Transforming potential of Src family kinases is limited by the cholesterol-enriched membrane microdomain

Abstract

The upregulation of Src family kinases (SFKs) has been implicated in cancer progression, but the molecular mechanisms regulating their transforming potentials remain unclear. Here we show that the transforming ability of all SFK members is suppressed by being distributed to the cholesterol-enriched membrane microdomain. All SFKs could induce cell transformation when overexpressed in C-terminal Src kinase (Csk)-deficient fibroblasts. However, their transforming abilities varied depending on their affinity for the microdomain. c-Src and Blk, with a weak affinity for the microdomain due to a single myristate modification at the N terminus, could efficiently induce cell transformation, whereas SFKs with both myristate and palmitate modifications were preferentially distributed to the microdomain and required higher doses of protein expression to induce transformation. In contrast, disruption of the microdomain by depleting cholesterol could induce a robust transformation in Csk-deficient fibroblasts in which only a limited amount of activated SFKs was expressed. Conversely, the addition of cholesterol or recruitment of activated SFKs to the microdomain via a transmembrane adaptor, Cbp/PAG1, efficiently suppressed SFK-induced cell transformation. These findings suggest that the membrane microdomain spatially limits the transforming potential of SFKs by sequestering them away from the transforming pathways.

FIG. 1.

Transforming abilities of SFKs. (A) Total cell lysates from Csk^−/− cells expressing each SFK were immunoblotted with the antibodies indicated. (B) The cell morphology of each cell clone was observed by phase-contrast microscopy (upper panel), and actin filaments were visualized by Alexa Fluor 488-phalloidin staining (lower panel). (C) Each cell clone was subjected to the soft-agar colony formation assay. Representative dishes from three independent experiments are shown. The mean numbers of colonies per cm² ± standard deviations (SD) are indicated. (D) The constitutively active form of c-Src, Fyn, c-Yes, or Lck, which has a Tyr-to-Phe replacement in its C-terminal regulatory site (c-SrcYF, FynYF, c-YesYF, or LckYF), was expressed in Csk^+/+ cells. Total cell lysates from these cells were immunoblotted with the antibodies indicated (left panel). Csk^+/+ cells expressing c-SrcYF, FynYF, c-YesYF, or LckYF were subjected to the soft-agar colony formation assay (right panel). Colonies were scored 7 days after plating. The mean numbers of colonies per cm² ± SD are indicated. (E) DRMs and non-DRMs of the indicated cell clones were separated on sucrose density gradients. Aliquots of the fractions were immunoblotted with the indicated antibodies. Shown are representative fractionation patterns obtained from more than three independent experiments. (F) The ratios of SFK distributions to DRMs (open bars) and non-DRMs (closed bars) were calculated by quantifying the SFK levels detected by immunoblotting. The mean ratios ± SD are indicated. (G) N-terminal sequences of SFKs. Myristoylation sites (G) and palmitoylation sites (C) are indicated by blue and red letters, respectively.

FIG. 2.

Localization of active SFK to nonraft compartments is associated with cell transformation. (A) Csk^−/− cells expressing different levels of Fyn, Lck, or Lyn were cloned, and their cell lysates were subjected to immunoblot analysis with the antibodies indicated. (B) Cell clones used in panel A were subjected to the soft-agar colony formation assay. Colonies were scored 11 days after plating. The mean numbers of colonies per cm² ± standard deviations obtained from three independent experiments are shown. (C) Expression levels of Fyn in the indicated clones were detected by immunoblotting with anti-Fyn, and the relative Fyn levels (Fyn/actin ratio) were determined by defining the Fyn/actin ratio in Csk^−/− cells (Mock) as 1. (D) Expression levels of c-Yes in the indicated clones were analyzed as described for Fyn (upper panels). Transforming activity of c-Yes clones was analyzed by soft-agar colony formation assay. The mean numbers of colonies per cm² ± standard deviations are indicated (lower panel). (E) Aliquots of the fractions in DRMs and non-DRMs from the indicated cell lines were immunoblotted with anti-pY418 antibody.

FIG. 3.

Disruption of the microdomain induces cell transformation in Csk^−/− cells. (A) Total cell lysates from MEFs, Csk^−/−, or Csk^−/−/Csk cells were immunoblotted with the antibodies indicated. (B) Csk^−/− cells were treated with or without 3 mM MβCD and were subjected to the cholesterol assay. (C) Cells used in panel A were treated with or without 3 mM MβCD or Csk^−/−/Src cells were treated with or without 50 μM PEG-cholesterol (PEG-Chol) and were subjected to the soft-agar colony formation assay. Colonies were scored 11 days after plating. Representative dishes from three independent experiments are shown. Cont, control. (D) Cells used in panel A were treated with the indicated concentrations of MβCD and subjected to the soft-agar colony formation assay. The mean numbers of colonies per cm² ± standard deviations obtained from three independent experiments are shown.

FIG. 4.

Disruption of the microdomain induces the relocation of activated SFKs to focal contacts. (A) Csk^−/− cells were untreated or treated with 5 mM MβCD. Actin filaments were visualized by Alexa Fluor 488-phalloidin staining (F-actin [green, left panel]). Intracellular localization of activated SFKs (pY418 [red in the left panel and green in the right panel]) in the indicated cells was analyzed by immunostaining. Localization of activated SFKs at focal contacts (vinculin [red, right panel]) was also analyzed. (B) Total cell lysates from Csk^−/− cells untreated or treated with 3 mM MβCD were immunoblotted with the indicated antibodies. Phosphorylation of the Src substrate, FAK, and activity of the downstream effector, ERK1/2, were determined by immunoblotting with the indicated antibodies (right panels). The mean values of relative activity of ERK1/2 ± standard deviations obtained from three independent experiments are shown. **, P < 0.01 by Student's t test.

FIG. 5.

Cbp serves as a common suppressor of SFK-mediated transformation. (A) DRMs from the cells used in Fig. 1 were subjected to immunoblotting with anti-Cbp. Caveolin was detected as a marker for DRMs. P.C., positive control. (B) Expression of Cbp mRNA in the indicated cells was analyzed by RT-PCR. (C) Total cell lysates from SFK-introduced Csk^−/− cells and those expressing Cbp were immunoblotted with the antibodies indicated. (D) Csk^−/− cell clones expressing the indicated SFK with (white) or without (black) Cbp expression were subjected to the soft-agar colony formation assay. Colonies were scored 11 days after plating. The mean numbers of colonies per cm² ± standard deviations obtained from three independent experiments are shown.

FIG. 6.

Cbp sequesters activated SFK into lipid rafts. (A) DRMs and non-DRMs from the indicated SFKs (left panels) and those expressing Cbp (right panels) were immunoblotted with the antibodies indicated. (B) DRMs and non-DRMs from the transformed Fyn#6 and Fyn#3 clones (left panels) and those expressing Cbp (right panels) were immunoblotted with the antibodies indicated. (C) Cell clones used in panel B were subjected to soft-agar colony formation assay. The mean numbers of colonies per cm² ± standard deviation are indicated. (D) Total cell lysates from the cells expressing Cbp were subjected to IP for Cbp and Myc (SFK). The immunoprecipitates were then analyzed by immunoblotting with the antibodies indicated.

FIG. 7.

LIME cannot suppress SFK-induced cell transformation. (A) Expression of mRNA for LIME, Cbp, or GAPDH in the indicated cells was analyzed by RT-PCR. (B) Cbp or LIME was expressed in c-Src- or Lck-transformed Csk^−/− cells, and the total cell lysates were immunoblotted with the antibodies indicated. (C) Cbp or LIME was expressed in Csk^−/− cells transformed by c-Src or Lck, and cell morphology was observed by phase-contrast microscopy. (D) Csk^−/− cells expressing c-Src or Lck with Cbp or LIME or without expression (Mock) were subjected to the soft-agar colony formation assay. Colonies were scored 11 days after plating. The mean numbers of colonies per cm² ± standard deviations obtained from three independent experiments are shown. (E) Csk^−/− cell clones expressing the indicated SFK without (Mock) or with LIME expression were subjected to the soft-agar colony formation assay. The mean numbers of colonies per cm² ± standard deviations obtained from three independent experiments are shown. (F) c-Src- or Lck-transformed Csk^−/− cells coexpressing with or without LIME were separated into DRMs and non-DRMs, and each fraction was immunoblotted with the antibodies indicated.