Many protein-modifying enzymes recognize their substrates via docking motifs, but the range of functionally permissible motif sequences is often poorly defined. During eukaryotic cell division, ...cyclin-specific docking motifs help cyclin-dependent kinases (CDKs) phosphorylate different substrates at different stages, thus enforcing a temporally ordered series of events. In budding yeast, CDK substrates with Leu/Pro-rich (LP) docking motifs are recognized by Cln1/2 cyclins in late G1 phase, yet the key sequence features of these motifs were unknown. Here, we comprehensively analyze LP motif requirements in vivo by combining a competitive growth assay with deep mutational scanning. We quantified the effect of all single-residue replacements in five different LP motifs by using six distinct G1 cyclins from diverse fungi including medical and agricultural pathogens. The results uncover substantial tolerance for deviations from the consensus sequence, plus requirements at some positions that are contingent on the favorability of other motif residues. They also reveal the basis for variations in functional potency among wild-type motifs, and allow derivation of a quantitative matrix that predicts the strength of other candidate motif sequences. Finally, we find that variation in docking motif potency can advance or delay the time at which CDK substrate phosphorylation occurs, and thereby control the temporal ordering of cell cycle regulation. The overall results provide a general method for surveying viable docking motif sequences and quantifying their potency in vivo, and they reveal how variations in docking strength can tune the degree and timing of regulatory modifications.
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•In vivo assay quantifies the functional effect of hundreds of docking motif variants•Hierarchical and contingent residue preferences define a spectrum of motif strengths•A quantitative matrix surpasses a simple consensus for predicting motif potency•Variation in docking motif potency modulates the degree and timing of CDK regulation
CDKs use cyclin-mediated docking interactions to recognize substrates, but the docking motifs lack a quantitative description of how sequence variations affect recognition. Bandyopadhyay et al. combine deep mutational scanning with an in vivo competitive growth assay to quantify the functional strength of motif variants and define recognition rules.
Linear motifs are short segments of multidomain proteins that provide regulatory functions independently of protein tertiary structure. Much of intracellular signalling passes through protein ...modifications at linear motifs. Many thousands of linear motif instances, most notably phosphorylation sites, have now been reported. Although clearly very abundant, linear motifs are difficult to predict de novo in protein sequences due to the difficulty of obtaining robust statistical assessments. The ELM resource at http://elm.eu.org/ provides an expanding knowledge base, currently covering 146 known motifs, with annotation that includes >1300 experimentally reported instances. ELM is also an exploratory tool for suggesting new candidates of known linear motifs in proteins of interest. Information about protein domains, protein structure and native disorder, cellular and taxonomic contexts is used to reduce or deprecate false positive matches. Results are graphically displayed in a 'Bar Code' format, which also displays known instances from homologous proteins through a novel 'Instance Mapper' protocol based on PHI-BLAST. ELM server output provides links to the ELM annotation as well as to a number of remote resources. Using the links, researchers can explore the motifs, proteins, complex structures and associated literature to evaluate whether candidate motifs might be worth experimental investigation.
Exploring the diversity of SPRY/B30.2-mediated interactions Perfetto, Livia; Gherardini, Pier Federico; Davey, Norman E. ...
Trends in biochemical sciences (Amsterdam. Regular ed.),
January 2013, 2013, 2013-Jan, 2013-1-00, 20130101, Letnik:
38, Številka:
1
Journal Article
Recenzirano
The SPla/Ryanodine receptor (SPRY)/B30.2 domain is one of the most common folds in higher eukaryotes. The human genome encodes 103 SPRY/B30.2 domains, several of which are involved in the immune ...response. Approximately 45% of human SPRY/B30.2-containing proteins are E3 ligases. The role and function of the majority of SPRY/B30.2 domains are still poorly understood, however, in several cases mutations in this domain have been linked to congenital disorders. The recent characterization of SPRY/B30.2-mediated protein interactions has provided evidence for a role of this domain as an adaptor module to assemble macromolecular complexes, analogous to Src homology (SH)2, SH3, and WW domains. However, functional and structural evidence suggests that SPRY/B30.2 is a more versatile fold, allowing a wide range of binding modes.
PP1 and PP2A-B56 are major serine/threonine phosphatase families that achieve specificity by colocalizing with substrates. At the kinetochore, however, both phosphatases localize to an almost ...identical molecular space and yet they still manage to regulate unique pathways and processes. By switching or modulating the positions of PP1/PP2A-B56 at kinetochores, we show that their unique downstream effects are not due to either the identity of the phosphatase or its precise location. Instead, these phosphatases signal differently because their kinetochore recruitment can be either inhibited (PP1) or enhanced (PP2A) by phosphorylation inputs. Mathematical modeling explains how these inverse phospho-dependencies elicit unique forms of cross-regulation and feedback, which allows otherwise indistinguishable phosphatases to produce distinct network behaviors and control different mitotic processes. Furthermore, our genome-wide analysis suggests that these major phosphatase families may have evolved to respond to phosphorylation inputs in opposite ways because many other PP1 and PP2A-B56-binding motifs are also phospho-regulated.
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•PP1 and PP2A-B56 can functionally substitute for each other at the kinetochore•The major difference is their ability to respond to phospho-inputs in opposite ways•This underlies their distinct phenotypic behaviors•Many other signaling pathways also select for these same key features
Smith et al. investigate PP1 and PP2A-B56 specificity at kinetochores and conclude that the main difference between these phosphatases is their ability to respond in opposite ways to phosphorylation inputs. This allows them to produce distinct network behaviors and control different mitotic processes. These unique features have likely been exploited by many other signaling pathways throughout evolution.
The identification of protein surfaces required for interaction with other biomolecules broadens our understanding of protein function, their regulation by post-translational modification, and the ...deleterious effect of disease mutations. Protein interaction interfaces are often identifiable as patches of conserved residues on a protein's surface. However, finding conserved accessible surfaces on folded regions requires an understanding of the protein structure to discriminate between functional and structural constraints on residue conservation. With the emergence of deep learning methods for protein structure prediction, high-quality structural models are now available for any protein. In this study, we introduce tools to identify conserved surfaces on AlphaFold2 structural models. We define autonomous structural modules from the structural models and convert these modules to a graph encoding residue topology, accessibility, and conservation. Conserved surfaces are then extracted using a novel eigenvector centrality-based approach. We apply the tool to the human proteome identifying hundreds of uncharacterised yet highly conserved surfaces, many of which contain clinically significant mutations. The xProtCAS tool is available as open-source Python software and an interactive web server.
Protein kinase B (AKT1) is a central node in a signaling pathway that regulates cell survival. The diverse pathways regulated by AKT1 are communicated in the cell via the phosphorylation of perhaps ...more than 100 cellular substrates. AKT1 is itself activated by phosphorylation at Thr-308 and Ser-473. Despite the fact that these phosphorylation sites are biomarkers for cancers and tumor biology, their individual roles in shaping AKT1 substrate selectivity are unknown. We recently developed a method to produce AKT1 with programmed phosphorylation at either or both of its key regulatory sites. Here, we used both defined and randomized peptide libraries to map the substrate selectivity of site-specific, singly and doubly phosphorylated AKT1 variants. To globally quantitate AKT1 substrate preferences, we synthesized three AKT1 substrate peptide libraries: one based on 84 “known” substrates and two independent and larger oriented peptide array libraries (OPALs) of ∼1011 peptides each. We found that each phospho-form of AKT1 has common and distinct substrate requirements. Compared with pAKT1T308, the addition of Ser-473 phosphorylation increased AKT1 activities on some, but not all of its substrates. This is the first report that Ser-473 phosphorylation can positively or negatively regulate kinase activity in a substrate-dependent fashion. Bioinformatics analysis indicated that the OPAL-activity data effectively discriminate known AKT1 substrates from closely related kinase substrates. Our results also enabled predictions of novel AKT1 substrates that suggest new and expanded roles for AKT1 signaling in regulating cellular processes.
The Database of Protein Disorder (DisProt, URL: www.disprot.org) has been significantly updated and upgraded since its last major renewal in 2007. The current release holds information on more than ...800 entries of IDPs/IDRs, i.e. intrinsically disordered proteins or regions that exist and function without a well-defined three-dimensional structure. We have re-curated previous entries to purge DisProt from conflicting cases, and also upgraded the functional classification scheme to reflect continuous advance in the field in the past 10 years or so. We define IDPs as proteins that are disordered along their entire sequence, i.e. entirely lack structural elements, and IDRs as regions that are at least five consecutive residues without well-defined structure. We base our assessment of disorder strictly on experimental evidence, such as X-ray crystallography and nuclear magnetic resonance (primary techniques) and a broad range of other experimental approaches (secondary techniques). Confident and ambiguous annotations are highlighted separately. DisProt 7.0 presents classified knowledge regarding the experimental characterization and functional annotations of IDPs/IDRs, and is intended to provide an invaluable resource for the research community for a better understanding structural disorder and for developing better computational tools for studying disordered proteins.
The ubiquitin protein ligase anaphase-promoting complex or cyclosome (APC/C) controls mitosis by promoting ordered degradation of securin, cyclins, and other proteins. The mechanisms underlying the ...timing of APC/C substrate degradation are poorly understood. We explored these mechanisms using quantitative fluorescence microscopy of GFP-tagged APC/C(Cdc20) substrates in living budding yeast cells. Degradation of the S cyclin, Clb5, begins early in mitosis, followed 6 min later by the degradation of securin and Dbf4. Anaphase begins when less than half of securin is degraded. The spindle assembly checkpoint delays the onset of Clb5 degradation but does not influence securin degradation. Early Clb5 degradation depends on its interaction with the Cdk1-Cks1 complex and the presence of a Cdc20-binding "ABBA motif" in its N-terminal region. The degradation of securin and Dbf4 is delayed by Cdk1-dependent phosphorylation near their Cdc20-binding sites. Thus, a remarkably diverse array of mechanisms generates robust ordering of APC/C(Cdc20) substrate destruction.
Short linear motifs (SLiMs) are protein interaction sites that play an important role in cell regulation by controlling protein activity, localization, and local abundance. The functionality of a ...SLiM can be modulated in a context-dependent manner to induce a gain, loss, or exchange of binding partners, which will affect the function of the SLiM-containing protein. As such, these conditional interactions underlie molecular decision-making in cell signaling. We identified multiple types of pre- and posttranslational switch mechanisms that can regulate the function of a SLiM and thereby control its interactions. The collected examples of experimentally characterized SLiM-based switch mechanisms were curated in the freely accessible switches.ELM resource (http://switches.elm.eu.org). On the basis of these examples, we defined and integrated rules to analyze SLiMs for putative regulatory switch mechanisms. We applied these rules to known validated SLiMs, providing evidence that more than half of these are likely to be pre- or posttranslationally regulated. In addition, we showed that posttranslationally modified sites are enriched around SLiMs, which enables cooperative and integrative regulation of protein interaction interfaces. We foresee switches.ELM complementing available resources to extend our knowledge of the molecular mechanisms underlying cell signaling.
Polyphosphates (polyP) are chains of inorganic phosphates found in all cells. Previous work has implicated these chains in diverse functions, but the mechanism of action is unclear. A recent study ...reports that polyP can be non-enzymatically and covalently attached to lysine residues on yeast proteins Nsr1 and Top1. One question emerging from this work is whether so-called “polyphosphorylation” is unique to these proteins or instead functions as a global regulator akin to other lysine post-translational modifications. Here, we present the results of a screen for polyphosphorylated proteins in yeast. We uncovered 15 targets including a conserved network of proteins functioning in ribosome biogenesis. Multiple genes contribute to polyphosphorylation of targets by regulating polyP synthesis, and disruption of this synthesis results in translation defects as measured by polysome profiling. Finally, we identify 6 human proteins that can be modified by polyP, highlighting the therapeutic potential of manipulating polyphosphorylation in vivo.
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•A strategy for identifying targets of lysine polyphosphorylation•15 yeast targets include a conserved network involved in ribosome biogenesis•Defects in polyP synthesis result in phenotypes related to translation•Demonstration of polyphosphorylation on 6 PASK-containing human proteins
Bentley-DeSousa et al. screen yeast for proteins that undergo covalent modification by polyphosphate. They describe 15 substrates enriched for functions related to ribosome biogenesis. Homologs of these and other human proteins containing certain motifs can be “polyphosphorylated” using an ectopic expression system, providing a method to explore polyphosphorylation beyond yeast.
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