The N-Terminal 24 Amino Acids of the p55 Gamma Regulatory Subunit of Phosphoinositide 3-Kinase Binds Rb and Induces Cell Cycle Arrest

Abstract

Although phosphoinositide 3-kinase (PI 3-kinase) is essential for cell cycle progression, the molecular mechanisms that regulate its diverse biological effects are poorly understood. We demonstrate here that Rb, a key regulator of cell cycle progression, associates with p55 kDa (p55α and p55γ) regulatory subunits of PI 3-kinase in vivo and in vitro. Both confocal microscopy and biochemical analysis demonstrated the presence of p55γ in the nucleus. The 24-amino-acid N-terminal end of p55γ, which is unique among PI 3-kinase regulatory subunits, was sufficient to bind Rb. Addition of serum or growth factors to quiescent cells triggered the dissociation of Rb from p55. Ectopic expression of the 24-amino-acid N-terminal end of p55γ inhibited cell cycle progression, as evidenced by induction of cell growth arrest at the G₀/G₁ phase, inhibition of DNA synthesis, inhibition of cyclin D and cyclin E promoter activity, and changes in the expression of cell cycle-related proteins. The inhibitory effects of the N-terminal end of p55γ on cell cycle progression depended on the presence of functional Rb. These data demonstrate for the first time an association of p55γ with Rb and show that modification of this association can lead to cell cycle arrest.

ACKNOWLEDGMENTS

We thank S. Mittnacht, J. Downward, R. Livingston, T. Kouzarides, C. Kahn, and R. Pestell for various plasmid constructs used in this study; T. Jacks for the Rb^−/− and Rb^+/+ MEFs; N. Wehman for help in cell cycle analysis and cell sorting; and A. Passaniti, X. Wang, and H. Cheng for critical reading of the manuscript.

This work was supported in part by NIH grant R01 CA76047 and University of Maryland DRIF funds (to A.W.H.).