Michael B. Yaffe

The Structural Basis for 14-3-3:Phosphopeptide Binding Specificity

The 14-3-3 family of proteins mediates signal transduction by binding to phosphoserine-containing proteins. Using phosphoserine-oriented peptide libraries to probe all mammalian and yeast 14-3-3s, we identified two different binding motifs, RSXpSXP and RXY/FXpSXP, present in nearly all known 14-3-3 binding proteins. The crystal structure of 14-3-3zeta complexed with the phosphoserine motif in polyoma middle-T was determined to 2.6 A resolution. The bound peptide is in an extended conformation, with a tight turn created by the pS +2 Pro in a cis conformation. Sites of peptide-protein interaction in the complex rationalize the peptide library results. Finally, we show that the 14-3-3 dimer binds tightly to single molecules containing tandem repeats of phosphoserine motifs, implicating bidentate association as a signaling mechanism with molecules such as Raf, BAD, and Cbl.

Scansite 2.0: proteome-wide prediction of cell signaling interactions using short sequence motifs

Scansite identifies short protein sequence motifs that are recognized by modular signaling domains, phosphorylated by protein Ser/Thr- or Tyr-kinases or mediate specific interactions with protein or phospholipid ligands. Each sequence motif is represented as a position-specific scoring matrix (PSSM) based on results from oriented peptide library and phage display experiments. Predicted domain-motif interactions from Scansite can be sequentially combined, allowing segments of biological pathways to be constructed in silico. The current release of Scansite, version 2.0, includes 62 motifs characterizing the binding and/or substrate specificities of many families of Ser/Thr- or Tyr-kinases, SH2, SH3, PDZ, 14-3-3 and PTB domains, together with signature motifs for PtdIns(3,4,5)P(3)-specific PH domains. Scansite 2.0 contains significant improvements to its original interface, including a number of new generalized user features and significantly enhanced performance. Searches of all SWISS-PROT, TrEMBL, Genpept and Ensembl protein database entries are now possible with run times reduced by approximately 60% when compared with Scansite version 1.0. Scansite 2.0 allows restricted searching of species-specific proteins, as well as isoelectric point and molecular weight sorting to facilitate comparison of predictions with results from two-dimensional gel electrophoresis experiments. Support for user-defined motifs has been increased, allowing easier input of user-defined matrices and permitting user-defined motifs to be combined with pre-compiled Scansite motifs for dual motif searching. In addition, a new series of Sequence Match programs for non-quantitative user-defined motifs has been implemented. Scansite is available via the World Wide Web at http://scansite.mit.edu.

RNF8 transduces the DNA-damage signal via histone ubiquitylation and checkpoint protein assembly.

DNA-damage signaling utilizes a multitude of posttranslational modifiers as molecular switches to regulate cell-cycle checkpoints, DNA repair, cellular senescence, and apoptosis. Here we show that RNF8, a FHA/RING domain-containing protein, plays a critical role in the early DNA-damage response. We have solved the X-ray crystal structure of the FHA domain structure at 1.35 A. We have shown that RNF8 facilitates the accumulation of checkpoint mediator proteins BRCA1 and 53BP1 to the damaged chromatin, on one hand through the phospho-dependent FHA domain-mediated binding of RNF8 to MDC1, on the other hand via its role in ubiquitylating H2AX and possibly other substrates at damage sites. Moreover, RNF8-depleted cells displayed a defective G2/M checkpoint and increased IR sensitivity. Together, our study implicates RNF8 as a novel DNA-damage-responsive protein that integrates protein phosphorylation and ubiquitylation signaling and plays a critical role in the cellular response to genotoxic stress.

MDC1 Directly Binds Phosphorylated Histone H2AX to Regulate Cellular Responses to DNA Double-Strand Breaks

Histone variant H2AX phosphorylation in response to DNA damage is the major signal for recruitment of DNA-damage-response proteins to regions of damaged chromatin. Loss of H2AX causes radiosensitivity, genome instability, and DNA double-strand-break repair defects, yet the mechanisms underlying these phenotypes remain obscure. Here, we demonstrate that mammalian MDC1/NFBD1 directly binds to phospho-H2AX (gammaH2AX) by specifically interacting with the phosphoepitope at the gammaH2AX carboxyl terminus. Moreover, through a combination of biochemical, cell-biological, and X-ray crystallographic approaches, we reveal the molecular details of the MDC1/NFBD1-gammaH2AX complex. These data provide compelling evidence that the MDC1/NFBD1 BRCT repeat domain is the major mediator of gammaH2AX recognition following DNA damage. We further show that MDC1/NFBD1-gammaH2AX complex formation regulates H2AX phosphorylation and is required for normal radioresistance and efficient accumulation of DNA-damage-response proteins on damaged chromatin. Thus, binding of MDC1/NFBD1 to gammaH2AX plays a central role in the mammalian response to DNA damage.

The mTOR-regulated phosphoproteome reveals a mechanism of mTORC1-mediated inhibition of growth factor signaling

A search for substrates of a growth-promoting kinase revealed a regulatory feedback loop involved in tumor suppression. The mammalian target of rapamycin (mTOR) protein kinase is a master growth promoter that nucleates two complexes, mTORC1 and mTORC2. Despite the diverse processes controlled by mTOR, few substrates are known. We defined the mTOR-regulated phosphoproteome by quantitative mass spectrometry and characterized the primary sequence motif specificity of mTOR using positional scanning peptide libraries. We found that the phosphorylation response to insulin is largely mTOR dependent and that mTOR exhibits a unique preference for proline, hydrophobic, and aromatic residues at the +1 position. The adaptor protein Grb10 was identified as an mTORC1 substrate that mediates the inhibition of phosphoinositide 3-kinase typical of cells lacking tuberous sclerosis complex 2 (TSC2), a tumor suppressor and negative regulator of mTORC1. Our work clarifies how mTORC1 inhibits growth factor signaling and opens new areas of investigation in mTOR biology.

Systematic discovery of in vivo phosphorylation networks

Protein kinases control cellular decision processes by phosphorylating specific substrates. Thousands of in vivo phosphorylation sites have been identified, mostly by proteome-wide mapping. However, systematically matching these sites to specific kinases is presently infeasible, due to limited specificity of consensus motifs, and the influence of contextual factors, such as protein scaffolds, localization, and expression, on cellular substrate specificity. We have developed an approach (NetworKIN) that augments motif-based predictions with the network context of kinases and phosphoproteins. The latter provides 60%-80% of the computational capability to assign in vivo substrate specificity. NetworKIN pinpoints kinases responsible for specific phosphorylations and yields a 2.5-fold improvement in the accuracy with which phosphorylation networks can be constructed. Applying this approach to DNA damage signaling, we show that 53BP1 and Rad50 are phosphorylated by CDK1 and ATM, respectively. We describe a scalable strategy to evaluate predictions, which suggests that BCLAF1 is a GSK-3 substrate.

The Molecular Basis for Phosphodependent Substrate Targeting and Regulation of Plks by the Polo-Box Domain

Polo-like kinases (Plks) perform crucial functions in cell-cycle progression and multiple stages of mitosis. Plks are characterized by a C-terminal noncatalytic region containing two tandem Polo boxes, termed the Polo-box domain (PBD), which has recently been implicated in phosphodependent substrate targeting. We show that the PBDs of human, Xenopus, and yeast Plks all recognize similar phosphoserine/threonine-containing motifs. The 1.9 A X-ray structure of a human Plk1 PBD-phosphopeptide complex shows that the Polo boxes each comprise beta6alpha structures that associate to form a 12-stranded beta sandwich domain. The phosphopeptide binds along a conserved, positively charged cleft located at the edge of the Polo-box interface. Mutations that specifically disrupt phosphodependent interactions abolish cell-cycle-dependent localization and provide compelling phenotypic evidence that PBD-phospholigand binding is necessary for proper mitotic progression. In addition, phosphopeptide binding to the PBD stimulates kinase activity in full-length Plk1, suggesting a conformational switching mechanism for Plk regulation and a dual functionality for the PBD.

How do 14-3-3 proteins work? – Gatekeeper phosphorylation and the molecular anvil hypothesis

14‐3‐3 proteins were the first signaling molecules to be identified as discrete phosphoserine/threonine binding modules. This family of proteins, which includes seven isotypes in human cells and up to 15 in plants, plays critical roles in cell signaling events that control progress through the cell cycle, transcriptional alterations in response to environmental cues, and programmed cell death. Despite over 30 years of research, distinct roles for most isotypes remain unknown. Though 14‐3‐3 proteins perform different functions for different ligands, general mechanisms of 14‐3‐3 action include changes in activity of bound ligands, altered association of bound ligands with other cellular components, and changes in intracellular localization of 14‐3‐3‐bound cargo. We present a speculative model where binding of 14‐3‐3 to multiple sites on some ligands results in global ligand conformational changes that mediate their biological effects. For these multi‐site ligands, one binding site is likely to function as a ‘gatekeeper’ whose phosphorylation is necessary for 14‐3‐3 binding but may not always be sufficient for full biological activity. If correct, then 14‐3‐3 may prove to be a bona fide phosphodependent signaling chaperone.

14-3-3 proteins and survival kinases cooperate to inactivate BAD by BH3 domain phosphorylation.

The Bcl-2 homology 3 (BH3) domain of prodeath Bcl-2 family members mediates their interaction with prosurvival Bcl-2 family members and promotes apoptosis. We report that survival factors trigger the phosphorylation of the proapoptotic Bcl-2 family member BAD at a site (Ser-155) within the BAD BH3 domain. When BAD is bound to prosurvival Bcl-2 family members, BAD Ser-155 phosphorylation requires the prior phosphorylation of Ser-136, which recruits 14-3-3 proteins that then function to increase the accessibility of Ser-155 to survival-promoting kinases. Ser-155 phosphorylation disrupts the binding of BAD to prosurvival Bcl-2 proteins and thereby promotes cell survival. These findings define a mechanism by which survival signals inactivate a proapoptotic Bcl-2 family member, and suggest a role for 14-3-3 proteins as cofactors that regulate sequential protein phosphorylation events.

A motif-based profile scanning approach for genome-wide prediction of signaling pathways

The rapid increase in genomic information requires new techniques to infer protein function and predict protein–protein interactions. Bioinformatics identifies modular signaling domains within protein sequences with a high degree of accuracy. In contrast, little success has been achieved in predicting short linear sequence motifs within proteins targeted by these domains to form complex signaling networks. Here we describe a peptide library-based searching algorithm, accessible over the World Wide Web, that identifies sequence motifs likely to bind to specific protein domains such as 14-3-3, SH2, and SH3 domains, or likely to be phosphorylated by specific protein kinases such as Src and AKT. Predictions from database searches for proteins containing motifs matching two different domains in a common signaling pathway provides a much higher success rate. This technology facilitates prediction of cell signaling networks within proteomes, and could aid in the identification of drug targets for the treatment of human diseases.