Regulation of the cell cycle by a stable nuclear-localized retinoblastoma tumor suppressor complex in Chlamydomonas
Bradley J.S.C. Olson, Michael Olberhozer and James G. Umen
The Salk Institute for Biological Studies, 10010 N. Torrey Pines Rd, La Jolla, CA 92037
The retinoblastoma (RB) tumor suppressor is a conserved cell cycle regulator that is thought to repress cell cycle progression when bound to E2F-DP transcription factor heterodimers. In the canonical model for the RB pathway phosphorylation of RB-related proteins by cyclin dependent kinases (CDKs) causes release from E2F-DP, thereby allowing cell cycle progression. However, there is now accumulating data that suggests that RB-E2F-DP ternary complexes can act both as activators and repressors. Chlamydomonas reinhardtii undergoes multiple fission where growth and division are uncoupled. During G1-phase, cells can grow several-fold their original size. The number of fission cycles that the cells goes through is proportional to the size the cell grew to during G1-phase. The RB-related protein MAT3 controls cell size by gating each entry and re-entry into S phase until cells have returned to the proper daughter cell size. In order to understand how RB controls S phase entry we examined the cell cycle dynamics of RB, E2F and DP in Chlamydomonas and found that Chlamydomonas MAT3/RB is a phosphoprotein; that E2F1-DP1 can bind to a consensus E2F site; and that all three proteins interact in vivo to form a complex that can be quantitatively immunopurified. We examined the abundance of MAT3/RB and E2F1-DP1 in highly synchronous cultures and found that they are synthesized and remain stably associated throughout the cell cycle with no detectable fraction of free E2F1-DP1. Consistent with their stable association, MAT3/RB and DP1 are constitutively nuclear and MAT3/RB does not require DP1-E2F1 for localization. Together, our data show that cell cycle regulation by RB can occur without dissociation from its E2F-DP subunits, and that other changes may be sufficient to convert RB-E2F-DP from a cell cycle repressor to an activator.
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