ABSTRACT
Isoform-specific signaling by Class IA PI 3-kinases depends in part on the interactions between distinct catalytic subunits and upstream regulatory proteins. From among the class IA catalytic subunits (p110α, p110ß, and p110δ), p110ß has unique properties. Unlike the other family members, p110ß directly binds to Gßγ subunits, downstream from activated G-protein coupled receptors, and to activated Rab5. Furthermore, the Ras-binding domain (RBD) of p110ß binds to Rac and Cdc42 but not to Ras. Defining mutations that specifically disrupt these regulatory interactions is critical for defining their role in p110ß signaling. This chapter describes the approach that was used to identify the Rab5 binding site in p110ß, and discusses methods for the analysis of p110ß-Rab5 interactions.
Subject(s)
Catalytic Domain , Phosphatidylinositol 3-Kinases/chemistry , Phosphatidylinositol 3-Kinases/metabolism , Protein Interaction Mapping/methods , rab5 GTP-Binding Proteins/metabolism , Guanosine 5'-O-(3-Thiotriphosphate)/chemistry , Guanosine Diphosphate/chemistry , HEK293 Cells , Humans , Immobilized Proteins/chemistry , Immobilized Proteins/genetics , Immobilized Proteins/isolation & purification , Immobilized Proteins/metabolism , Phosphatidylinositol 3-Kinases/genetics , Protein Binding , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , rab5 GTP-Binding Proteins/chemistry , rab5 GTP-Binding Proteins/genetics , rab5 GTP-Binding Proteins/isolation & purificationABSTRACT
Tumor suppressor gene DBC2 stops growth of tumor cells through regulation of CCND1. Interference of CCND1 down-regulation prevented growth arrest caused by DBC2 [T. Yoshihara, D. Collado, M. Hamaguchi, Cyclin D1 down-regulation is essential for DBC2's tumor suppressor function, Biochemical and biophysical research communications 358 (2007) 1076-1079]. It was also noted that DBC2 resistant cells eventually arose after repeated induction of DBC2 with muristerone A treatment [M. Hamaguchi, J.L. Meth, C. Von Klitzing, W. Wei, D. Esposito, L. Rodgers, T. Walsh, P. Welcsh, M.C. King, M.H. Wigler, DBC2, a candidate for a tumor suppressor gene involved in breast cancer, Proc. Natl. Acad. Sci. USA 99 (2002) 13647-13652]. In order to elucidate the mechanism of resistance acquisition, we analyzed DBC2 sensitive and resistant cells derived from the same progenitor cells (T-47D). We discovered that DBC2 protein was abundantly expressed in the sensitive cells when DBC2 was induced. In contrast, it was undetectable by western blot analysis in the resistant cells. We confirmed that the inducible gene expression system was responsive in both cells by detecting induced GFP. Additionally, inhibition of 26S proteasome by MG132 revealed production of DBC2 protein in the resistant cells. These findings indicate that the resistant T-47D cells survive DBC2 induction by rapid destruction of DBC2 through 26S proteasome-mediated protein degradation.
Subject(s)
Breast Neoplasms/metabolism , GTP-Binding Proteins/genetics , GTP-Binding Proteins/metabolism , Gene Expression Regulation, Neoplastic/genetics , Proteasome Endopeptidase Complex/metabolism , Tumor Suppressor Proteins/genetics , Tumor Suppressor Proteins/metabolism , Blotting, Western , Breast Neoplasms/drug therapy , Breast Neoplasms/genetics , Cell Line , Cysteine Proteinase Inhibitors/pharmacology , Drug Resistance, Neoplasm/genetics , Ecdysterone/analogs & derivatives , Ecdysterone/pharmacology , Female , Gene Expression Regulation, Neoplastic/drug effects , Gene Transfer Techniques , Humans , Leupeptins/pharmacology , Proteasome Inhibitors , TransgenesABSTRACT
The expression of tumor suppressor gene DBC2 causes certain breast cancer cells to stop growing [M. Hamaguchi, J.L. Meth, C. Von Klitzing, W. Wei, D. Esposito, L. Rodgers, T. Walsh, P. Welcsh, M.C. King, M.H. Wigler, DBC2, a candidate for a tumor suppressor gene involved in breast cancer, Proc. Natl. Acad. Sci. USA 99 (2002) 13647-13652]. Recently, DBC2 was found to participate in diverse cellular functions such as protein transport, cytoskeleton regulation, apoptosis, and cell cycle control [V. Siripurapu, J.L. Meth, N. Kobayashi, M. Hamaguchi, DBC2 significantly influences cell cycle, apoptosis, cytoskeleton, and membrane trafficking pathways. J. Mol. Biol. 346 (2005) 83-89]. Its tumor suppression mechanism, however, remains unclear. In this paper, we demonstrate that DBC2 suppresses breast cancer proliferation through down-regulation of Cyclin D1 (CCND1). Additionally, the constitutional overexpression of CCND1 prevented the negative impact of DBC2 expression on their growth. Under a CCND1 promoter, the expression of CCNE1 exhibited the same protective effect. Our results indicate that the down-regulation of CCND1 is an essential step for DBC2's growth suppression of cancer cells. We believe that this discovery contributes to a better understanding of DBC2's tumor suppressor function.
