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Raymond J. Deshaies - One of the best experts on this subject based on the ideXlab platform.
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mechanism of lysine 48 linked ubiquitin chain synthesis by the cullin ring ubiquitin ligase complex scf cdc34
Cell, 2005Co-Authors: Matthew D. Petroski, Raymond J. DeshaiesAbstract:Summary Ubiquitin chains linked via lysine 48 (K48) of ubiquitin mediate recognition of ubiquitinated proteins by the proteasome. However, the mechanisms underlying polymerization of this targeting signal on a substrate are unknown. Here we dissect this process using the cyclin-dependent kinase inhibitor Sic1 and its ubiquitination by the cullin-RING ubiquitin ligase SCF Cdc4 and the ubiquitin-conjugating enzyme Cdc34. We show that Sic1 ubiquitination can be separated into two steps: attachment of the first ubiquitin, which is rate limiting, followed by rapid elongation of a K48-linked ubiquitin chain. Mutation of an acidic loop conserved among Cdc34 orthologs has no effect on attachment of the first ubiquitin onto Sic1 but compromises the processivity and linkage specificity of ubiquitin-chain synthesis. We propose that the acidic loop favorably positions K48 of a substrate-linked ubiquitin to attack SCF bound Cdc34∼ubiquitin thioester and thereby enables processive synthesis of K48-linked ubiquitin chains by SCF-Cdc34.
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mechanism of lysine 48 linked ubiquitin chain synthesis by the cullin ring ubiquitin ligase complex scf cdc34
Cell, 2005Co-Authors: Matthew D. Petroski, Raymond J. DeshaiesAbstract:Ubiquitin chains linked via lysine 48 (K48) of ubiquitin mediate recognition of ubiquitinated proteins by the proteasome. However, the mechanisms underlying polymerization of this targeting signal on a substrate are unknown. Here we dissect this process using the cyclin-dependent kinase inhibitor Sic1 and its ubiquitination by the cullin-RING ubiquitin ligase SCF(Cdc4) and the ubiquitin-conjugating enzyme Cdc34. We show that Sic1 ubiquitination can be separated into two steps: attachment of the first ubiquitin, which is rate limiting, followed by rapid elongation of a K48-linked ubiquitin chain. Mutation of an acidic loop conserved among Cdc34 orthologs has no effect on attachment of the first ubiquitin onto Sic1 but compromises the processivity and linkage specificity of ubiquitin-chain synthesis. We propose that the acidic loop favorably positions K48 of a substrate-linked ubiquitin to attack SCF bound Cdc34 approximately ubiquitin thioester and thereby enables processive synthesis of K48-linked ubiquitin chains by SCF-Cdc34.
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Analysis of Polyubiquitin Conjugates Reveals That the Rpn10 Substrate Receptor Contributes to the Turnover of Multiple Proteasome Targets
Molecular & cellular proteomics : MCP, 2005Co-Authors: Thibault Mayor, J. Russell Lipford, Johannes Graumann, Geoffrey T. Smith, Raymond J. DeshaiesAbstract:The polyubiquitin receptor Rpn10 targets ubiquitylated Sic1 to the 26S proteasome for degradation. In contrast, turnover of at least one ubiquitin-proteasome system (UPS) substrate, CPY*, is impervious to deletion of RPN10. To distinguish whether RPN10 is involved in the turnover of only a small set of cell cycle regulators that includes Sic1 or plays a more general role in the UPS, we sought to develop a general method that would allow us to survey the spectrum of ubiquitylated proteins that selectively accumulate in rpn10 cells. Polyubiquitin conjugates from yeast cells that express hexahistidine-tagged ubiquitin (H6-ubiquitin) were first enriched on a polyubiquitin binding protein affinity resin. This material was then denatured and subjected to IMAC to retrieve H6-ubiquitin and proteins to which it may be covalently linked. Using this approach, we identified 127 proteins that are candidate substrates for the 26S proteasome. We then sequenced ubiquitin conjugates from cells lacking Rpn10 (rpn10) and identified 54 proteins that were uniquely recovered from rpn10 cells. These include two known targets of the UPS, the cell cycle regulator Sic1 and the transcriptional activator Gcn4. Our approach of comparing the ubiquitin conjugate proteome in wild-type and mutant cells has the resolving power to identify even an extremely inabundant transcriptional regulatory protein and should be generally applicable to mapping enzyme substrate networks in the UPS.
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In Vitro Reconstitution of SCF Substrate Ubiquitination with Purified Proteins
Methods in enzymology, 2005Co-Authors: Matthew D. Petroski, Raymond J. DeshaiesAbstract:The development of in vitro systems to monitor ubiquitin ligase activity with highly purified proteins has allowed for new insights into the mechanisms of protein ubiquitination to be uncovered. This chapter describes the methodologies employed to reconstitute ubiquitination of the budding yeast cyclin‐dependent kinase inhibitor Sic1 by the evolutionarily conserved ubiquitin ligase SCFCdc4 and its ubiquitin‐conjugating enzyme Cdc34. Based on our experience in reconstituting Sic1 ubiquitination, we suggest some parameters to consider that should be generally applicable to the study of different SCF complexes and other ubiquitin ligases.
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Context of multiubiquitin chain attachment influences the rate of Sic1 degradation.
Molecular cell, 2003Co-Authors: Matthew D. Petroski, Raymond J. DeshaiesAbstract:Abstract The ubiquitin-dependent targeting of proteins to the proteasome is an essential mechanism for regulating eukaryotic protein stability. Here we define the minimal signal for the degradation of the S phase CDK inhibitor Sic1. Of 20 lysines scattered throughout Sic1, 6 N-terminal lysines serve as major ubiquitination sites. Sic1 lacking these lysines (K0N) is stable in vivo, but readdition of any one restores turnover. Nevertheless, ubiquitin chains attached at different N-terminal lysines specify degradation in vitro at markedly different rates. Moreover, although K0N can be ubiquitinated by SCF Cdc4 /Cdc34 in vitro in the absence (but not in the presence) of S-CDK, it is degraded slowly. Our results reveal that a single multiubiquitin chain can sustain a physiological turnover rate, but that chain position plays an unexpectedly significant role in the rate of proteasomal proteolysis.
Mike Tyers - One of the best experts on this subject based on the ideXlab platform.
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Composite low affinity interactions dictate recognition of the cyclin-dependent kinase inhibitor Sic1 by the SCFCdc4 ubiquitin ligase
Proceedings of the National Academy of Sciences of the United States of America, 2012Co-Authors: Xiaojing Tang, Veronika Csizmok, Julie D. Forman-kay, Tanja Mittag, Stephen Orlicky, Frank Sicheri, Tony Pawson, Mike TyersAbstract:The ubiquitin ligase SCFCdc4 (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.
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The Role of Dynamic Protein Complexes in the Ubiquitin-Proteasome Pathway
Biophysical Journal, 2011Co-Authors: Tanja Mittag, Stephen Orlicky, Frank Sicheri, Mike Tyers, Xiaojing Tang, Julie D. Forman-kayAbstract:Intrinsically disordered proteins (IDPs) have been implicated in the regulation of many important cellular processes, such as regulation of cell cycle progression, of transcription and of translation. IDPs are thought to function primarily in the mediation of protein-protein interactions, but detailed analysis of the molecular mechanisms that render disorder beneficial in protein interactions is required. According to a common view, IDPs are usually thought to undergo disorder-to-order transitions upon interaction with their binding partners. It is becoming increasingly clear that predominantly disordered protein complexes are functionally relevant. We have shown previously that the intrinsically disordered cyclin dependent kinase (CDK) inhibitor Sic1 interacts with the substrate adapter, Cdc4, of its ubiquitin ligase via multiple phosphorylated binding motifs in a dynamic complex. Cdc4 is the substrate recognition subunit of a culin ubiquitin ligase and targets Sic1 for degradation at the G1/S phase transition of the yeast cell cycle. Individual binding motifs in Sic1 are ordered transiently without a global disorder-to-order transition upon binding Cdc4. The dynamic complex allows for engagement of several phosphorylation sites in a dynamic interface resulting in an affinity that depends on the number of phosphorylation sites in an ultrasensitive manner. The dynamic nature of the complex allows for ‘counting’ of phosphorylation sites via largely electrostatic interactions. The Sic1-Cdc4 interaction therefore acts as a sensor of the concentration of active kinase and the cell cycle status. We continue to use NMR spectroscopy and other biophysical methods to study dynamic interactions in the ubiquitin proteasome pathway with the objective of unraveling nature's repertoire of disorder in protein function. The combination of disorder with multi-site phosphorylation may serve as a general means to set thresholds in regulated protein-protein interactions.
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New insight into the role of the Cdc34 ubiquitin-conjugating enzyme in cell cycle regulation via Ace2 and Sic1.
Genetics, 2010Co-Authors: Ross Cocklin, Mike Tyers, Josh Heyen, Tolonda R. Larry, Mark G. GoeblAbstract:The Cdc34 ubiquitin-conjugating enzyme plays a central role in progression of the cell cycle. Through analysis of the phenotype of a mutant missing a highly conserved sequence motif within the catalytic domain of Cdc34, we discovered previously unrecognized levels of regulation of the Ace2 transcription factor and the cyclin-dependent protein kinase inhibitor Sic1. In cells carrying the Cdc34tm mutation, which alters the conserved sequence, the cyclin-dependent protein kinase inhibitor Sic1, an SCFCdc4 substrate, has a shorter half-life, while the cyclin Cln1, an SCFGrr1 substrate, has a longer half-life than in wild-type cells. Expression of the Sic1 gene cluster, which is regulated by Swi5 and Ace2 transcription factors, is induced in CDC34tm cells. Levels of Swi5, Ace2, and the SCFGrr1 targets Cln1 and Cln2 are elevated in Cdc34tm cells, and loss of Grr1 causes an increase in Ace2 levels. Sic1 levels are similar in CDC34tm ace2Δ and wild-type cells, explaining a paradoxical increase in the steady-state level of Sic1 protein despite its reduced half-life. A screen for mutations that interact with CDC34tm uncovered novel regulators of Sic1, including genes encoding the polyubiquitin chain receptors Rad23 and Rpn10.
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Structure/Function Implications in a Dynamic Complex of the Intrinsically Disordered Sic1 with the Cdc4 Subunit of an SCF Ubiquitin Ligase
Structure (London England : 1993), 2010Co-Authors: Tanja Mittag, Joseph A. Marsh, Alexander Grishaev, Stephen Orlicky, Hong Lin, Frank Sicheri, Mike Tyers, Julie D. Forman-kayAbstract:Intrinsically disordered proteins can form highly dynamic complexes with partner proteins. One such dynamic complex involves the intrinsically disordered Sic1 with its partner Cdc4 in regulation of yeast cell cycle progression. Phosphorylation of six N-terminal Sic1 sites leads to equilibrium engagement of each phosphorylation site with the primary binding pocket in Cdc4, the substrate recognition subunit of a ubiquitin ligase. ENSEMBLE calculations using experimental nuclear magnetic resonance and small-angle X-ray scattering data reveal significant transient structure in both phosphorylation states of the isolated ensembles (Sic1 and pSic1) that modulates their electrostatic potential, suggesting a structural basis for the proposed strong contribution of electrostatics to binding. A structural model of the dynamic pSic1-Cdc4 complex demonstrates the spatial arrangements in the ubiquitin ligase complex. These results provide a physical picture of a protein that is predominantly disordered in both its free and bound states, enabling aspects of its structure/function relationship to be elucidated.
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structure function implications in a dynamic complex of the intrinsically disordered Sic1 with the cdc4 subunit of an scf ubiquitin ligase
Structure, 2010Co-Authors: Tanja Mittag, Joseph A. Marsh, Alexander Grishaev, Stephen Orlicky, Hong Lin, Frank Sicheri, Mike Tyers, Julie D FormankayAbstract:Intrinsically disordered proteins can form highly dynamic complexes with partner proteins. One such dynamic complex involves the intrinsically disordered Sic1 with its partner Cdc4 in regulation of yeast cell cycle progression. Phosphorylation of six N-terminal Sic1 sites leads to equilibrium engagement of each phosphorylation site with the primary binding pocket in Cdc4, the substrate recognition subunit of a ubiquitin ligase. ENSEMBLE calculations using experimental nuclear magnetic resonance and small-angle X-ray scattering data reveal significant transient structure in both phosphorylation states of the isolated ensembles (Sic1 and pSic1) that modulates their electrostatic potential, suggesting a structural basis for the proposed strong contribution of electrostatics to binding. A structural model of the dynamic pSic1-Cdc4 complex demonstrates the spatial arrangements in the ubiquitin ligase complex. These results provide a physical picture of a protein that is predominantly disordered in both its free and bound states, enabling aspects of its structure/function relationship to be elucidated.
Julie D. Forman-kay - One of the best experts on this subject based on the ideXlab platform.
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Conformational Ensembles of an Intrinsically Disordered Protein Consistent with NMR, SAXS, and Single-Molecule FRET.
Journal of the American Chemical Society, 2020Co-Authors: Gregory-neal W. Gomes, Julie D. Forman-kay, Tanja Mittag, Mickael Krzeminski, Ashley Namini, Erik W. Martin, Teresa Head-gordon, Claudiu C. GradinaruAbstract:Intrinsically disordered proteins (IDPs) have fluctuating heterogeneous conformations, which makes their structural characterization challenging. Although challenging, characterization of the conformational ensembles of IDPs is of great interest, since their conformational ensembles are the link between their sequences and functions. An accurate description of IDP conformational ensembles depends crucially on the amount and quality of the experimental data, how it is integrated, and if it supports a consistent structural picture. We used integrative modeling and validation to apply conformational restraints and assess agreement with the most common structural techniques for IDPs: Nuclear Magnetic Resonance (NMR) spectroscopy, Small-angle X-ray Scattering (SAXS), and single-molecule Forster Resonance Energy Transfer (smFRET). Agreement with such a diverse set of experimental data suggests that details of the generated ensembles can now be examined with a high degree of confidence. Using the disordered N-terminal region of the Sic1 protein as a test case, we examined relationships between average global polymeric descriptions and higher-moments of their distributions. To resolve apparent discrepancies between smFRET and SAXS inferences, we integrated SAXS data with NMR data and reserved the smFRET data for independent validation. Consistency with smFRET, which was not guaranteed a priori, indicates that, globally, the perturbative effects of NMR or smFRET labels on the Sic1 ensemble are minimal. Analysis of the ensembles revealed distinguishing features of Sic1, such as overall compactness and large end-to-end distance fluctuations, which are consistent with biophysical models of Sic1's ultrasensitive binding to its partner Cdc4. Our results underscore the importance of integrative modeling and validation in generating and drawing conclusions from IDP conformational ensembles.
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Integrating multiple experimental data to determine conformational ensembles of an intrinsically disordered protein
2020Co-Authors: Gregory-neal W. Gomes, Julie D. Forman-kay, Tanja Mittag, Mickael Krzeminski, Ashley Namini, Erik W. Martin, Teresa Head-gordon, Claudiu C. GradinaruAbstract:Abstract Intrinsically disordered proteins (IDPs) have fluctuating heterogeneous conformations, which makes structural characterization challenging. Although challenging, characterizing the conformational ensembles of IDPs is of great interest, since their conformational ensembles are the link between their sequences and functions. An accurate description of IDP conformational ensembles depends crucially on the amount and quality of the experimental data used in their calculations. We used integrative modelling to understand and implement conformational restraints imposed by the most common structural techniques for IDPs: Nuclear Magnetic Resonance (NMR) spectroscopy, Small-angle X-ray Scattering (SAXS), and single-molecule Forster Resonance Energy Transfer (smFRET). Agreement with such a diverse set of experimental data suggests that details of the generated ensembles can be examined with a high degree of confidence. Using the disordered N-terminal region of the Sic1 protein as a test case, we examined relationships between average global polymeric descriptions and higher-moments of their distributions. To resolve apparent discrepancies between smFRET and SAXS inferences, we integrated SAXS data with non-smFRET (NMR) data and reserved the smFRET data as an independent validation. Consistency with smFRET, which was not guaranteed a priori, indicates that, globally, the perturbative effects of NMR or smFRET labels on the Sic1 ensemble are minimal. Analysis of the integrative ensembles revealed distinguishing features of Sic1, such as overall compactness and large end-to-end distance fluctuations, which are consistent with biophysical models of Sic1’s ultrasensitive binding to its partner Cdc4. Our results underscore the importance of integrative modelling in calculating and drawing conclusions from IDP conformational ensembles.
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Single Molecule FRET Investigation of the Dimensions and Dynamics in Highly Cooperative Sic1-WD40 Binding
Biophysical Journal, 2017Co-Authors: Gregory W. Gomes, Veronika Csizmok, Jianhui Song, Hue-sun Chan, Julie D. Forman-kay, Claudiu C. GradinaruAbstract:Sic1 is a cyclin-dependent kinase inhibitor which must be phosphorylated on at least six sites (termed Cdc4 phosphodegrons, CPDs) to allow its recognition by the WD40 binding domain of Cdc4. The highly-cooperative switch-like dependence on the number of phosphorylated sites on Sic1 cannot be accounted for by traditional thermodynamic models of cooperativity. Further experimental attention is necessary to determine the physicochemical/mechanistic basis of its highly cooperative binding.We used single molecule fluorescence techniques to study the dimensions and dynamics of Sic1's N-terminal targeting region (residues 1-90, henceforth Sic1), phosphorylated Sic1 (pSic1), and the pSic1-WD40 dynamic complex.Previous single molecule Fӧrster Resonance Energy Transfer (smFRET) measurements [Liu, 2014] observed end-to-end reconfiguration on timescales larger than ∼1ms; resulting in FRET histograms with multiple conformational sub-ensembles. Sic1, pSic1, and the pSic1-WD40 complex are examined using smFRET to study the dynamics and dimensions of the various sub-ensembles. In a refinement to the conventional approaches for inferring dimensions from smFRET experiments, we use distance distributions from Monte Carlo simulations which extensively sample coarse-grained protein conformations. The application of polymer physics theory/simulation towards smFRET data interpretation, and towards IDP binding, contributes to the growing toolkit for understanding the diverse behaviours of IDPs.
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Dimensions and Dynamics of Highly Cooperative Sic1-WD40 Binding: smFRET through a Polymer Physics Lens
Biophysical Journal, 2016Co-Authors: Gregory W. Gomes, Veronika Csizmok, Jianhui Song, Hue-sun Chan, Julie D. Forman-kay, Claudiu C. GradinaruAbstract:Sic1 is a cyclin-dependent kinase inhibitor which must be phosphorylated on at least six sites (termed Cdc4 phosphodegrons, CPDs) to allow its recognition by the WD40 binding domain of Cdc4. The highly-cooperative switch-like dependence on the number of phosphorylated sites on Sic1 cannot be accounted for by traditional thermodynamic models of cooperativity. Further experimental attention is necessary to determine the physicochemical/mechanistic basis of its highly cooperative binding.We used single molecule fluorescence techniques to study the dimensions and dynamics of Sic1's N-terminal targeting region (residues 1-90, henceforth Sic1), phosphorylated Sic1 (pSic1), and the pSic1-WD40 dynamic complex. Using time-resolved fluorescence anisotropy, we find local segment specific rotational correlation times which are complexly modulated by chain compactness and electrostatics (charge screening and phosphorylation).Previous single molecule Fӧrster Resonance Energy Transfer (smFRET) measurements [1] observed end-to-end reconfiguration on timescales larger than ∼1ms; resulting in FRET histograms with multiple conformational sub-ensembles. These sub-ensembles and their dynamics are further explored by trapping single Sic1, pSic1 and WD40 in nanometer sized surface-tethered lipid vesicles and modulating their electrostatics and compactness. In a refinement to the conventional approaches for inferring dimensions from smFRET experiments, we use distance distributions from Monte Carlo simulations which extensively sample coarse-grained protein conformations. The application of polymer physics theory/simulation towards smFRET data interpretation, and towards IDP binding, contributes to the growing toolkit for understanding the diverse behaviours of IDPs.[1] Liu B. et al., J. Phys. Chem. B. 2014 118(15):4088-97.
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The Effect of Intrachain Electrostatic Repulsion on Conformational Disorder and Dynamics of the Sic1 Protein
The journal of physical chemistry. B, 2014Co-Authors: Baoxu Liu, Veronika Csizmok, Julie D. Forman-kay, Patrick Farber, Darius Chia, Claudiu C. GradinaruAbstract:The yeast cyclin-dependent kinase inhibitor Sic1 is a disordered protein that, upon multisite phosphorylation, forms a dynamic complex with the Cdc4 subunit of an SCF ubiquitin ligase. To understand the multisite phosphorylation dependence of the Sic1:Cdc4 interaction, which ultimately leads to a sharp cell cycle transition, the conformational properties of the disordered Sic1 N-terminal targeting region were studied using single-molecule fluorescence spectroscopy. Multiple conformational populations with different sensitivities to charge screening were identified by performing experiments in nondenaturing salts and ionic denaturants. Both the end-to-end distance and the hydrodynamic radius decrease monotonically with increasing the salt concentration, and a rollover of the chain dimensions in high denaturant conditions is observed. The data were fit to the polyelectrolyte binding-screening model, yielding parameters such as the excluded volume of the uncharged chain and the binding constant to denaturant...
Lilia Alberghina - One of the best experts on this subject based on the ideXlab platform.
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A surface-activated chemical ionization approach allows quantitative phosphorylation analysis of the cyclin-dependent kinase inhibitor Sic1 phosphorylated on Ser201.
Rapid communications in mass spectrometry : RCM, 2012Co-Authors: Claudia Cirulli, Lilia Alberghina, Paola Coccetti, Farida TripodiAbstract:RATIONALE Quantitative phosphoproteomics represents a front line for functional proteomics and hence for systems biology. Here we present a new application of the surface-activated chemical ionization (SACI) technology for quantitative phosphoproteomics analysis. The main advantages of SACI-MS technology are high sensitivity, quantitative accuracy and matrix effect reduction, which allow quantitative estimations. METHODS A SACI-MS approach was used to investigate the quantitative in vivo phosphorylation of the cyclin-dependent kinase inhibitor Sic1, a low-abundance protein of Saccharomyces cerevisiae, which is phosphorylated on Ser201 by casein kinase 2 (CK2) and compared its phosphorylation status in cells growing in two different carbon sources (glucose or ethanol). RESULTS Our relative quantification indicated that the Sic1-Ser201 phosphorylation level is about 2-fold higher in ethanol- than in glucose-growing cells, proportional to the Sic1 protein level. This finding is coherent with results of western blot analysis using anti-phospho-Ser201-specific antibody, validating the results obtained with this new SACI approach. CONCLUSIONS The findings presented in this paper indicate that the innovative LC/SACI-MS method, coupled with immunoprecipitation, is a powerful device to obtain quantitative information on the phosphorylation state of low abundance proteins.
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Order propensity of an intrinsically disordered protein, the cyclin-dependent-kinase inhibitor Sic1.
Proteins, 2009Co-Authors: Stefania Brocca, Maria Šamalikova, Marina Lotti, Marco Vanoni, Lilia Alberghina, Vladimir N. Uversky, Rita GrandoriAbstract:Intrinsically disordered proteins (IDPs) carry out important biological functions and offer an instructive model system for folding and binding studies. However, their structural characterization in the absence of interactors is hindered by their highly dynamic conformation. The cyclin-dependent-kinase inhibitor (Cki) Sic1 from Saccharomyces cerevisiae is a key regulator of the yeast cell cycle, which controls entrance into S phase and coordination between cell growth and proliferation. Its last 70 out of 284 residues display functional and structural homology to the inhibitory domain of mammalian p21 and p27. Sic1 has escaped systematic structural characterization until now. Here, complementary biophysical methods are applied to the study of conformational properties of pure Sic1 in solution. Based on sequence analysis, gel filtration, circular dichroism (CD), electrospray-ionization mass spectrometry (ESI-MS), and limited proteolysis, it can be concluded that the whole molecule exists in a highly disordered state and can, therefore, be classified as an IDP. However, the results of these experiments indicate, at the same time, that the protein displays some content in secondary and tertiary structure, having properties similar to those of molten globules or pre-molten globules. Proteolysis-hypersensitive sites cluster at the N-terminus and in the middle of the molecule, while the most structured region resides at the C-terminus, including part of the inhibitory domain and the casein-kinase-2 (CK2) phosphorylation target S201. The mutations S201A and S201E, which are known to affect Sic1 function, do not have significant effects on the conformational properties of the pure protein.
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In CK2 inactivated cells the cyclin dependent kinase inhibitor Sic1 is involved in cell-cycle arrest before the onset of S phase.
Biochemical and biophysical research communications, 2007Co-Authors: Farida Tripodi, Vittoria Zinzalla, Marco Vanoni, Lilia Alberghina, Paola CoccettiAbstract:Protein kinase CK2 is a heterotetramer composed of two catalytic and two regulatory subunits. In Saccharomyces cerevisiae the catalytic subunits (alpha and alpha') are encoded by the CKA1, CKA2 genes. cka1Deltacka2(ts) mutants arrest cell cycle in both G1 and G2/M at 37 degrees C. Hence, it has been proposed that CK2 plays an important role in cell-cycle progression and several cell-cycle proteins have been reported to be CK2 substrates. We have previously shown that Sic1, the inhibitor of Clb5-Cdc28 complexes required for the G1/S transition, is a physiologically relevant CK2 substrate. Here we show that CK2 inactivation up-regulates Sic1 level resulting in severe down-regulation of Clb5-Cdc28 kinase activity. Concurrent inactivation of Sic1 and CK2 leads to accumulation of cells with a post-synthetic DNA content and short/elongated spindles, typical of cells arrested in mitosis. These findings indicate that Sic1 plays a major role during G1 arrest of CK2-inactivated cells.
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Rapamycin-mediated G1 arrest involves regulation of the Cdk inhibitor Sic1 in Saccharomyces cerevisiae.
Molecular microbiology, 2007Co-Authors: Vittoria Zinzalla, Andrea Mastriani, Marco Vanoni, Michele Graziola, Lilia AlberghinaAbstract:The rapamycin-sensitive (TOR) signalling pathway in Saccharomyces cerevisiae controls growth and cell proliferation in response to nutrient availability. Rapamycin treatment causes cells to arrest growth in G1 phase. The mechanism by which the inhibition of the TOR pathway regulates cell cycle progression is not completely understood. Here we show that rapamycin causes G1 arrest by a dual mechanism that comprises downregulation of the G1-cyclins Cln1-3 and upregulation of the Cdk inhibitor protein Sic1. The increase of Sic1 level is mostly independent of the downregulation of the G1 cyclins, being unaffected by ectopic CLN2 expression, but requires Sic1 phosphorylation of Thr173, because it is lost in cells expressing Sic1(T173A). Rapamycin-mediated Sic1 upregulation involves nuclear accumulation of a more stable, non-ubiquitinated protein. Either Sic1 deletion or CLN3 overexpression results in non-cell-cycle-specific arrest upon rapamycin treatment and makes cells sensitive to a sublethal dose of rapamycin and to nutrient starvation. In conclusion, our data indicate that Sic1 is involved in rapamycin-induced G1 arrest and that deregulated entrance into S phase severely decreases the ability of a cell to cope with starvation conditions induced by nutrient depletion or which are mimicked by rapamycin treatment.
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Sic1 is phosphorylated by CK2 on Ser201 in budding yeast cells.
Biochemical and biophysical research communications, 2006Co-Authors: Paola Coccetti, Vittoria Zinzalla, Marco Vanoni, Oriano Marin, Lorenzo A. Pinna, Gabriella Tedeschi, Gian Luigi Russo, Sonia Fantinato, Lilia AlberghinaAbstract:We have previously identified Ser201 of Sic1, a yeast cyclin-dependent kinase inhibitor, as an in vitro target of protein kinase CK2. Here we present new evidence, by using specific anti-P-Ser201 antibodies and 2-D gel electrophoresis coupled to MALDI mass spectrometry analysis, that Sic1 is phosphorylated in vivo on Ser201 shortly after its de novo synthesis, during late anaphase in glucose-grown cells. This phosphorylation is also detected in Sic1 immunopurified from G1 cells. In agreement with these data we also show that the catalytic alpha' subunit of CK2, whose function is required for cell cycle progression, is detected in Sic1 immunopurified complexes, and that phosphorylation on Ser201 is reduced after CK2 inactivation at the non-permissive temperature in a cka1delta cka2(ts) yeast strain. These data strongly support the notion that CK2 phosphorylates Sic1 in vivo.
Frank Sicheri - One of the best experts on this subject based on the ideXlab platform.
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Composite low affinity interactions dictate recognition of the cyclin-dependent kinase inhibitor Sic1 by the SCFCdc4 ubiquitin ligase
Proceedings of the National Academy of Sciences of the United States of America, 2012Co-Authors: Xiaojing Tang, Veronika Csizmok, Julie D. Forman-kay, Tanja Mittag, Stephen Orlicky, Frank Sicheri, Tony Pawson, Mike TyersAbstract:The ubiquitin ligase SCFCdc4 (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.
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The Role of Dynamic Protein Complexes in the Ubiquitin-Proteasome Pathway
Biophysical Journal, 2011Co-Authors: Tanja Mittag, Stephen Orlicky, Frank Sicheri, Mike Tyers, Xiaojing Tang, Julie D. Forman-kayAbstract:Intrinsically disordered proteins (IDPs) have been implicated in the regulation of many important cellular processes, such as regulation of cell cycle progression, of transcription and of translation. IDPs are thought to function primarily in the mediation of protein-protein interactions, but detailed analysis of the molecular mechanisms that render disorder beneficial in protein interactions is required. According to a common view, IDPs are usually thought to undergo disorder-to-order transitions upon interaction with their binding partners. It is becoming increasingly clear that predominantly disordered protein complexes are functionally relevant. We have shown previously that the intrinsically disordered cyclin dependent kinase (CDK) inhibitor Sic1 interacts with the substrate adapter, Cdc4, of its ubiquitin ligase via multiple phosphorylated binding motifs in a dynamic complex. Cdc4 is the substrate recognition subunit of a culin ubiquitin ligase and targets Sic1 for degradation at the G1/S phase transition of the yeast cell cycle. Individual binding motifs in Sic1 are ordered transiently without a global disorder-to-order transition upon binding Cdc4. The dynamic complex allows for engagement of several phosphorylation sites in a dynamic interface resulting in an affinity that depends on the number of phosphorylation sites in an ultrasensitive manner. The dynamic nature of the complex allows for ‘counting’ of phosphorylation sites via largely electrostatic interactions. The Sic1-Cdc4 interaction therefore acts as a sensor of the concentration of active kinase and the cell cycle status. We continue to use NMR spectroscopy and other biophysical methods to study dynamic interactions in the ubiquitin proteasome pathway with the objective of unraveling nature's repertoire of disorder in protein function. The combination of disorder with multi-site phosphorylation may serve as a general means to set thresholds in regulated protein-protein interactions.
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Structure/Function Implications in a Dynamic Complex of the Intrinsically Disordered Sic1 with the Cdc4 Subunit of an SCF Ubiquitin Ligase
Structure (London England : 1993), 2010Co-Authors: Tanja Mittag, Joseph A. Marsh, Alexander Grishaev, Stephen Orlicky, Hong Lin, Frank Sicheri, Mike Tyers, Julie D. Forman-kayAbstract:Intrinsically disordered proteins can form highly dynamic complexes with partner proteins. One such dynamic complex involves the intrinsically disordered Sic1 with its partner Cdc4 in regulation of yeast cell cycle progression. Phosphorylation of six N-terminal Sic1 sites leads to equilibrium engagement of each phosphorylation site with the primary binding pocket in Cdc4, the substrate recognition subunit of a ubiquitin ligase. ENSEMBLE calculations using experimental nuclear magnetic resonance and small-angle X-ray scattering data reveal significant transient structure in both phosphorylation states of the isolated ensembles (Sic1 and pSic1) that modulates their electrostatic potential, suggesting a structural basis for the proposed strong contribution of electrostatics to binding. A structural model of the dynamic pSic1-Cdc4 complex demonstrates the spatial arrangements in the ubiquitin ligase complex. These results provide a physical picture of a protein that is predominantly disordered in both its free and bound states, enabling aspects of its structure/function relationship to be elucidated.
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structure function implications in a dynamic complex of the intrinsically disordered Sic1 with the cdc4 subunit of an scf ubiquitin ligase
Structure, 2010Co-Authors: Tanja Mittag, Joseph A. Marsh, Alexander Grishaev, Stephen Orlicky, Hong Lin, Frank Sicheri, Mike Tyers, Julie D FormankayAbstract:Intrinsically disordered proteins can form highly dynamic complexes with partner proteins. One such dynamic complex involves the intrinsically disordered Sic1 with its partner Cdc4 in regulation of yeast cell cycle progression. Phosphorylation of six N-terminal Sic1 sites leads to equilibrium engagement of each phosphorylation site with the primary binding pocket in Cdc4, the substrate recognition subunit of a ubiquitin ligase. ENSEMBLE calculations using experimental nuclear magnetic resonance and small-angle X-ray scattering data reveal significant transient structure in both phosphorylation states of the isolated ensembles (Sic1 and pSic1) that modulates their electrostatic potential, suggesting a structural basis for the proposed strong contribution of electrostatics to binding. A structural model of the dynamic pSic1-Cdc4 complex demonstrates the spatial arrangements in the ubiquitin ligase complex. These results provide a physical picture of a protein that is predominantly disordered in both its free and bound states, enabling aspects of its structure/function relationship to be elucidated.
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Dynamic equilibrium engagement of a polyvalent ligand with a single-site receptor.
Proceedings of the National Academy of Sciences of the United States of America, 2008Co-Authors: Tanja Mittag, Stephen Orlicky, Hong Lin, Frank Sicheri, Mike Tyers, Xiaojing Tang, Wing-yiu Choy, Lewis E. Kay, Julie D. Forman-kayAbstract:Intrinsically disordered proteins play critical but often poorly understood roles in mediating protein interactions. The interactions of disordered proteins studied to date typically entail structural stabilization, whether as a global disorder-to-order transition or minimal ordering of short linear motifs. The disordered cyclin-dependent kinase (CDK) inhibitor Sic1 interacts with a single site on its receptor Cdc4 only upon phosphorylation of its multiple dispersed CDK sites. The molecular basis for this multisite-dependent interaction with a single receptor site is not known. By NMR analysis, we show that multiple phosphorylated sites on Sic1 interact with Cdc4 in dynamic equilibrium with only local ordering around each site. Regardless of phosphorylation status, Sic1 exists in an intrinsically disordered state but is surprisingly compact with transient structure. The observation of this unusual binding mode between Sic1 and Cdc4 extends the understanding of protein interactions from predominantly static complexes to include dynamic ensembles of intrinsically disordered states.