Identification of a new interaction mode between the Src homology 2 domain of C-terminal Src kinase (Csk) and Csk-binding protein/phosphoprotein associated with glycosphingolipid microdomains

This research was originally published in Journal of Biological Chemistry. Hiroaki Tanaka, Ken-ichi Akagi, Chitose Oneyama, Masakazu Tanaka, Yuichi Sasaki, Takashi Kanou, Young-Ho Lee, Daisuke Yokogawa, Marc-Werner Dobenecker, Atsushi Nakagawa, Masato Okada and Takahisa Ikegami. Identification of a new interaction mode between the Src homology 2 domain of C-terminal Src kinase (Csk) and Csk-binding protein/phosphoprotein associated with glycosphingolipid microdomains. Journal of Biological Chemistry. 2013; 288, 15240-15254. © the American Society for Biochemistry and Molecular Biology

Oncogenic mutation or overexpression of oncogenic KRAS or BRAF is not sufficient to confer oncogene addiction.

Oncogene addiction is a cellular property by which cancer cells become highly dependent on the expression of oncogenes for their survival. Oncogene addiction can be exploited to design molecularly targeted drugs that kill only cancer cells by inhibiting the specific oncogenes. Genes and cell lines exhibiting oncogene addiction, as well as the mechanisms by which cell death is induced when addicted oncogenes are suppressed, have been extensively studied. However, it is still not fully understood how oncogene addiction is acquired in cancer cells. Here, we take a synthetic biology approach to investigate whether oncogenic mutation or oncogene expression suffices to confer the property of oncogene addiction to cancer cells. We employed human mammary epithelium-derived MCF-10A cells expressing the oncogenic KRAS or BRAF. MCF-10A cells harboring an oncogenic mutation in a single-allele of KRAS or BRAF showed weak transformation activity, but no characteristics of oncogene addiction. MCF-10A cells overexpressing oncogenic KRAS demonstrated the transformation activity, but MCF-10A cells overexpressing oncogenic BRAF did not. Neither cell line exhibited any oncogene addiction properties. These results indicate that the introduction of oncogenic mutation or the overexpression of oncogenes is not sufficient for cells to acquire oncogene addiction, and that oncogene addiction is not associated with transformation activity

Efficient Epstein-Barr Virus Progeny Production Mediated by Cancer-Derived LMP1 and Virally-Encoded microRNAs

Epstein-Barr virus (EBV) genomes, particularly their latent genes, are heterogeneous among strains. The heterogeneity of EBV-encoded latent membrane protein 1 (LMP1) raises the question of whether there are functional differences between LMP1 expressed by cancer-associated EBV and that by non-cancerous strains. Here, we used bacterial artificial chromosome (BAC)-cloned EBV genomes retaining all virally encoded microRNA (miRNA) genes to investigate the functions of cancer-derived LMP1 in the context of the EBV genome. HEK293 cells were stably transfected with EBV-BAC clone DNAs encoding either nasopharyngeal carcinoma (NPC)-derived CAO-LMP1 (LMP1CAO) or LMP1 from a prototype B95-8 strain of EBV (LMP1B95-8). When an EBV-BAC clone DNA encoding LMP1CAO was stably transfected into HEK293 cells, it generated many more stable transformants than the control clone encoding LMP1B95-8. Furthermore, stably transfected HEK293 cells exhibited highly efficient production of progeny virus. Importantly, deletion of the clustered viral miRNA genes compromised the ability to produce progeny viruses. These results indicate that cancer-derived LMP1 and viral miRNAs together are necessary for efficient production of progeny virus, and that the resulting increase in efficiency contributes to EBV-mediated epithelial carcinogenesis

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

Roles of Raft-Anchored Adaptor Cbp/PAG1 in Spatial Regulation of c-Src Kinase

<div><p>The tyrosine kinase c-Src is upregulated in numerous human cancers, implying a role for c-Src in cancer progression. Previously, we have shown that sequestration of activated c-Src into lipid rafts via a transmembrane adaptor, Cbp/PAG1, efficiently suppresses c-Src-induced cell transformation in Csk-deficient cells, suggesting that the transforming activity of c-Src is spatially regulated via Cbp in lipid rafts. To dissect the molecular mechanisms of the Cbp-mediated regulation of c-Src, a combined analysis was performed that included mathematical modeling and <i>in vitro</i> experiments in a c-Src- or Cbp-inducible system. c-Src activity was first determined as a function of c-Src or Cbp levels, using focal adhesion kinase (FAK) as a crucial c-Src substrate. Based on these experimental data, two mathematical models were constructed, the sequestration model and the ternary model. The computational analysis showed that both models supported our proposal that raft localization of Cbp is crucial for the suppression of c-Src function, but the ternary model, which includes a ternary complex consisting of Cbp, c-Src, and FAK, also predicted that c-Src function is dependent on the lipid-raft volume. Experimental analysis revealed that c-Src activity is elevated when lipid rafts are disrupted and the ternary complex forms in non-raft membranes, indicating that the ternary model accurately represents the system. Moreover, the ternary model predicted that, if Cbp enhances the interaction between c-Src and FAK, Cbp could promote c-Src function when lipid rafts are disrupted. These findings underscore the crucial role of lipid rafts in the Cbp-mediated negative regulation of c-Src-transforming activity, and explain the positive role of Cbp in c-Src regulation under particular conditions where lipid rafts are perturbed.</p></div

The mTOR pathway controls cell proliferation by regulating the FoxO3a transcription factor via SGK1 kinase.

The mechanistic target of rapamycin (mTOR) functions as a component of two large complexes, mTORC1 and mTORC2, which play crucial roles in regulating cell growth and homeostasis. However, the molecular mechanisms by which mTOR controls cell proliferation remain elusive. Here we show that the FoxO3a transcription factor is coordinately regulated by mTORC1 and mTORC2, and plays a crucial role in controlling cell proliferation. To dissect mTOR signaling, mTORC1 was specifically inactivated by depleting p18, an essential anchor of mTORC1 on lysosomes. mTORC1 inactivation caused a marked retardation of cell proliferation, which was associated with upregulation of cyclin-dependent kinase inhibitors (CDKIs). Although Akt was activated by mTORC1 inactivation, FoxO3a was upregulated via an epigenetic mechanism and hypophosphorylated at Ser314, which resulted in its nuclear accumulation. Consistently, mTORC1 inactivation induced downregulation of serum- and glucocorticoid-inducible kinase 1 (SGK1), the kinase responsible for Ser314 phosphorylation. Expression of FoxO3a mutated at Ser314 suppressed cell proliferation by inducing CDKI expression. SGK1 overexpression suppressed CDKI expression in p18-deficient cells, whereas SGK1 knockdown induced CDKI expression in wild-type cells, resulting in the suppression of cell proliferation. These results suggest that mTORC1, in coordination with mTORC2, controls cell proliferation by regulating FoxO3a gene expression and SGK1-mediated phosphorylation of FoxO3a at Ser314