Transforming Potential of Src Family Kinases Is Limited by the Cholesterol-Enriched Membrane Microdomain

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.Src family kinases (SFKs) are membrane-associated, non-receptor protein tyrosine kinases involved in a variety of intracellular signaling pathways (5). SFKs are comprised of eight members in mammals: c-Src, Fyn, c-Yes, Lyn, Lck, Hck, c-Fgr and Blk. Among these, c-Src, Fyn, and c-Yes are ubiquitously expressed, whereas the others are relatively concentrated in hematopoietic cell lineages. The intracellular distribution of each SFK also varies depending on their unique N-terminal sequences and acyl modifications (5, 27). These distinctive features of SFKs suggest that each SFK member plays a unique role in particular tissues or cells. In contrast, it is also known that SFKs have redundant and pleiotropic functions in regulating critical cellular events, such as cell division, motility, adhesion, angiogenesis, and survival (26). In a variety of human cancers, protein levels and/or specific activities of c-Src and c-Yes are frequently upregulated (13, 35). Upregulation of Lyn, Lck, Hck, c-Fgr, or Blk is also observed in some leukemias and lymphomas (10, 16, 26). These observations imply a role for SFKs in cell transformation, tumorigenesis, and metastasis (31). However, because SFK genes are rarely mutated in human cancers (31), the mechanisms underlying their upregulation in these cancers remain unclear. Furthermore, the distinctive expression patterns and functional redundancy among SFK members have hampered concurrent analyses of their intrinsic transforming abilities and contribution to cancer progression.In normal cells, the kinase activity of SFKs is negatively regulated by the phosphorylation of its C-terminal regulatory Tyr residue by C-terminal Src kinase (Csk) (21, 22). The cytoplasmic Csk requires Csk-binding scaffold proteins to gain efficient access to membrane-bound SFKs. Previously, we identified a transmembrane adaptor protein, Cbp (also known as PAG1), as a specific Csk-binding protein. Cbp/PAG1 is exclusively localized to a membrane microdomain enriched by cholesterol and sphingolipids and plays a scaffolding role for Cbp/PAG1 in Csk-mediated negative regulation of SFKs (3, 15). We also reported that expression of Cbp/PAG1 is noticeably downregulated by c-Src transformation and in some human cancer cells and that reexpression of Cbp/PAG1 can suppress c-Src-induced transformation and tumorigenesis (23). In addition, we showed that Cbp/PAG1 suppressed c-Src function independently of Csk by directly sequestering activated c-Src in the membrane microdomain. These findings suggest a potential role for Cbp/PAG1 as a suppressor for c-Src-mediated cancer progression. However, whether Cbp/PAG1 would serve as a suppressor for other SFK members and whether other microdomain adaptors, such as LIME (4, 11), would also contribute to the suppression of SFK-mediated transformation have yet to be examined.The membrane microdomain has been regarded as a signaling platform that harbors various signaling molecules and positively transduces cell signaling evoked by activated receptors (29). This model has been best exemplified in immunoreceptor-mediated signaling (8). Moreover, it was reported that SFKs could function positively when bound to Cbp/PAG1 in the microdomain (30, 32). Such positive roles of the microdomain in cell signaling are apparently inconsistent with its suppressive role in Src-mediated transformation. However, this discrepancy rather raises the possibility that the membrane microdomain would function to segregate or protect the normal signaling pathway from the transforming pathways. To prove this hypothesis, more extensive analysis of the role of the membrane microdomain in controlling cell transformation remains to be performed (28).To elucidate the role of the membrane microdomain in regulating the functions of SFKs, we first compared the transforming abilities of all SFK members using Csk-deficient cells, a reconstitution system in which wild-type SFKs can induce cell transformation (24), and we evaluated the relevance of the membrane distribution of SFKs to their transforming activities. We then investigated the role of the microdomain by disrupting or enhancing its function using methyl-β-cyclodextrin (MβCD) and a microdomain-specific adaptor, Cbp/PAG1, respectively. Our results show that the membrane microdomain and Cbp/PAG1 spatially limit the oncogenic potential of SFKs by sequestering them away from the transforming pathways.