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The human tumor suppressor arf interacts with spinophilin/neurabin II, a type 1 protein-phosphatase-binding protein
M Vivo1, R A Calogero, F Sansone
1Department of Genetics, General and Molecular Biology, University of Naples "Federico II," via Mezzocannone 8, Napoli 80134, Italy.
Abstract:
The INK4a gene, one of the most often disrupted loci in human cancer, encodes two unrelated proteins, p16(INK4a) and p14(ARF) (ARF) both capable of inducing cell cycle arrest. Although it has been clearly demonstrated that ARF inhibits cell cycle via p53 stabilization, very little is known about the involvement of ARF in other cell cycle regulatory pathways, as well as on the mechanisms responsible for activating ARF following oncoproliferative stimuli. In search of factors that might associate with ARF to control its activity or its specificity, we performed a yeast two-hybrid screen. We report here that the human homologue of spinophilin/neurabin II, a regulatory subunit of protein phosphatase 1 catalytic subunit specifically interacts with ARF, both in yeast and in mammalian cells. We also show that ectopic expression of spinophilin/neurabin II inhibits the formation of G418-resistant colonies when transfected into human and mouse cell lines, regardless of p53 and ARF status. Moreover, spinophilin/ARF coexpression in Saos-2 cells, where ARF ectopic expression is ineffective, somehow results in a synergic effect. These data demonstrate a role for spinophilin in cell growth and suggest that ARF and spinophilin could act in partially overlapping pathways.
Insights
The tumor suppressor p14(ARF) interacts with spinophilin, a protein phosphatase 1 regulatory subunit. This interaction suggests spinophilin plays a role in cell growth and may function in pathways overlapping with p14(ARF).
Area of Science:
- Oncology
- Molecular Biology
- Cell Cycle Regulation
Background:
- The INK4a gene locus is frequently disrupted in human cancers, encoding p16(INK4a) and p14(ARF), both cell cycle arrest inducers.
- While p14(ARF)'s role in cell cycle arrest via p53 stabilization is known, its broader pathway involvement and activation mechanisms remain unclear.
Purpose of the Study:
- To identify novel interacting partners of p14(ARF) to elucidate its regulatory mechanisms.
- To investigate the functional consequences of p14(ARF) and its interacting partners on cell proliferation.
Main Methods:
- Yeast two-hybrid screening to identify ARF-interacting proteins.
- Co-immunoprecipitation assays in yeast and mammalian cells to confirm protein interactions.
- Cell-based assays measuring colony formation after transfection with spinophilin and/or ARF.
Main Results:
- The human homologue of spinophilin/neurabin II was identified as a direct interactor of p14(ARF).
- Spinophilin/neurabin II expression inhibited colony formation in human and mouse cell lines, independent of p53 and ARF status.
- Coexpression of spinophilin and ARF showed synergistic inhibition of colony formation in Saos-2 cells, where ARF alone was ineffective.
Conclusions:
- Spinophilin plays a significant role in regulating cell growth.
- ARF and spinophilin may function in partially overlapping cell cycle regulatory pathways.
- The interaction between ARF and spinophilin offers new insights into cancer development and potential therapeutic targets.