p85 and Grb2 co-IP with Axl from O4+ and O4– protein extracts prepared from post-natal day 10 mouse brain. Cells from total brain, O4– flow through, and O4+ immunopanning were stimulated in suspension with rhGas6 for 30 min at 37°C. Homogenates from post-natal day 10 mouse brain (a) total brain; (b) O4+ and O4– Homogenates, were incubated with either an Axl or DT-12 (irrelevant IgG1 negative control antibody) mAb. Total homogenates and IP’ed proteins were separated by SDS/PAGE and transferred to nitrocellulose. Western blot analysis of total homogenates (40 μg) with the Axl pAb (1 : 1000) confirms the presence of Axl in total brain, O4+, and O4– populations. The blot was cut and immunoblot analysis was performed using Axl pAb (1 : 1000), p85 pAb (1 : 500), and Grb2 mAb (1 : 5000). A faint background band only in the O4+ DT-12 control lanes was consistently observed and most likely is a result of an interaction between residual O4 antibody (IgM) used during immunopanning and the DT-12 antibody (IgG1) used in the control IP. IP buffer with O4 and IgG1 antibody, but no protein shows a similar faint background band, and pre-clearing with Protein-l agarose beads did not eliminate the faint background (data not shown). Visualization was by enhanced chemiluminescence, except for Grb2 which was visualized by Super-signal west femto (Pierce, Rockford, IL, USA).

A 1-min rhGas6 stimulation of WT Axl transfected COS7 cells results in a 2.9-fold increase in Akt phosphorylation at Thr308 relative to unstimulated WT Axl expressing cells; there was no increase in rhGas6 stimulated I,QI mutant Axl transfected COS7 cells relative to its unstimulated counterpart. COS7 cells were transfected with WT or I,QI mutant Axl, serum starved overnight, left unstimulated or stimulated for 1 min with rhGas6. After 1 min, the media was removed, replaced by 1X Tris-buffered saline, and protein homogenates were prepared immediately. (a) Representative experiment of western blots; p-Akt T308 (1 : 1000), Axl (1 : 1000), total Akt (1 : 1000), and β-actin (1 : 5000, load control). (b) Values are means ± SEM combined from three experiments. Asterisk represents significant increase (p = 0.022) of pAkt Thr308 levels in 1 min rhGas6 stimulated WT Axl transfected COS7 cells versus unstimulated WT Axl transfected COS7 cells. There is no significant increase between stimulated or unstimulated I,QI mutant Axl transfected COS7 cells. Significance is based on three experiments.

Pull-downs demonstrate that p85 directly binds to the SH3 domain of Grb2 via its second proline-rich region. (a) Full-length myc-tagged Grb2 was transfected into COS7 cells and pull-down assays were performed on protein homogenates with p85(1–333)-GST fusion proteins spanning both proline-rich regions [amino acids 1–333; GST-fusion protein containing p85Δpro1, p85Δpro2-GST, and p85Δpro1 + 2-GST; or GST only protein (negative control)]. Western blot analysis performed with a myc mAb (1 : 20 000) confirmed the presence of Grb2 in the total homogenates (b) COS7 cells were transfected with HA-tagged p85, and pull-down assays were performed on the protein homogenates using full-length or the N- or C-terminal SH3 domain of Grb2 fused to GST, or GST only protein (negative control). Western blot analysis performed with an HA mAb (1 : 1000) confirmed the presence of p85. (c) Pull-down assays were performed using either Grb2-SH3N-GST or GST only incubated with either thrombin cut p85(1–433), or uncut p85(1–433) GST. The p85(1–433) constructs span both proline-rich regions and the first SH2 domain of p85. The presence of p85 on the blot was detected using a pAb generated to a p85-GST fusion protein. The lower panels (a–c) show GST with or without the expressed fusion protein, using a GST mAb (DT-12, 1 : 100).

p85 and Grb2 pull-down and co-IP with Axl. (a) Pull-downs were performed from Axl transfected COS7 cell homogenates using GST-SH2 domain fusion proteins. Confirmation of Axl in the pull-down was performed by western blot analysis using an Axl pAb (1 : 1000). Total Homog refers to the 140 kDa Axl protein in Axl transfected protein homogenate. Visualization was by enhanced chemiluminescence. (b) Axl and ARG co-transfected COS7 cell homogenates were IP’ed with DT-12, an irrelevant IgG1 mAb (negative control), an ARG pAb, or an Axl mAb. Western blot analysis was performed using an ARG pAb (1 : 1000). (c) Axl and p85 co-transfected COS7 cell homogenates were IP’ed with either the Axl mAb or the irrelevant IgG1 mAb as described in (b). Western blot analysis confirms the presence of Axl (c-i; Axl pAb, 1 : 1000), p85 (c-ii; p85 pAb, 1 : 400), and Grb2 (c-iii; Grb2 mAb, 1 : 5000). (d) Pull-downs were performed on GST-SH2 domain fusion proteins and protein homogenates from WT or YVQM mutant Axl transfected COS7 cells. Confirmation of Axl in the pull-down was performed by western blot analysis using an Axl pAb (1 : 1000). The lower panel shows the 26 kDa GST-protein and the GST-expressed fusion protein using a GST mAb (DT-12, 1 : 100). (e) Axl and p85 co-transfected cells were serum starved overnight, washed and administered DMEM plus or minus 200 ng/mL rhGas6 for 30 min. IP's were performed as detailed above. Blots were incubated with either an Axl pAb or a Grb2 mAb. The amount of IP’ed p85 and Grb2 was normalized to the total amount of the respective protein expressed in 20 μg of total protein (lighter exposure used for Grb2 normalization not shown) relative to β-actin and the amount of IP’ed Axl (top panel). (f) PI3 kinase activity increased following rhGas6 stimulation of Axl transfected COS7 cells. T75 flasks of subconfluent COS7 cells were transfected with Axl, serum starved, and stimulated for 1 min plus and minus rhGas6. Total homogenates were IP’ed with an Axl mAb (+Gas6, No Gas6), or DT-12 (IgG Control), and PI3 kinase activity assays were performed. Western blot analysis using an Axl pAb and b-actin mAb on 20 lg of total homogenates shows equal transfection efficiency (f-i), and using an Axl pAb shows the ability of the Axl mAb to IP Axl (f-ii). f-iii shows a two-fold increase in activity in +Gas6 samples relative to the unstimulated samples. Values are means ± SEM combined from three independent experiments (n = 3 for No Gas6 and +Gas6; n = 2 for IgG control for each experiment). Asterisk represents significance (p = 0.023) between No Gas6 and +Gas6.

Transfection of WT and Axl mutant constructs into COS7 cells, followed by pull-downs and IPs, demonstrate that p85 binding to Axl is both a direct and indirect interaction. (a) Schematic showing the three tyrosine residues (779, 821, and 866) on Axl that are autophosphorylated following Gas6 binding to the extracellular IgG domains. Seven mutant Axl constructs were generated and sequenced. The single Axl mutants (YVNI; YALI; and M,QM); double Axl mutants (I,I; I, QM; and M,QI); and the triple Axl mutant (I,QI) are shown. For details see Experimental procedures. (b) WT and Axl mutant constructs were transfected into COS7 cells and protein homogenates were incubated with SH2-p85-GST fusion protein, or GST-only protein (negative control). Western blot analysis confirmed the presence of Axl in the WT Axl Total homogenate. DT-12, an IgG1 antibody to GST (1 : 100), identifies GST proteins and the GST fusion proteins. The M,QI and I,QI Axl mutant pull-downs were done on a different blot than the rest of the Axl mutants, and therefore, have a separate WT positive control. (c) Protein homogenates from serum-starved COS7 cells co-transfected with Axl constructs and HA-tagged full-length p85 were IP’ed with the Axl mAb, or DT-12. Western blot analysis was performed with an HA mAb (top; 1 : 1000). The lower panel was incubated with an Axl pAb (1 : 1000) and an HA mAb confirming the expression of Axl and HA-p85 in the total protein homogenates (30 μg); β-actin (1 : 5000) was used as a loading control. (d) Serum starved COS7 cells were co-transfected with HA-tagged full-length p85, and WT or mutant Axl constructs. Protein homogenates were IP’ed with an Axl mAb or DT-12. The blot was cut and western blot analysis was performed with an Axl pAb (1 : 1000), HA mAb (1 : 1000), and a Grb2 mAb (1 : 5000). The Axl pAb confirms the presence of Axl in the total homogenates (5 μg) and in the IP. Because of the low amount of protein loaded in the total homogenate lanes the exposure time for the total homogenate lanes with the HA mAb was 90 s whereas the exposure time was 5 s for the IP lanes (Axl and control). All other antibodies had the same exposure time for their respective total homogenates and IP lanes. (e) Serum starved COS7 cells were transfected with WT or mutant Axl constructs, or empty vector controls. Protein homogenates were IP’ed with an Axl mAb or DT-12. Blots were incubated with GST-p85(1-433) fusion protein and subsequently incubated with a GST mAb (1 : 100), to detect p85-GST bound to Axl at 140 kDa. Visualization was by enhanced chemiluminescence.

Transfection of WT, or mutant Axl constructs, plus and minus full-length p85, show a competition between p85 and Grb2 for the pYVNM site on Axl. WT and Axl mutant constructs were co-transfected into serum starved COS7 cells with HA-tagged p85 (+p85) or empty vector (–p85) cDNA constructs. Protein homogenates were IP’ed with an Axl mAb. Western blot analysis was performed using the Grb2 mAb (1 : 5000). In the total homogenates, the presence of Axl was determined using an Axl pAb (1 : 1000).

Conserved amino acid alignment of the human Axl, Tyro3, and Mer autophosphorylation sites and schematic representation of the proposed Gas6/Axl downstream cell survival signaling pathway. (a) The number represents the position of the amino acid within the respective protein. Note that the spacing between the first YXXM motif and the second YXN motif is conserved amongst all three receptors. The accession number for human Axl is BC050914; human Rse (Tyro3) is U05682, and human c-Mer is U08023. (b) Gas6 stimulation of Axl results in autophosphorylation at Y779ALM, Y821VNM, and Y866VLC. Direct recruitment of p85 can occur via its SH2 domains binding to one or both of the pY779ALM, pY821VNM motifs, whereas direct recruitment of Grb2, via its SH2 domain, can only bind to the pY821VN motif. Indirect association of p85 and Axl can occur through Grb2 via the second proline-rich region of p85 binding to one of the SH3 domains of Grb2. The direct or indirect recruitment of p85/p110 PI3 kinase can signal downstream to activate Akt resulting in enhanced cell survival.