Fig. 3: Rb is first phosphorylated at T373 before its C-terminal sites.
From: An intermediate Rb–E2F activity state safeguards proliferation commitment
a, Domain architecture of Rb, E2F1 and DP1. Rb consists of a structured amino-terminal domain (RbN) and RbP. Its RbC is disordered except for a short sequence (RbC(core)) that adopts a structure after E2F binding. CC, coiled-coil domain; DBD, DNA-binding domain. b, Left, Rb–E2F1–DP1 complex prediction with AlphaFold. Rb(ΔCDK(43–928)) (CDK phosphorylation sites mutated to alanines to mimic unphosphorylated Rb), E2F1(200–437), and DP1(198–410) were used to predict the structure of the complex. Right, two interaction sites between Rb and E2F1–DP1: (1) RbP interacts with E2F(TD) and (2) RbC interacts with E2F(MB)–DP(MB). c,d, PSP plots showing single-cell correlation of Rb phosphorylation (phos-Rb) between two different sites 16 h after release with starvation medium + EGF (20 ng ml–1) + CDK4/6i (20 nM) (c) or release after starvation medium + EGF (20 ng ml–1) + CDK4/6i (1 µM) (d). Each phosphorylation signal was normalized by the total Rb antibody signal in the same cell and each axis was adjusted to the average phosphorylation signal in S phase of 1 (when Rb is hyperphosphorylated). A red line shows fitting with a preferential relative phosphorylation–dephosphorylation rate between the two sites (PSPcoeff) (see Methods for more details). c, Rb phosphorylation at T373 (pT373), S608 (pS608), S780 (pS780) and T826 (pT826) plotted against S807/S811 (pS807/S811). n = 2,734, 2,159, 2,684 and 2203 cells for T373, S608, S780 and T826, respectively; 1 out of 3 biological replicates. d, Rb phosphorylation at T373 plotted against S807/S811. Cells were fixed 8, 16, 24 and 48 h after release. n = 2,531, 2,655, 2,713 and 2,774 cells for 8 h, 16 h, 24 h and 48 h, respectively; 1 out of 2 biological replicates for 8 h, 3 biological replicates for 16 h, 4 biological replicates for 24 and 48 h. e, Model for phosphorylation and inactivation of Rb in a two-step process. First, Rb is phosphorylated at T373 and S608/S612, which probably disrupts (1) the RbP–E2F(TD) interaction. Second, Rb phosphorylation at C-terminal sites disrupts (2) the RbC and E2F(MB)–DP(MB) interaction, leading to full release of Rb from E2F.
