Proteome analysis of NIH3T3 cells transformed by activated Galpha12: regulation of leukemia-associated protein SET.
Galpha(12), the alpha-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 Galpha(12) (Galpha(12)Q229L or Galpha(12)QL) 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 Galpha(12)QL-transformed NIH3T3, we carried out a two-dimensional differential proteome analysis of Galpha(12)QL-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 Galpha(12)QL cells were validated by immunoblot analysis. Furthermore, transient transfection studies with NIH3T3 cells indicated that the expression of activated Galpha(12) 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 Galpha(12). Our results indicate that the nuclear phosphatase activity is inhibited by greater than 50% in Galpha(12)QL 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 Galpha(12)-mediated signaling pathways and a role for Galpha(12) in the regulation of the leukemia-associated SET-protein expression.
Kumar
Rashmi N
RN
Fels Institute for Cancer Research and Molecular Biology, Temple University School of Medicine, Philadelphia, PA 19140, USA.
Radhakrishnan
Rangasudhakar
R
Ha
Ji Hee
JH
Dhanasekaran
N
N
D000818:Animals; D002467:Cell Nucleus / Q000737:chemistry; D002471:Cell Transformation, Neoplastic / Q000235:genetics*; D002868:Chromosomal Proteins, Non-Histone / Q000032:analysis / Q000235:genetics*; D044365:GTP-Binding Protein alpha Subunits, G12-G13 / Q000502:physiology*; D015972:Gene Expression Regulation, Neoplastic; D051379:Mice; D041681:NIH 3T3 Cells; D010749:Phosphoprotein Phosphatases / Q000378:metabolism; D018809:Proliferating Cell Nuclear Antigen / Q000032:analysis / Q000235:genetics; D020543:Proteome / Q000235:genetics*; D015398:Signal Transduction; D014157:Transcription Factors / Q000032:analysis / Q000235:genetics*
D002868:Chromosomal Proteins, Non-Histone; D018809:Proliferating Cell Nuclear Antigen; D020543:Proteome; D014157:Transcription Factors; D010749:Phosphoprotein Phosphatases; D044365:GTP-Binding Protein alpha Subunits, G12-G13