panel).</b> After normalization of tissue extracts for protein concentration, Tyro3, Axl and Mer were immunoprecipitated (IPed) (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036800#pone-0036800-g001" target="_blank">Fig. 1A</a> for antibodies). SDS-PAGE was performed using 8% gels followed by Western blot analysis. The membranes were probed against Tyro3 <b>(top panel),</b> Axl <b>(center panel),</b> and Mer <b>(bottom panel),</b> as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036800#pone-0036800-g001" target="_blank">Fig. 1A</a>. The observed differences in Tyro3 molecular weight depending on the source are due in part by N-linked glycosylation as shown in <b>panel C.</b> Detergent extracts were prepared from Rat2/T3V5 cells (<b>Rat/T3, lanes 1 and 2</b>), and adult rat brain tissue (<b>brain</b>, <b>lanes 3 and 4)</b>. 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.</p