Biomedical subjects

Mark A Lemmon

Publications and source records attributed to Mark A Lemmon.

34 records · Page 2Linked to original sources

PubMed2004

Genome-wide analysis of membrane targeting by S. cerevisiae pleckstrin homology domains.

Pleckstrin homology (PH) domains are small protein modules known for their ability to bind phosphoinositides and to drive membrane recruitment of their host proteins. We investigated phosphoinositide binding (in vitro and in vivo) and subcellular localization, and we modeled the electrostatic properties for all 33 PH domains encoded in the S. cerevisiae genome. Only one PH domain (from Num1p) binds phosphoinositides with high affinity and specificity. Six bind phosphoinositides with moderate affinity and little specificity and are membrane targeted in a phosphoinositide-dependent manner. Although all of the remaining 26 yeast PH domains bind phosphoinositides very weakly or not at all, three were nonetheless efficiently membrane targeted. Our proteome-wide analysis argues that membrane targeting is important for only approximately 30% of yeast PH domains and is defined by binding to both phosphoinositides and other targets. These findings have significant implications for understanding the function of proteins that contain this common domain.

Binding Sites↗

PubMed2004

The p21-activated protein kinase-related kinase Cla4 is a coincidence detector of signaling by Cdc42 and phosphatidylinositol 4-phosphate.

Signal transduction pathways that co-regulate a given biological process often are organized into networks by molecules that act as coincidence detectors. Phosphoinositides and the Rho-type GTPase Cdc42 regulate overlapping processes in all eukaryotic cells. However, the coincidence detectors that link these pathways into networks remain unknown. Here we show that the p21-activated protein kinase-related kinase Cla4 of yeast integrates signaling by Cdc42 and phosphatidylinositol 4-phosphate (PI4P). We found that the Cla4 pleckstrin homology (PH) domain binds in vitro to several phosphoinositide species. To determine which phosphoinositides regulate Cla4 in vivo, we analyzed phosphatidylinositol kinase mutants (stt4, mss4, and pik1). This indicated that the plasma membrane pool of PI4P, but not phosphatidylinositol 4,5-bisphosphate or the Golgi pool of PI4P, is required for localization of Cla4 to sites of polarized growth. A combination of the Cdc42-binding and PH domains of Cla4 was necessary and sufficient for localization to sites of polarized growth. Point mutations affecting either domain impaired the ability of Cla4 to regulate cell morphogenesis and the mitotic exit network (localization of Lte1). Therefore, Cla4 must retain the ability to bind both Cdc42 and phosphoinositides, the hallmark of a coincidence detector. PI4P may recruit Cla4 to the plasma membrane where Cdc42 activates its kinase activity and refines its localization to cortical sites of polarized growth. In mammalian cells, the myotonic dystrophy-related Cdc42-binding kinase possesses p21-binding and PH domains, suggesting that this kinase may be a coincidence detector of signaling by Cdc42 and phosphoinositides.

Alleles↗

PubMed2004

A structure-based model for ligand binding and dimerization of EGF receptors.

On the basis of the 3D structures of the extracellular ligand-binding domains of the epidermal growth factor (EGF) receptor (EGFR) and ErbB3, a mechanism has been proposed for how the extracellular region of the EGFR is maintained in an autoinhibited configuration and for how EGF binding induces EGFR dimerization and activation. We have attempted to derive a mathematical model for EGF binding to the EGFR and for ligand-induced receptor dimerization and activation that uses this structural information and can explain the characteristic concave-up curvilinear Scatchard plots seen when EGF binding to intact EGFR is studied in living cells. We show that these curvilinear plots cannot be accounted for by simply ascribing different affinities to the autoinhibited and extended (dimeric) configurations of the receptor seen in structural studies. Concave-up plots can only be obtained by including in the mathematical model an additional binding event in which occupied EGFR dimers bind to an "external site." The external site may represent receptor interactions with coated-pit regions in the cell membrane or with other cellular components involved in receptor endocytosis and turnover. We conclude in this study and in the accompanying article that the active extended EGFR configuration binds EGF 5- to 20-fold more strongly than the autoinhibited monomeric receptor configuration. However, these extended receptors do not correspond directly with the "high-affinity" EGF-binding sites seen in EGF-binding studies on intact cells.

Dimerization↗

PubMed2004

The tethered configuration of the EGF receptor extracellular domain exerts only a limited control of receptor function.

Quantitative epidermal growth factor (EGF)-binding experiments have shown that the EGF-receptor (EGFR) is displayed on the surface of intact cells in two forms, a minority of high-affinity and a majority of low-affinity EGFRs. On the basis of the three-dimensional structure of the extracellular ligand binding domain of the EGFR, it was proposed that the intramolecularly tethered and autoinhibited configuration corresponds to the low-affinity receptor, whereas the extended configuration accounts for the high-affinity EGFRs on intact cells. Here we test this model by analyzing the properties of EGFRs mutated in the specific regions responsible for receptor autoinhibition and dimerization, respectively. Our results show that mutagenic disruption of the autoinhibitory tether in EGFR results in a decrease in the dissociation rate of EGF without a detectable change in EGFR activation and signaling through EGFR even in response to stimulation with low concentrations of EGF. Mutagenic disruption of the dimerization arm, on the other hand, increased the rate of EGF dissociation and impaired EGFR activation and signaling via the EGFR. This study demonstrates that the extended configuration of EGFR does not account for the apparent high-affinity EGF-binding to EGFR on intact cells. Furthermore, the autoinhibition conferred by the tethered configuration of the extracellular ligand-binding domain provides only a limited control of EGFR function.

Cell Line↗

PubMed2004

Signaling by the sea.

In a spectacular location nestled in the hills on Croatia's coast, a group of 300 scientists from around the world gathered to listen to recent advances in cellular signaling and to gaze across the Adriatic Sea during discussions that surely put this work into perspective. Topics discussed ranged from precise structural details of signaling events to animal models for understanding signaling disorders.

Animals↗

PubMed2003

SH2 and PTB domains in tyrosine kinase signaling.

Intracellular signaling pathways that involve protein tyrosine kinases (PTKs) are critical for the control of most cellular processes. Dysfunctions in PTKs, or in the signaling pathways that they regulate, result in a variety of diseases such as cancer, diabetes, immune deficiency, and many others. SH2 (Src homology region 2) and PTB (phosphotyrosine-binding) domains are small protein modules that mediate protein-protein interactions involved in many signal transduction pathways. Both domains were initially identified as modules that recognize phosphorylated tyrosines in receptor tyrosine kinases and other signaling proteins. Subsequent studies have shown that, while binding of SH2 domains to their target proteins is strictly regulated by tyrosine phosphorylation, most PTB domains actually bind to their (nonphosphorylated) targets constitutively. The functions of SH2 and PTB domains include targeting of their host proteins to different cellular compartments, assembly of key components of signaling pathways in response to extracellular signals, and the control of autoinhibition, activation and dimerization of their host proteins. The information flow from the cell surface to different cellular compartments to regulate the cell cycle, cell shape and movement, cell proliferation, differentiation and cell survival are all controlled in part by SH2 and PTB domains that can recognize phosphotyrosine or particular amino acid sequence motifs in a wide variety of target molecules.

Animals↗

PubMed2003

Loss of phosphatidylinositol 3-phosphate binding by the C-terminal Tiam-1 pleckstrin homology domain prevents in vivo Rac1 activation without affecting membrane targeting.

Dbl family guanine nucleotide exchange factors (GEFs) for Rho family small GTPases invariably contain a pleckstrin homology (PH) domain that immediately follows their Dbl homology (DH) domain. Although the DH domain is responsible for GEF activity, the role of the PH domain is less clear. We previously reported that PH domains from several Dbl family members bind phosphoinositides with very low affinity (K(d) values in the 10 microM range). This suggests that, unlike several other PH domains, those from Dbl proteins will not function as independent membrane-targeting modules. To determine the functional relevance of low affinity phosphoinositide binding, we mutated the corresponding PH domain from Tiam-1 to abolish its weak, specific binding to phosphatidylinositol 3-phosphate. We first confirmed in vitro that phosphoinositide binding by the isolated DH/PH domain was impaired by the mutations but that intrinsic GEF activity was unaffected. We then introduced the PH domain mutations into full-length Tiam-1 and found that its ability to activate Rac1 or serum response factor in vivo was abolished. Immunofluorescence studies showed that membrane targeting of Tiam-1 was essentially unaffected by mutations in the C-terminal PH domain. Our studies therefore indicate that low affinity phosphatidylinositol 3-phosphate binding by the C-terminal PH domain may be critical for in vivo regulation and activity of Tiam-1 but that the PH domain exerts its regulatory effects without altering membrane targeting. We suggest instead that ligand binding to the PH domain induces conformational and/or orientational changes at the membrane surface that are required for maximum exchange activity of its adjacent DH domain.

Animals↗

PubMed2003

EGF activates its receptor by removing interactions that autoinhibit ectodomain dimerization.

Epidermal growth factor (EGF) receptor is the prototype of the ErbB (HER) family receptor tyrosine kinases (RTKs), which regulate cell growth and differentiation and are implicated in many human cancers. EGF activates its receptor by inducing dimerization of the 621 amino acid EGF receptor extracellular region. We describe the 2.8 A resolution crystal structure of this entire extracellular region (sEGFR) in an unactivated state. The structure reveals an autoinhibited configuration, where the dimerization interface recently identified in activated sEGFR structures is completely occluded by intramolecular interactions. To activate the receptor, EGF binding must promote a large domain rearrangement that exposes this dimerization interface. This contrasts starkly with other RTK activation mechanisms and suggests new approaches for designing ErbB receptor antagonists.

Crystallography, X-Ray↗

PubMed2003

An open-and-shut case? Recent insights into the activation of EGF/ErbB receptors.

Recent crystallographic studies have provided significant new insight into how receptor tyrosine kinases from the EGF receptor or ErbB family are regulated by their growth factor ligands. EGF receptor dimerization is mediated by a unique dimerization arm, which becomes exposed only after a dramatic domain rearrangement is promoted by growth factor binding. ErbB2, a family member that has no ligand, has its dimerization arm constitutively exposed, and this explains several of its unique properties. We outline a mechanistic view of ErbB receptor homo- and heterodimerization, which suggests new approaches for interfering with these processes when they are implicated in human cancers.

Animals↗

PubMed2003

Genome-wide analysis of signaling domain function.

Approximately 2.5% of human gene products contain one or more small domains that drive interactions between proteins and other cellular components in cell signaling processes. The many interactions driven by these relatively simple domains are thought to cooperate with one another to yield complex signaling networks that allow very fine control of cell function. In principle, if we can understand all domain-mediated interactions it should be possible to model these networks. Genome-wide analysis of signaling domain interactions represents a first step in this direction, and several advances of this sort in yeast have been reported over the past year. These reports suggest, for some domains at least, that the prospect of generating 'wiring diagrams' with this simple approach is feasible.

Amino Acid Sequence↗

PubMed2003

Phosphoinositide recognition domains.

Domains or modules known to bind phosphoinositides have increased dramatically in number over the past few years, and are found in proteins involved in intracellular trafficking, cellular signaling, and cytoskeletal remodeling. Analysis of lipid binding by these domains and its structural basis has provided significant insight into the mechanism of membrane recruitment by the different cellular phosphoinositides. Domains that target only the rare (3-phosphorylated) phosphoinositides must bind with very high affinity, and with exquisite specificity. This is achieved solely by headgroup interactions in the case of certain pleckstrin homology (PH) domains [which bind PtdIns(3,4,5)P3 and/or PtdIns(3,4)P2], but requires an additional membrane-insertion and/or oligomerization component in the case of the PtdIns(3)P-targeting phox homology (PX) and FYVE domains. Domains that target PtdIns(4,5)P2, which is more abundant by some 25-fold, do not require the same stringent affinity and specificity characteristics, and tend to be more diverse in structure. The mode of phosphoinositide binding by different domains also appears to reflect their distinct functions. For example, pleckstrin homology domains that serve as simple targeting domains recognize only phosphoinositide headgroups. By contrast, certain other domains, notably the epsin ENTH domain, appear to promote bilayer curvature by inserting into the membrane upon binding.

Binding Sites↗

PubMed2003

The EGF receptor family as therapeutic targets in breast cancer.

Many studies have suggested an association between overexpression of receptor tyrosine kinases (RTKs) from the EGF receptor (or ErbB) family and breast cancer. The orphan RTK ErbB2/HER2/Neu is highly overexpressed in up to 30% of human breast cancer cases as a consequence of gene amplification. ErbB2/HER2/Neu can be activated by simple overexpression, and its signaling is thought to play an important role in initiation and progression of ErbB2-positive breast cancers. In support of this, ErbB2/HER2/Neu-targeted therapy (the Herceptin antibody) has proven valuable in many of these cases. Other studies also correlate EGF receptor (EGFR) expression with poor prognosis in breast cancer, but follow-up studies suggest that this association is much less robust than with ErbB2, and requires more careful analysis. A particular problem is that EGFR, unlike ErbB2/HER2/Neu, cannot be activated simply by overexpression, its signaling remaining growth factor-dependent under these conditions. It is therefore critical to analyze EGFR signaling activity itself, rather than simply EGFR expression, in order to establish causal links and to identify patients for ErbB-targeted therapies. This distinction between ErbB2/HER2/Neu and EGFR is satisfyingly explained by recent crystallographic studies of ErbB receptor family members that are reviewed here. These structures also provide new insight into how ErbB2/HER2/Neu-targeted and EGFR-targeted therapeutic agents function, and suggest approaches for the development of novel mechanism-based ErbB inhibitors.

Journal Article↗

PubMed2002

Phosphoinositide binding by the pleckstrin homology domains of Ipl and Tih1.

The Ipl protein consists of a single pleckstrin homology (PH) domain with short N- and C-terminal extensions. This protein is highly conserved among vertebrates, and it acts to limit placental growth in mice. However, its biochemical function is unknown. The closest paralogue of Ipl is Tih1, another small PH domain protein. By sequence comparisons, Ipl and Tih1 define an outlying branch of the PH domain superfamily. Here we describe phosphatidylinositol phosphate (PIP) binding by these proteins. Ipl and Tih1 bind to immobilized PIPs with moderate affinity, but this binding is weaker and more promiscuous than that of prototypical PH domains from the general receptor for phosphoinositides (GRP1), phospholipase C delta1, and dual adaptor for phosphoinositides and phosphotyrosine 1. In COS7 cells exposed to epidermal growth factor, green fluorescent protein (GFP)-Ipl and GFP-Tih1 accumulate at membrane ruffles without clearing from the cytoplasm, whereas control GFP-GRP1 translocates rapidly to the plasma membrane and clears from the cytoplasm. Ras*-Ipl and Ras*-Tih1 fusion proteins both rescue cdc25ts Saccharomyces cerevisiae, but Ras*-Ipl rescues more efficiently in the presence of phosphatidylinositol 3-kinase (PI3K), whereas PI3K-independent rescue is more efficient with Ras*-Tih1. Site-directed mutagenesis defines amino acids in the beta1-loop1-beta2 regions of Ipl and Tih1 as essential for growth rescue in this assay. Thus, Ipl and Tih1 are bona fide PH domain proteins, with broad specificity and moderate affinity for PIPs.

Amino Acid Sequence↗

PubMed2002

Pleckstrin homology domains and the cytoskeleton.

Pleckstrin homology (PH) domains are 100-120 amino acid protein modules best known for their ability to bind phosphoinositides. All possess an identical core beta-sandwich fold and display marked electrostatic sidedness. The binding site for phosphoinositides lies in the center of the positively charged face. In some cases this binding site is well defined, allowing highly specific and strong ligand binding. In several of these cases the PH domains specifically recognize 3-phosphorylated phosphoinositides, allowing them to drive membrane recruitment in response to phosphatidylinositol 3-kinase activation. Examples of these PH domain-containing proteins include certain Dbl family guanine nucleotide exchange factors, protein kinase B, PhdA, and pleckstrin-2. PH domain-mediated membrane recruitment of these proteins contributes to regulated actin assembly and cell polarization. Many other PH domain-containing cytoskeletal proteins, such as spectrin, have PH domains that bind weakly, and to all phosphoinositides. In these cases, the individual phosphoinositide interactions may not be sufficient for membrane association, but appear to require self-assembly of their host protein and/or cooperation with other anchoring motifs within the same molecule to drive membrane attachment.

Animals↗

PubMed2002

Normalization of nomenclature for peptide motifs as ligands of modular protein domains.

We propose a normalization of symbols and terms used to describe, accurately and succinctly, the detailed interactions between amino acid residues of pairs of interacting proteins at protein:protein (or protein:peptide) interfaces. Our aim is to unify several diverse descriptions currently in use in order to facilitate communication in the rapidly progressing field of signaling by protein domains. In order for the nomenclature to be convenient and widely used, we also suggest a parallel set of symbols restricted to the ASCII format allowing accurate parsing of the nomenclature to a computer-readable form. This proposal will be reviewed in the future and will therefore be open for the inclusion of new rules, modifications and changes.

Binding Sites↗

PubMed2001

The single transmembrane domains of ErbB receptors self-associate in cell membranes.

Members of the epidermal growth factor receptor, or ErbB, family of receptor tyrosine kinases have a single transmembrane (TM) alpha-helix that is usually assumed to play a passive role in ligand-induced dimerization and activation of the receptor. However, recent studies with the epidermal growth factor receptor (ErbB1) and the erythropoietin receptor have indicated that interactions between TM alpha-helices do contribute to stabilization of ligand-independent and/or ligand-induced receptor dimers. In addition, not all of the expected ErbB receptor ligand-induced dimerization events can be recapitulated using isolated extracellular domains, suggesting that other regions of the receptor, such as the TM domain, may contribute to dimerization in vivo. Using an approach for analyzing TM domain interactions in Escherichia coli cell membranes, named TOXCAT, we find that the TM domains of ErbB receptors self-associate strongly in the absence of their extracellular domains, with the rank order ErbB4-TM > ErbB1-TM equivalent to ErbB2-TM > ErbB3-TM. A limited mutational analysis suggests that dimerization of these TM domains involves one or more GXXXG motifs, which occur frequently in the TM domains of receptor tyrosine kinases and are critical for stabilizing the glycophorin A TM domain dimer. We also analyzed the effect of the valine to glutamic acid mutation in ErbB2 that constitutively activates this receptor. Contrary to our expectations, this mutation reduced rather than increased ErbB2-TM dimerization. Our findings suggest a role for TM domain interactions in ErbB receptor function, possibly in stabilizing inactive ligand-independent receptor dimers that have been observed by several groups.

Amino Acid Motifs↗