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Multisite phosphorylation of proteins has been proposed to transform a graded protein kinase signal into an ultrasensitive switch-like response. Although many multiphosphorylated targets have been identified, the dynamics and sequence of individual phosphorylation events within the multisite phosphorylation process have never been thoroughly studied. In(More)
The retinoblastoma (Rb) protein negatively regulates the G1-S transition by binding to the E2F transcription factors, until cyclin-dependent kinases phosphorylate Rb, causing E2F release. The Rb pocket domain is necessary for E2F binding, but the Rb C-terminal domain (RbC) is also required for growth suppression. Here we demonstrate a high-affinity(More)
Inactivation of the RB protein is one of the most fundamental events in cancer. Coming to a molecular understanding of its function in normal cells and how it impedes cancer development has been challenging. Historically, the ability of RB to regulate the cell cycle placed it in a central role in proliferative control, and research focused on RB regulation(More)
The order and timing of cell-cycle events is controlled by changing substrate specificity and different activity thresholds of cyclin-dependent kinases (CDKs). However, it is not understood how a single protein kinase can trigger hundreds of switches in a sufficiently time-resolved fashion. We show that cyclin-Cdk1-Cks1-dependent phosphorylation of(More)
Cyclin-dependent kinase (Cdk) phosphorylation of the Retinoblastoma protein (Rb) drives cell proliferation through inhibition of Rb complexes with E2F transcription factors and other regulatory proteins. We present the first structures of phosphorylated Rb that reveal the mechanism of its inactivation. S608 phosphorylation orders a flexible "pocket" domain(More)
Cks is an evolutionarily conserved protein that regulates cyclin-dependent kinase (CDK) activity. Clarifying the underlying mechanisms and cellular contexts of Cks function is critical because Cks is essential for proper cell growth, and its overexpression has been linked to cancer. We observe that budding-yeast Cks associates with select phosphorylated(More)
Yeast telomeres comprise irregular TG₁₋₃ DNA repeats bound by the general transcription factor Rap1. Rif1 and Rif2, along with Rap1, form the telosome, a protective cap that inhibits telomerase, counteracts SIR-mediated transcriptional silencing, and prevents inadvertent recognition of telomeres as DNA double-strand breaks. We provide a molecular,(More)
Eukaryotic cell division is controlled by cyclin-dependent kinases (CDKs), which require phosphorylation by a CDK-activating kinase (CAK) for full activity. Chemical genetics uncovered requirements for the metazoan CAK Cdk7 in determining cyclin specificity and activation order of Cdk2 and Cdk1 during S and G2 phases. It was unknown if Cdk7 also activates(More)
The retinoblastoma tumor suppressor (RB) is a central cell cycle regulator and tumor suppressor. RB cellular functions are known to be regulated by a diversity of post-translational modifications such as phosphorylation and acetylation, raising the possibility that RB may also be methylated in cells. Here we demonstrate that RB can be methylated by SMYD2 at(More)
DNA replication fork stalling poses a major threat to genome stability. This is counteracted in part by the intra-S phase checkpoint, which stabilizes arrested replication machinery, prevents cell-cycle progression and promotes DNA repair. The checkpoint kinase Mec1/ATR and RecQ helicase Sgs1/BLM contribute synergistically to fork maintenance on hydroxyurea(More)