Figure 4. The role of SRC-C3G-RAP1 signaling in transformation induced by CRKL.

(A) Immunoblot of CRKL in AALE cell lines overexpressing a control vector or CRKL, as compared to NSCLC cells that harbored CRKL amplifications. Relative intensity of bands is determined.

(B) Anchorage independent growth of AALE cell lines stably overexpressing a control vector, wild-type or mutant CRKL. Colony number indicates colonies greater than 0.2 mm in diameter 4 weeks after plating. Data represent mean + s.d. of six replicate determinations from two independent experiments. Wild-type CRKL and CRKL mutants that induce anchorage independent growth are marked in red.

(C) Overexpression of CRKL in AALE cells increased the phosphorylation of SRC. Immunoblot of phospho-Y419 SRC proteins in AALE cells overexpressing a control vector, CRKL or CRKL^W160L mutant.

(D) Immunoblot of phospho-T185/Y187 ERK1/2 and phospho-S473 AKT in AALE cell lines overexpressing wildtype or mutant CRKL. Wild-type CRKL and CRKL mutants that induced anchorage independent growth in (B) are marked in red.

(E) Effect of CRKL overexpression on CRKL-C3G interaction and phosphorylation of C3G in AALE cells. CRKL immune complexes in AALE cells expressing indicated constructs were isolated and immunoblotting for phosphorylated C3G and total C3G proteins was performed using specific antibodies.

(F) Overexpression of CRKL in AALE cells increased RAP1 activity. Pull-down assay for GTP-bound RAP1 proteins were performed followed by immunoblotting for RAP1 proteins. Immunoblot of RAP1 proteins in total lysates before pull-down assay was used as loading control. Relative intensity of bands is determined.

(G) Effect of CRKL suppression on RAP1 activity in NSCLC cells that harbored amplification of CRKL. Pull-down assays were performed to assess the RAP1-GTP levels in NSCLC cells 4 d after infection with a control shRNA targeting GFP or a CRKL-specific shRNA (shCRKL#1) followed by immunoblotting for RAP1. Immunoblot of RAP1 proteins in total lysates was shown. Relative intensity of bands is shown.

(H) Effect of expressing RAP1 on cell proliferation of H1755 cells after CRKL suppression. Each H1755 cell line expressing a control vector, or wildtype RAP1A or RAP1B or constitutively active mutants, RAP1A^63E or RAP1B^V12, was infected with control shRNAs or a CRKL-specific shRNA (shCRKL#3). Cell proliferation was measured 6 d after infection with shRNA. Data represent mean + s.d. of six replicate measurements. * indicates p<0.0001 as compared to cells expressing a control vector and shCRKL#3.

(I) Effect of suppression of SRC, C3G or RAP1 on CRKL-induced anchorage independent growth. Left, Immunoblots of SRC, C3G or RAP1 proteins in CRKL-overexpressing AALE cells expressing a control shRNA targeting GFP or each gene-specific shRNA. The shRNAs that suppressed the target protein the best were marked in red color. The antibody for RAP1 detects both RAP1A and RAP1B. Right, Anchorage independent growth of AALE cells expressing indicated constructs. Colony number indicates colonies greater than 0.2 mm in diameter 4 weeks after plating. Data represent mean + s.d. of six replicate determinations from two independent experiments. * indicates p<0.001 as compared to cells expressing CRKL and shGFP.

(J) Effect of suppression of SRC, C3G or RAP1 on phospho-ERK1/2 levels in CRKL-overexpressing AALE cells. Immunoblots of phospho-ERK1/2 in CRKL-overexpressing AALE cells expressing indicated shRNAs. The shRNAs marked in red were validated in (I). (K) Effect of dasatinib treatment on anchorage independent growth of CRKL-overexpressing AALE cells. Anchorage independent growth of CRKL-overexpressing AALE cells that expressed wildtype SRC or mutant SRC or a control vector in the presence of dasatinib at indicated concentration. Colony number indicates colonies greater than 0.2 mm in diameter 4 weeks after plating. Mean + s.d. are shown. * indicates p<0.001 compared to cells expressing a control vector.

(L) Effect of dasatinib treatment on phospho-SRC and phospho-ERK1/2 in CRKL-overexpressing AALE cells. AALE cells expressing indicated constructs were exposed to dasatinib at indicated concentration for 6 h followed by immunoblotting.