Epidermal growth factor-induced rapid retinoblastoma phosphorylation at Ser⁷⁸⁰ and Ser⁷⁹⁵ is mediated by ERK1/2 in small intestine epithelial cells

The retinoblastoma protein Rb is critical for the regulation of mammalian cell cycle entry. Hypophosphorylated Rb is considered to be the active form and directs G₁ arrest, while hyperphosphorylated Rb permits the transition from G₁ to S phase for cell proliferation. Upon stimulation by various growth factors, Rb appears to be phosphorylated by a cascade of phosphorylation events mediated mainly by kinases associated with cyclins D and E. Here we report that in prototype small intestine crypt stem cells (RIEC-6), stimulation with either epidermal growth factor or fetal bovine serum results in an unexpected rapid and sustained Rb phosphorylation at sites Ser⁷⁸⁰, Ser⁷⁹⁵, and Thr⁸²¹ which precedes cyclin D1 expression, cyclin D1/cdk4 complex formation, and cdk4 kinase activity. Rb phosphorylation at Ser⁷⁸⁰ and Ser⁷⁹⁵ is prevented by MEK, but not phosphatidylinositol 3-kinase, inhibitors. In vitro, Rb is directly phosphorylated by active ERK1/2 as shown by [γ- ³²P]ATP labeling. The phosphorylation sites are further directed to Ser⁷⁸⁰ and Ser⁷⁹⁵ by kinase assays using recombined active ERK1/2 or immunoprecipitated phospho-ERK1/2 from mitogen stimulated cells. Pull-down assays revealed that Rb interacts with active ERK1/2 but not their inactive unphosphorylated forms. Upon EGF stimulation, phosphorylated ERK1/2 co-immunoprecipitates together with phosphorylated Rb. Collectively, these results demonstrate a novel rapid Rb phosphorylation at specific sites induced by mitogen stimulation in epithelial cells of the small intestine. These data specifically identify ERK1/2 as the kinase responsible for Rb phosphorylation targeted to sites Ser⁷⁸⁰ and Ser⁷⁹⁵. It appears that ERK1/2 could be an important link between a mitogenic signal directly to Rb, thereby providing a rapid response mechanism between mitogen stimulation and cell cycle machinery.