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Michael B Yaffe

Publications and source records attributed to Michael B Yaffe.

At least 37 records · Page 2Linked to original sources
PubMed2005

A structural basis for 14-3-3sigma functional specificity.

The 14-3-3 family of proteins includes seven isotypes in mammalian cells that play numerous diverse roles in intracellular signaling. Most 14-3-3 proteins form homodimers and mixed heterodimers between different isotypes, with overlapping roles in ligand binding. In contrast, one mammalian isoform, 14-3-3sigma, expressed primarily in epithelial cells, appears to play a unique role in the cellular response to DNA damage and in human oncogenesis. The biological and structural basis for these 14-3-3sigma-specific functions is unknown. We demonstrate that endogenous 14-3-3sigma preferentially forms homodimers in cells. We have solved the x-ray crystal structure of 14-3-3sigma bound to an optimal phosphopeptide ligand at 2.4 angstroms resolution. The structure reveals the presence of stabilizing ring-ring and salt bridge interactions unique to the 14-3-3sigma homodimer structure and potentially destabilizing electrostatic interactions between subunits in 14-3-3sigma-containing heterodimers, rationalizing preferential homodimerization of 14-3-3sigma in vivo. The interaction of the phosphopeptide with 14-3-3 reveals a conserved mechanism for phospho-dependent ligand binding, implying that the phosphopeptide binding cleft is not the critical determinant of the unique biological properties of 14-3-3sigma. Instead, the structure suggests a second ligand binding site involved in 14-3-3sigma-specific ligand discrimination. We have confirmed this by site-directed mutagenesis of three sigma-specific residues that uniquely define this site. Mutation of these residues to the alternative sequence that is absolutely conserved in all other 14-3-3 isotypes confers upon 14-3-3sigma the ability to bind to Cdc25C, a ligand that is known to bind to other 14-3-3 proteins but not to sigma.

14-3-3 Proteins↗
PubMed2005

Structure and function of Polo-like kinases.

Polo-like kinases play critical roles during multiple stages of cell cycle progression. All Polo-like kinases contain an N-terminal Ser/Thr kinase catalytic domain and a C-terminal region that contains one or two Polo-boxes. For Polo-like kinase 1, 2, and 3, and their homologs, the entire C-terminal region, including both Polo-boxes, functions as a single modular phosphoserine/threonine-binding domain known as the Polo-box domain (PBD). In the absence of a bound substrate, the PBD inhibits the basal activity of the kinase domain. Phosphorylation-dependent binding of the PBD to its ligands releases the kinase domain, while simultaneously localizing Polo-like kinases to specific subcellular structures. These observations suggest two different models for how the PBD integrates signals arising from other mitotic kinases to target the activated kinase towards distinct substrates. The recent X-ray crystal structures of the PBD provide insights into the structural basis for PBD function and kinase regulation. Molecular modelling of the structure of the isolated kinase domain reveals a potential basis for motif-dependent substrate specificity.

Cell Cycle Proteins↗
PubMed2005

MAPKAP kinase-2 is a cell cycle checkpoint kinase that regulates the G2/M transition and S phase progression in response to UV irradiation.

The cellular response to DNA damage is mediated by evolutionarily conserved Ser/Thr kinases, phosphorylation of Cdc25 protein phosphatases, binding to 14-3-3 proteins, and exit from the cell cycle. To investigate DNA damage responses mediated by the p38/stress-activated protein kinase (SAPK) axis of signaling, the optimal phosphorylation motifs of mammalian p38alpha SAPK and MAPKAP kinase-2 were determined. The optimal substrate motif for MAPKAP kinase-2, but not for p38 SAPK, closely matches the 14-3-3 binding site on Cdc25B/C. We show that MAPKAP kinase-2 is directly responsible for Cdc25B/C phosphorylation and 14-3-3 binding in vitro and in response to UV-induced DNA damage within mammalian cells. Downregulation of MAPKAP kinase-2 eliminates DNA damage-induced G2/M, G1, and intra S phase checkpoints. We propose that MAPKAP kinase-2 is a new member of the DNA damage checkpoint kinase family that functions in parallel with Chk1 and Chk2 to integrate DNA damage signaling responses and cell cycle arrest in mammalian cells.

14-3-3 Proteins↗
PubMed2005

SIRT1 shows no substrate specificity in vitro.

SIR2 is a key regulator of the aging process in many model organisms. The human ortholog SIRT1 plays a pivotal role in the regulation of cellular differentiation, metabolism, cell cycle, and apoptosis. SIRT1 is an NAD(+)-dependent deacetylase, and its enzymatic activity may be regulated by cellular energy. There is a growing number of known SIRT1 substrates that contain epsilon-acetyl lysine but for which no obvious consensus sequence has been defined. In this study, we developed a novel unbiased method to identify deacetylase sequence specificity using oriented peptide libraries containing acetylated lysine. Following incubation with SIRT1, the subset of deacetylated peptides was selectively captured using a photocleavable N-hydroxysuccinimide (NHS)-biotin linker and streptavidin beads and analyzed using mass spectrometry and Edman degradation. These studies revealed that substrate recognition by SIRT1 does not depend on the amino acid sequence proximate to the acetylated lysine. This result brings us one step closer to understanding how SIRT1 and possibly other protein deacetylases chose their substrate.

Amino Acid Sequence↗
PubMed2004

A computational method for the analysis and prediction of protein:phosphopeptide-binding sites.

Phosphopeptide-binding domains, including the FHA, SH2, WW, WD40, MH2, and Polo-box domains, as well as the 14-3-3 proteins, exert control functions in important processes such as cell growth, division, differentiation, and apoptosis. Structures and mechanisms of phosphopeptide binding are generally diverse, revealing few general principles. A computational method for analysis of phosphopeptide-binding domains was therefore developed to elucidate the physical and chemical nature of phosphopeptide binding, given this lack of structural similarity. The surfaces of nine phosphopeptide-binding proteins, representing seven distinct classes of phosphopeptide-binding modules, were discretized, and encoded with information about amino acid identity, surface curvature, and electrostatic potential at every point on the surface in order to identify local surface properties enriched in phosphoresidue contact sites. Cross-validation indicated that propensities corresponding to this enrichment calculated from a subset of the training data could be used to predict the phosphoresidue contact site on proteins not used in training with no false negative results, and with few unconfirmed positive predictions. The locations of phosphoresidue contact sites were then predicted on the surfaces of the checkpoint kinase Chk1 and the BRCA1 BRCT repeat domain, and these predictions are consistent with recent experimental evidence.

Binding Sites↗
PubMed2004

Cytosolic phospholipase A2-alpha is necessary for platelet-activating factor biosynthesis, efficient neutrophil-mediated bacterial killing, and the innate immune response to pulmonary infection: cPLA2-alpha does not regulate neutrophil NADPH oxidase activity.

The role of a cytosolic phospholipase A(2)-alpha (cPLA(2)-alpha) in neutrophil arachidonic acid release, platelet-activating factor (PAF) biosynthesis, NADPH oxidase activation, and bacterial killing in vitro, and the innate immune response to bacterial infection in vivo was examined. cPLA(2)-alpha activity was blocked with the specific cPLA(2)-alpha inhibitor, Pyrrolidine-1 (human cells), or by cPLA(2) -alpha gene disruption (mice). cPLA(2)-alpha inhibition or gene disruption led to complete suppression of neutrophil arachidonate release and PAF biosynthesis but had no effect on neutrophil NADPH oxidase activation, FcgammaII/III or CD11b surface expression, primary or secondary granule secretion, or phagocytosis of Escherichia coli in vitro. In contrast, cPLA(2)-alpha inhibition or gene disruption diminished neutrophil-mediated E. coli killing in vitro, which was partially rescued by exogenous arachidonic acid or PAF but not leukotriene B(4). Following intratracheal inoculation with live E. coli in vivo, pulmonary PAF biosynthesis, inflammatory cell infiltration, and clearance of E. coli were attenuated in cPLA(2)-alpha(-/-) mice compared with wild type littermates. These studies identify a novel role for cPLA(2)-alpha in the regulation of neutrophil-mediated bacterial killing and the innate immune response to bacterial infection.

Animals↗
PubMed2004

Caged phosphopeptides reveal a temporal role for 14-3-3 in G1 arrest and S-phase checkpoint function.

Using classical genetics to study modular phosphopeptide-binding domains within a family of proteins that are functionally redundant is difficult when other members of the domain family compensate for the product of the knocked-out gene. Here we describe a chemical genetics approach that overcomes this limitation by using UV-activatable caged phosphopeptides. By incorporating a caged phosphoserine residue within a consensus motif, these reagents simultaneously and synchronously inactivate all phosphoserine/phosphothreonine-binding domain family members in a rapid and temporally regulated manner. We applied this approach to study the global function of 14-3-3 proteins in cell cycle control. Activation of the caged phosphopeptides by UV irradiation displaced endogenous proteins from 14-3-3-binding, causing premature cell cycle entry, release of G1 cells from interphase arrest and loss of the S-phase checkpoint after DNA damage, accompanied by high levels of cell death. This class of reagents will greatly facilitate molecular dissection of kinase-dependent signaling pathways when applied to other phosphopeptide-binding domains including SH2, Polo-box and tandem BRCT domains.

14-3-3 Proteins↗
PubMed2004

14-3-3 family members act coordinately to regulate mitotic progression.

The mitosis promoting phosphatase, cdc25C, is a target of both the DNA replication and DNA damage checkpoint pathways. These pathways regulate cdc25C function, in part, by promoting the association of cdc25C with 14-3-3 proteins, which results in the retention of cdc25C in the cytoplasm. To determine which 14-3-3 proteins were required to regulate cdc25C function, we tested the ability of various 14-3-3 family members to form a complex with and negatively regulate cdc25C in human cells. Two 14-3-3 family members, 14-3-3epsilon and 14-3-3gamma specifically formed a complex with cdc25C but not with the 14-3-3 binding defective cdc25C mutant, S216A. In addition, 14-3-3epsilon and 14-3-3gamma inhibited the ability of cdc25C, but not the S216A mutant, to induce premature chromatin condensation (PCC) in U-2OS cells. These results suggested that the reduction in PCC by 14-3-3epsilon and 14-3-3gamma was due to inhibition of cdc25C function. In contrast, 14-3-3sigma was unable to form a complex with cdc25C, but was able to inhibit the ability of both wild type cdc25C and S216A to induce PCC. This suggests that 14-3-3sigma regulates entry into mitosis independently of cdc25C and 14-3-3epsilon and 14-3-3gamma. Thus, specific members of the 14-3-3 family of proteins may act coordinately to maintain the DNA replication checkpoint by regulating the activity of different cell cycle proteins.

14-3-3 Proteins↗
PubMed2004

Structure and mechanism of BRCA1 BRCT domain recognition of phosphorylated BACH1 with implications for cancer.

Germline mutations in the BRCA1 tumor suppressor gene often result in a significant increase in susceptibility to breast and ovarian cancers. Although the molecular basis of their effects remains largely obscure, many mutations are known to target the highly conserved C-terminal BRCT repeats that function as a phosphoserine/phosphothreonine-binding module. We report the X-ray crystal structure at a resolution of 1.85 A of the BRCA1 tandem BRCT domains in complex with a phosphorylated peptide representing the minimal interacting region of the DEAH-box helicase BACH1. The structure reveals the determinants of this novel class of BRCA1 binding events. We show that a subset of disease-linked mutations act through specific disruption of phospho-dependent BRCA1 interactions rather than through gross structural perturbation of the tandem BRCT domains.

BRCA1 Protein↗
PubMed2004

Distinct ligand-dependent roles for p38 MAPK in priming and activation of the neutrophil NADPH oxidase.

In response to certain cytokines and inflammatory mediators, the activity of the neutrophil NADPH oxidase enzyme is primed for enhanced superoxide production when the cells receive a subsequent oxidase-activating stimulus. The relative role of p38 MAPK in the priming and activation processes is incompletely understood. We have developed a 2-step assay that allows the relative contributions of p38 MAPK activity in priming to be distinguished from those involved in oxidase activation. Using this assay, together with in vitro kinase assays and immunochemical studies, we report that p38 MAPK plays a critical role in TNFalpha priming of the human and porcine NADPH oxidase for superoxide production in response to complement-opsonized zymosan (OpZ), but little, if any, role in neutrophil priming by platelet-activating factor (PAF) for OpZ-dependent responses. The OpZ-mediated activation process per se is independent of p38 MAPK activity, in contrast to oxidase activation by fMLP, where 70% of the response is eliminated by p38 MAPK inhibitors regardless of the priming agent. We further report that incubation of neutrophils with TNFalpha results in the p38 MAPK-dependent phosphorylation of a subpopulation of p47(phox) and p67(phox) molecules, whereas PAF priming results in phosphorylation only of p67(phox). Despite these phosphorylations, TNFalpha priming does not result in significant association of either of these oxidase subunits with neutrophil membranes, demonstrating that the molecular basis for priming does not appear to involve preassembly of the NADPH oxidase holoenzyme/cytochrome complex prior to oxidase activation.

Animals↗
PubMed2004

Master of all things phosphorylated.

Serine/threonine phosphorylation plays a central role in cellular regulation, either by altering a protein's activity directly or by inducing specific protein-protein interactions, which, in turn, affect localization, binding specificity or activity. One group of molecules that bind to phosphoserine/phosphothreonine-containing sequences are the 14-3-3 proteins, which regulate a wide range of cellular targets. A new analysis of the 14-3-3 phosphoproteome using affinity chromatography has revealed many previously unknown 14-3-3 ligands whose binding to 14-3-3 proteins is phosphorylation-dependent. This study paves the way for future work on these important 14-3-3-interacting proteins.

14-3-3 Proteins↗
PubMed2004

TAZ interacts with TTF-1 and regulates expression of surfactant protein-C.

Thyroid transcription factor-1 (TTF-1/Nkx-2.1) is required for formation of the lung and differentiation of peripheral respiratory epithelial cells. TTF-1 activates transcription of target genes, including the surfactant proteins critical for lung function. A recently identified protein TAZ (transcriptional co-activator with PDZ-binding motif) contains a WW domain and a COOH-terminal PDZ-binding motif that are proposed to mediate its interactions with various transcriptional proteins. To determine the role of TAZ in the regulation of gene expression in the lung, the sites of TAZ expression and the role of TAZ in the regulation of respiratory epithelial gene expression were assessed. TAZ mRNA was detected in immortalized mouse lung epithelial cells, primary isolates of mouse alveolar type II epithelial cells, and epithelial cells of fetal lung. Sites of TAZ mRNA and protein overlapped with those of TTF-1 and surfactant protein C (SP-C) in the respiratory epithelial cells of the mouse lung. In the presence of TTF-1, TAZ synergistically activated the expression of mouse SP-C-luciferase reporter constructs. Mammalian two-hybrid assays and pull-down experiments demonstrated that the TAZ directly interacted with TTF-1. Further, deletion analysis demonstrated that TAZ binds to the NH(2)-terminal domain of TTF-1. TAZ binds to TTF-1, increasing the transcriptional activity of TTF-1 on the SP-C promoter. Developmental and cell-selective regulation of TAZ provides a mechanism by which the activity of TTF-1 on target genes is modulated.

Acyltransferases↗
PubMed2004

Lamellipodin, an Ena/VASP ligand, is implicated in the regulation of lamellipodial dynamics.

Lamellipodial protrusion is regulated by Ena/VASP proteins. We identified Lamellipodin (Lpd) as an Ena/VASP binding protein. Both proteins colocalize at the tips of lamellipodia and filopodia. Lpd is recruited to EPEC and Vaccinia, pathogens that exploit the actin cytoskeleton for their own motility. Lpd contains a PH domain that binds specifically to PI(3,4)P2, an asymmetrically localized signal in chemotactic cells. Lpd's PH domain can localize to ruffles in PDGF-treated fibroblasts. Lpd overexpression increases lamellipodial protrusion velocity, an effect observed when Ena/VASP proteins are overexpressed or artificially targeted to the plasma membrane. Conversely, knockdown of Lpd expression impairs lamellipodia formation, reduces velocity of residual lamellipodial protrusion, and decreases F-actin content. These phenotypes are more severe than loss of Ena/VASP, suggesting that Lpd regulates other effectors of the actin cytoskeleton in addition to Ena/VASP.

Actins↗
PubMed2004

14-3-3 Proteins--a focus on cancer and human disease.

14-3-3 Proteins are a ubiquitous family of molecules that participate in protein kinase signaling pathways within all eukaryotic cells. Functioning as phosphoserine/phosphothreonine-binding modules, 14-3-3 proteins participate in phosphorylation-dependent protein-protein interactions that control progression through the cell cycle, initiation and maintenance of DNA damage checkpoints, activation of MAP kinases, prevention of apoptosis, and coordination of integrin signaling and cytoskeletal dynamics. In this review, we discuss the regulation of 14-3-3 structure and ligand binding, with a focus on the role of 14-3-3 proteins in human disease, particularly cancer. We discuss the latest data on the role of different 14-3-3 isotypes, the interaction of 14-3-3 proteins with Raf, Cdc25, and various integrin family members, and the likelihood that 14-3-3 proteins could be useful therapeutic targets in the treatment of human disease.

14-3-3 Proteins↗
PubMed2004

The use of in vitro peptide-library screens in the analysis of phosphoserine/threonine-binding domain structure and function.

Phosphoserine/threonine-binding domains integrate intracellular signal transduction events by forming multiprotein complexes with substrates of protein serine/threonine kinases. These phosphorylation-dependent molecular recognition events are responsible for coordinating the precise temporal and spatial response of cells to a wide range of stimuli, particularly those involved in cell cycle control and the response to DNA damage. The known families of phosphoserine/threonine-binding modules include 14-3-3 proteins, WW domains, FHA domains, WD40 repeats, and the Polo-box domains of Polo-like kinases. Peptide-library experiments reveal the optimal sequence motifs recognized by these domains, and facilitate high-resolution structural studies elucidating the mechanisms of phospho-dependent binding and the molecular basis for domain function within intricate signaling networks. Information emerging from these studies is critical for the design of novel experimental and therapeutic tools aimed at altering signal transduction cascades in normal and diseased cells.

Amino Acid Motifs↗