Proteome Analysis of NIH3T3 Cells Transformed by Activated Gα₁₂:  Regulation of Leukemia-Associated Protein SET

Gα₁₂, the α-subunit of the G12 family of heterotrimeric G proteins is involved in the regulation of cell proliferation and neoplastic transformation. GTPase-deficient, constitutively activated mutant of Gα₁₂ (Gα₁₂Q229L or Gα₁₂QL) has been previously shown to induce oncogenic transformation of NIH3T3 cells promoting serum- and anchorage-independent growth. Reduced growth-factor dependent, autonomous cell growth forms a critical defining point at which a normal cell turns into an oncogenic one. To identify the underlying mechanism involved in such growth-factor/serum independent growth of Gα₁₂QL-transformed NIH3T3, we carried out a two-dimensional differential proteome analysis of Gα₁₂QL-transformed NIH3T3 cells and cells expressing vector control. This analysis revealed a total of 22 protein-spots whose expression was altered by more than 3-folds. Two of these spots were identified by MALDI−MS analysis as proliferating cell nuclear antigen (PCNA) and myeloid-leukemia-associated SET protein. The increased expressions of these proteins in Gα₁₂QL cells were validated by immunoblot analysis. Furthermore, transient transfection studies with NIH3T3 cells indicated that the expression of activated Gα₁₂ readily increased the expression of SET protein by 24 h. As SET has been previously reported to be an inhibitor of phosphatase PP2A, the nuclear phosphatase activity was monitored in cells expressing activated Gα₁₂. Our results indicate that the nuclear phosphatase activity is inhibited by greater than 50% in Gα₁₂QL cells compared to vector control cells. Thus, our results from differential proteome analysis presented here report for the first time a role for SET in Gα₁₂-mediated signaling pathways and a role for Gα₁₂ in the regulation of the leukemia-associated SET-protein expression.

Keywords: G protein • Gα₁₂ • SET • PP2A • PCNA • oncogene • cell transformation • cell proliferation