Multisite Phosphorylation of the
            <i>Saccharomyces cerevisiae</i>
            Filamentous Growth Regulator Tec1 Is Required for its Recognition by the E3 Ubiquitin Ligase Adaptor Cdc4 and Its Subsequent Destruction In Vivo

The ubiquitin ligase SCF Cdc4 (Skp1/Cul1/F-box protein) recognizes its substrate, the cyclin-dependent kinase inhibitor Sic1, in a multisite phosphorylation-dependent manner. Although short diphosphorylated peptides derived from Sic1 can bind to Cdc4 with high affinity, through systematic mutagenesis and quantitative biophysical analysis we show that individually weak, dispersed Sic1 phospho sites engage Cdc4 in a dynamic equilibrium. The affinities of individual phosphoepitopes serve to tune the overall phosphorylation site threshold needed for efficient recognition. Notably, phosphoepitope affinity for Cdc4 is dramatically weakened in the context of full-length Sic1, demonstrating the importance of regional environment on binding interactions. The multisite nature of the Sic1-Cdc4 interaction confers cooperative dependence on kinase activity for Sic1 recognition and ubiquitination under equilibrium reaction conditions. Composite dynamic interactions of low affinity sites may be a general mechanism to establish phosphorylation thresholds in biological responses.

Section: Discussionmentioning

confidence: 95%

Composite low affinity interactions dictate recognition of the cyclin-dependent kinase inhibitor Sic1 by the SCF^Cdc4ubiquitin ligase

Tang

Orlicky

Mittag

et al. 2012

“…Cdc4 often recognizes its substrates through a phosphodegron containing two phosphorylation sites spaced closely together (20,21,23), although data on Sic1 in budding yeast suggests that this spacing may not be strictly required (36). We find that maximal degradation of Hst3 requires two such phosphodegrons, and each phospho-degron contributes to the increased turnover in response to replication stress.…”

Section: Hst3 Turnover Is Involved In Proper Regulation Of Acetylatedmentioning

confidence: 84%

“…SCF Cdc4 recognizes its substrates through a phospho-degron (20)(21)(22). To understand how Hst3 was targeted for turnover both during an unperturbed cell cycle and after treatment with HU, we mapped replication stress-dependent and -independent phosphorylation sites on Hst3 by mass spectrometry.…”

Section: Resultsmentioning

confidence: 99%

“…1D, wild-type Hst3 binds to Cdc4ΔF but not to GST alone (lanes 7 and 5). Cdc4 often binds its substrates through a Cdc4 phospho-degron containing closely spaced phosphorylation sites (20)(21)(22). Within the Hst3 C terminus are two small clusters of phosphorylation sites that correspond to the di-phosphorylated consensus (20,21,23), which in the subsequent analysis we called "degron 1" (D1) and "degron 2" (D2) (shown in Fig.…”

mentioning

confidence: 99%

See 1 more Smart Citation

Hst3 is turned over by a replication stress-responsive SCF ^Cdc4 phospho-degron

Edenberg

Vashisht

Topacio

et al. 2014

Hst3 is the histone deacetylase that removes histone H3K56 acetylation. H3K56 acetylation is a cell-cycle-and damage-regulated chromatin marker, and proper regulation of H3K56 acetylation is important for replication, genomic stability, chromatin assembly, and the response to and recovery from DNA damage. Understanding the regulation of enzymes that regulate H3K56 acetylation is of great interest, because the loss of H3K56 acetylation leads to genomic instability. HST3 is controlled at both the transcriptional and posttranscriptional level. Here, we show that Hst3 is targeted for turnover by the ubiquitin ligase SCF Cdc4 after phosphorylation of a multisite degron. In addition, we find that Hst3 turnover increases in response to replication stress in a Rad53-dependent way. Turnover of Hst3 is promoted by Mck1 activity in both conditions. The Hst3 degron contains two canonical Cdc4 phospho-degrons, and the phosphorylation of each of these is required for efficient turnover both in an unperturbed cell cycle and in response to replication stress.H istone H3K56 acetylation is a cell-cycle-and damageregulated histone modification (1). In Saccharomyces cerevisiae, K56 acetylation is important for replication, genomic stability, chromatin assembly, and the response and recovery from DNA damage (1-6). Because of the critical nature of this chromatin mark, the enzymes that control it are themselves very important.In the budding yeast S. cerevisiae, Hst3 is a sirtuin histone deacetylase that, along with its close homolog Hst4, removes K56 acetylation (5, 7, 8). Overexpression of Hst3 or deletion of Hst3, along with related deacetylase Hst4, sensitizes cells to replication stress, suggesting that cells must control Hst3 levels precisely (5, 7). Hst3 is regulated both transcriptionally and by protein turnover. During an unperturbed cell cycle, HST3 transcript levels cycle, and Hst3 protein turns over very quickly (5,9). In response to DNA damage, transcription of HST3 is turned down, and Hst3 protein turnover is even faster (5,8,(10)(11)(12).Many cell-cycle-regulated proteins are targeted for proteasomal degradation after ubiquitination by a member of the SCF E3 ubiquitin ligase family. SCF ligases are multisubunit ubiquitin ligases composed of Skp1, a cullin (Cdc53), a RING-finger protein (Rbx1), and an F-box protein (13). The F-box protein is the substrate recognition module that confers substrate specificity to SCF ligases, with different F-box proteins recognizing different sets of substrates. The essential F-box protein Cdc4 regulates many cell-cycle-regulated proteins after their phosphorylation, including Sic1, Eco1, Cln3, and many others (14-19).Here, we have characterized the mechanism of Hst3 protein turnover. We find that Hst3 is targeted for degradation by SCF Cdc4 through a multisite phospho-degron. We find that Hst3 turnover is increased in response to replication stress in a Rad53-dependent way. Finally, we show that stabilizing Hst3 leads to misregulation of K56 acetylation in response to DNA damage and that...

“…The yeast mating pathway is one of the best-studied signal transduction networks in eukaryotes, and our work capitalized on the current molecular understanding of fast-timescale regulation by its associated MAP kinase, Fus3. The transcription factor Tec1 is a Fus3 (MAPK) substrate that, when phosphorylated, binds the WD40 domain of Cdc4 (an SCF ubiquitin ligase complex adaptor protein) (25). To generate a minimal WD40 phospho-binding motif, we combined a short, 11-residue peptide from Tec1 that, when phosphorylated, binds to the WD40 domain, with a well-characterized Fus3 docking motif (from the MAPKK Ste7) (26) to form a 49-residue phospho-regulon tag.…”

Section: Significancementioning

confidence: 99%

Engineering dynamical control of cell fate switching using synthetic phospho-regulons

Gordley

Williams

Bashor

et al. 2016

Many cells can sense and respond to time-varying stimuli, selectively triggering changes in cell fate only in response to inputs of a particular duration or frequency. A common motif in dynamically controlled cells is a dual-timescale regulatory network: although longterm fate decisions are ultimately controlled by a slow-timescale switch (e.g., gene expression), input signals are first processed by a fast-timescale signaling layer, which is hypothesized to filter what dynamic information is efficiently relayed downstream. Directly testing the design principles of how dual-timescale circuits control dynamic sensing, however, has been challenging, because most synthetic biology methods have focused solely on rewiring transcriptional circuits, which operate at a single slow timescale. Here, we report the development of a modular approach for flexibly engineering phosphorylation circuits using designed phospho-regulon motifs. By then linking rapid phospho-feedback with slower downstream transcription-based bistable switches, we can construct synthetic dualtimescale circuits in yeast in which the triggering dynamics and the end-state properties of the ON state can be selectively tuned. These phospho-regulon tools thus open up the possibility to engineer cells with customized dynamical control.L ong-term cell fates can often be selectively triggered by specific temporal patterns (dynamics) of stimulation (Fig. 1A). Relatively few cellular systems that "decode" time-varying inputs have been characterized in detail, but recurrent network motifs are beginning to emerge (1, 2). One key feature that is often observed in such systems is the interlinking of circuits that operate on distinct timescales (3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14). In perhaps the best example of a biological "dynamic gate," the synaptic remodeling of neurons is mediated by two layers of regulation ( Fig. 1B): first, an upstream circuit of rapid but transient allosteric and posttranslational changes detects incoming stimuli and filters for high-frequency pulses; second, the signal is transmitted to downstream circuits regulated by slower processes (gene expression, trafficking, and morphological changes), which ultimately can yield stable alterations in receptor localization and synaptic function (4, 6). This common motif suggests that a simple solution for achieving tunable dynamic control systems is to link fast and slow subnetworks, whereby the upstream fast system processes how the intrinsically slow downstream switch receives and responds to external dynamic inputs.To test this hypothesis, we engineered synthetic cellular circuits based on linked fast (phosphorylation)-and slow (gene expression)-timescale modules (Fig. 1C). We first developed a versatile method for building fast-timescale signaling circuits in yeast using modular phospho-regulons. We then linked engineered phospho-feedback circuits with an intrinsically slow downstream transcription-based bistable switch, and were thereby able to generate a dynamic cell fate switch in yeast ...