Anaplastic lymphoma kinase activates the small GTPase Rap1 via the Rap1-specific GEF C3G in both neuroblastoma and PC12 cells

Abstract

Many different types of cancer originate from aberrant signaling from the anaplastic lymphoma kinase (ALK) receptor tyrosine kinase (RTK), arising through different translocation events and overexpression. Further, activating point mutations in the ALK domain have been recently reported in neuroblastoma. To characterize signaling in the context of the full-length receptor, we have examined whether ALK is able to activate Rap1 and contribute to differentiation/proliferation processes. We show that ALK activates Rap1 via the Rap1-specific guaninenucleotide exchange factor C3G, which binds in a constitutive complex with CrkL to activated ALK. The activation of the C3G/Rap1 pathway results in neurite outgrowth of PC12 cells, which is inhibited by either overexpression of Rap1GAP or siRNA-mediated knockdown of Rap1 itself or the guanine nucleotide exchange factor C3G. Significantly, this pathway also appears to function in the regulation of proliferation of neuroblastoma cells such as SK-N-SH and SH-SY5Y, because abrogation of Rap1 activity by Rap1-specific siRNA or overexpression of Rap1GAP reduces cellular growth. These results suggest that ALK activation of Rap1 may contribute to cell proliferation and oncogenesis of neuroblastoma driven by gain-of-function mutant ALK receptors.

Anaplastic Lymphoma Kinase (ALK) regulates initiation of transcription of MYCN in neuroblastoma cells

“…These results were confirmed in clonally-derived PC12 cells, which inducibly express wild-type or mutant ALK, allowing comparison of the activity of these mutants in a controlled manner. 35,37 In agreement with our previous results we observed that PC12 cells expressing wild-type ALK displayed a fivefold increase in MYCNP-luciferase upon stimulation of ALK with mAb46 (Figure 1c). PC12 clones expressing ALK gain-of-function human ALK F1174S and mouse ALK R1279Q (which is equivalent to the human ALK R1275Q mutation), also increased the expression of the MYCNPluciferase reporter 2 --3 fold.…”

“…In addition, the PC12hALK wt cells were stimulated with 1 mg/ml of the activating mAb46 as indicated. 37,48,49 Cells were lysed in 120 ml cell culture lysis reagent (Promega, Madison, WI, USA), of which 20 ml were used for luciferase assay according to manufacturer's protocol (Promega). Samples were measured in a TD-20/20 turner design luminometer (Turner Designs, Sunnyvale, CA, USA).…”

“…Cell lysis, immunoprecipitation and immunoblotting were performed according to the protocols described in. 37 (c) Luciferase assay of ALK-expressing doxycyline-inducible PC12 cell lines transfected with MYCNP-luciferase and empty pGL2 vector as a control, respectively. Stable PC12 Tet-on clones expressing human or mouse pTTP-ALK mut were generated and grown as described previously.…”

“…Stable PC12 Tet-on clones expressing human or mouse pTTP-ALK mut were generated and grown as described previously. 37 2 Â 10 6 doxycycline-inducible hALK wt hALK F1174S and mALK R1279Q PC12 cell lines 35 were transfected with 1.6 mg of either pGL2 (ctrl) or MYCNP-luciferase, as described in (a). Cells were then serum starved, and induced to express ALK employing doxycycline as indicated.…”

“…Cells were lysed in SDS sample buffer and whole-cell lysates were prepared for western blot analysis as described. 37 Samples were assessed by western blot with antibodies against p-ALK(Y1278), MYCN, Aurora A, p-PKB/AKT, p-ERK (all from Cell Signaling Technology), ALK and pan-ERK (BD Transduction Laboratories) was used to show equal loading. Three independent experiments with similar results were performed and representative blot are shown.…”