Volume 30, Issue 4, Pages 426-436 (May 2008) The Lipid Raft-Anchored Adaptor Protein Cbp Controls the Oncogenic Potential of c-Src  Chitose Oneyama, Tomoya Hikita, Kengo Enya, Marc-Werner Dobenecker, Kazunobu Saito, Shigeyuki Nada, Alexander Tarakhovsky, Masato Okada  Molecular Cell  Volume 30, Issue 4, Pages 426-436 (May 2008) DOI: 10.1016/j.molcel.2008.03.026 Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 1 Cbp Is a Suppressor of Src-Mediated Transformation (A) Raft fractions of Csk−/− cells expressing the indicated constructs were subjected to immunoblotting with anti-Cbp. (B) Expression of Cbp mRNA was analyzed by RT-PCR (upper) and real-time PCR (lower). Relative values ± SD were obtained from three independent assays. (C) Cell morphology of Csk−/− cells expressing c-Src and c-Src/Cbp was observed by phase-contrast microscopy (top). Actin filaments were visualized by Alexa 594-phalloidin staining (bottom). (D) Csk−/− cells expressing c-Src, c-SrcYF, v-Src, and H-RasV12 (top) and these cells coexpressing Cbp (bottom) were subjected to the soft-agar colony-formation assay. Representative dishes from three independent experiments are shown. (E) Csk−/− cells expressing the indicated constructs (1 × 106) were inoculated s.c. into nude mice. Means ± SD of tumor volume (cm3) obtained from five mice are plotted versus days after inoculation. (F) Parental MEF (Cbp+/+), cloned Cbp-deficient MEF lines (Cbp−/− 13 and 18), and Cbp−/− cells re-expressing Cbp (Cbp−/− + Cbp) were infected with retrovirus expressing v-Src, and cell morphology (upper) and soft-agar colony-formation activity (lower) were analyzed. The mean numbers of colonies per cm2 ± SD obtained from three independent experiments are shown. (G) Cbp+/+, Cbp−/− 13, and Cbp−/− 18 cells expressing v-Src (0.2 × 106) were inoculated s.c. into nude mice. Means ± SD of tumor volume (cm3) obtained from four mice are plotted versus days after inoculation. Molecular Cell 2008 30, 426-436DOI: (10.1016/j.molcel.2008.03.026) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 2 Cbp Sequesters Activated c-Src into Lipid Rafts (A) Total cell lysates from c-Src-transformed cells (c-Src) and those expressing Cbp (c-Src/Cbp) were immunoblotted with the indicated antibodies. (B) Phosphorylation of Src substrates and activity of downstream effectors in the cells used in Figure 1A and c-Src/Cbp cells were analyzed by immunoblotting with the indicated antibodies. (C) Raft and nonraft fractions of the indicated cells were separated on sucrose density gradients. Aliquots of the fractions were immunoblotted with the indicated antibodies. The transferrin receptor (TfR) and GM1 ganglioside (B subunit of cholera toxin, CTX, sensitive) were detected as markers of nonraft and raft fractions, respectively. (D) Intracellular localization of Cbp (green) and activated Src (Src pY416: red) in the indicated cells were analyzed by immunostaining. Localization of activated c-Src at focal contacts (vinculin: green) was also analyzed. Molecular Cell 2008 30, 426-436DOI: (10.1016/j.molcel.2008.03.026) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 3 Interaction between Cbp and c-Src (A) Cell lysates from c-Src-transformed cells and c-Src/Cbp cells were fractionated into raft and nonraft fractions, and each fraction was immunoblotted with the indicated antibodies (left). The solubilized raft and nonraft fractions were then subjected to immunoprecipitation (IP) with anti-Src, followed by immunoblotting with the indicated antibodies (middle). Samples from c-Src/Cbp cells were immunoprecipitated with anti-Cbp, followed by immunoblotting with anti-Src and anti-Cbp (right). (B) Schematic structure of Cbp and mutants. M, membrane. (C) c-Src-transformed cells expressing the indicated constructs were subjected to the soft-agar colony-formation assay. Colonies were scored 11 days after plating. The mean numbers of colonies per cm2 ± SD obtained from three independent experiments are shown. (D) c-Src-transformed cells expressing Cbp (left) or a Cbp(C39/42A) mutant (right) were subjected to sucrose gradient fractionation, and each fraction was immunoblotted with the indicated antibodies. (E) Csk−/− cells expressing the indicated constructs were subjected to the soft-agar colony-formation assay. The mean numbers of colonies per cm2 ± SD were obtained from three independent experiments. (F) Cbp was immunoprecipitated from the indicated cells, followed by immunoblotting with the indicated antibodies (left). P.C., positive control; Ig hc, immunoglobulin heavy chain. c-Src was immunoprecipitated from the indicated cells, followed by immunoblotting with the indicated antibodies (right). Molecular Cell 2008 30, 426-436DOI: (10.1016/j.molcel.2008.03.026) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 4 Role of Lipid Raft in Src Transformation (A) Lipid raft-anchored c-Src (CbpN-Src) and wild-type c-Src were expressed in Csk−/− cells, and total cell lysates were immunoblotted with the indicated antibodies. (B) Raft and nonraft fractions of cells expressing CbpN-Src were separated on a sucrose gradient, and each fraction was immunoblotted with the indicated antibodies. (C) The indicated cells were analyzed by the soft-agar colony-formation assay. Representative results from three independent experiments are shown. (D) Wild-type Fyn and its mutants (CA, KN, C3A, and Δ16) were expressed in Csk−/− cells, and total cell lysates were immunoblotted with the indicated antibodies. (E) Transforming activity of these cells was analyzed by the soft-agar colony-formation assay. Representative results from three independent experiments are shown. (F) Raft and nonraft fractions of Csk−/− cells expressing wild-type Fyn (top) and Fyn C3A (bottom) were separated on sucrose density gradients, and each fraction was immunoblotted with the indicated antibodies. (G) c-Src/Cbp cells were treated with the indicated concentrations of MβCD and cholesterol and were subjected to the soft-agar colony-formation assay. Representative results from three independent experiments are shown. The mean numbers of colonies per cm2 ± SD are indicated. (H) Cell lysates were separated into raft and nonraft fractions, and each fraction was immunoblotted with the indicated antibodies. (I) c-Src transformed cells were treated with cholesterol at the indicated concentrations and subjected to morphological analysis (top) and the soft-agar colony-formation assay (bottom). The mean numbers of colonies per cm2 ± SD obtained from three independent experiments are indicated. (J) Total cell lysates (left), or nonraft and raft fractions (right), from cholesterol treated cells were immunoblotted with the indicated antibodies. (K) MEFs transformed by an oncogenic H-RasV12 were treated with the indicated concentrations of cholesterol, followed by the soft-agar colony-formation assay. The mean numbers of colonies per cm2 ± SD obtained from three independent experiments are indicated. Molecular Cell 2008 30, 426-436DOI: (10.1016/j.molcel.2008.03.026) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 5 Dissection of Csk-Dependent and -Independent Roles of Cbp (A) Wild-type Csk or CskKN was expressed in c-Src/Cbp cells, and total cell lysates were immunoblotted with the indicated antibodies. The slower migration of Cbp in Csk expressing cells is due to the lower levels of Cbp phosphorylation. (B) Raft and nonraft fractions of cells expressing Csk and CskKN were separated on sucrose density gradients, and each fraction was immunoblotted with the indicated antibodies. (C) Cbp was immunoprecipitated from the raft and nonraft fractions in (B), and the immunoprecipitates were immunoblotted with the indicated antibodies. Molecular Cell 2008 30, 426-436DOI: (10.1016/j.molcel.2008.03.026) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 6 Role of Cbp in Human Cancer Cells (A) Raft fractions prepared from the indicated human cell lines were immunoblotted with anti-Cbp. Total cell lysates from these cells were used for immunoblotting with additional antibodies, as indicated. (B) The expression of Cbp mRNA in human colon tumors (T) and the corresponding normal tissue (N) from individual patients (1–5) was analyzed by RT-PCR. (C) HT29 and HCT116 cells with (black) or without (white) Cbp expression were subjected to the soft-agar colony-formation assay, and the mean numbers of colonies per cm2 ± SD from three independent experiments are plotted. ∗∗p < 0.01, by Student's t test. (D) HT29 and HT29/Cbp cells were inoculated s.c. into nude mice. Means ± SD of tumor volume (cm3) obtained from four mice are plotted versus days after inoculation. Excised tumors are shown in the lower panel. Molecular Cell 2008 30, 426-436DOI: (10.1016/j.molcel.2008.03.026) Copyright © 2008 Elsevier Inc. Terms and Conditions

Figure 7 Schematic Models of the Roles of Cbp/Csk and Lipid Rafts in Regulating the Transforming Potential of c-Src (A) In c-Src-transformed cells, the expression of Cbp is downregulated by unknown mechanisms, and activated c-Src is thereby constitutively retained in nonraft compartments, i.e., focal contacts, and interacts with critical targets such as FAK and Cas to induce cell transformation. (B) When Cbp is expressed, phosphorylated Cbp (pY165/183 or pY381/409) directly binds to activated c-Src and sequesters it in lipid rafts, resulting in efficient suppression of c-Src function. Excessive cholesterol can also sequester activated c-Src into lipid rafts. (C) When Csk is present, Csk is recruited to lipid rafts via binding to Cbp at pY314 and phosphorylates c-Src Y527 to inactivate the catalytic activity of c-Src. The inactivated c-Src then relocates to nonraft compartments. Molecular Cell 2008 30, 426-436DOI: (10.1016/j.molcel.2008.03.026) Copyright © 2008 Elsevier Inc. Terms and Conditions