Figure 6.

Ligand dependency of ARMS, CrkL and C3G interactions. (A) The polyproline-rich region of ARMS contains consensus sequences (PXXP) for binding to SH3 domains. Sequences within the polyproline-rich region of ARMS (P¹⁰⁵⁷–L¹¹⁵¹) present three PXXP motifs (bold) that are consensus for binding of SH3 domain-containing molecules (Feller, 2001) and are predicted by website sequence analysis: http://scansite.mit.edu/. (B) In vitro interaction between ARMS and CrkL, but not c-CrkII. GST–ARMS recombinant proteins were incubated with HEK293 cell extracts and subjected to Western blotting analysis with anti-c-Crk II or anti-CrkL antisera. A Coomassie-stained gel of the input GST fusion proteins is shown (bottom panel). (C) PC12 cells were stably transfected with FLAG-ARMS and FLAG-ARMSΔP. Individual clones were analyzed for the ectopic expression of ARMS or ARMSΔP proteins using anti-FLAG antibodies. Reprobing the blot with anti-ARMS antibody revealed that the overexpression levels for Flag-ARMS and Flag-ARMSΔP are approximately three-fold. (D) Expression of ARMSΔP abolishes Rap1 but not Ras activation elicited by NGF. PC12 clones stably expressing FLAG-ARMS or FLAG-ARMSΔP were serum starved for 16 h, lysates were prepared and subjected to incubation with 10 μg of GST-RalGDS RBD or the Raf-1 Ras-binding domain protein (GST-Raf RBD) in pull-down assays (Hermann et al, 1996) to detect active Rap1 and Ras, respectively. Immunoblots using anti-Rap1 and anti-Ras were carried out as indicated. (E) Expression of FLAG-ARMSΔP in PC12 cells impairs sustained MAP kinase activation elicited by NGF. Cell extracts from PC12 cells or PC12 clones described above (C), nontreated or treated with NGF for 5 or 40 min, were obtained and Western blots were performed with p-MAPK and p-Akt antibodies. A reduction in MAPK activation in PC12 clones expressing FLAG-ARMSΔP was seen at 40 min of NGF treatment, but no differences were observed in the activation of Akt. (F) Neurite outgrowth of PC12 cells transiently transfected with GFP, FLAG-ARMS or FLAG-ARMSΔP. At 24 h after transfection with the indicated plasmids, cells were treated with NGF for 2 additional days. PC12 transfectants were scored as positive as described in Figure 4A (^*P<0.02; ^**P<0.001). (G) Cortical neurons transfected with GFP/FLAG-ARMSΔP showed a reduced response to BDNF. Cells were transfected with GFP or GFP/FLAG-ARMSΔP. After 2 days, cultures were stimulated with BDNF for 40 min and staining for p-MAPK was performed. The statistical significance by Student's t-test was demonstrated for (F) and (G) (^*P<0.02; ^**P<0.001).