Biomedical subjects

Michael J Eck

Publications and source records attributed to Michael J Eck.

At least 19 recordsLinked to original sources

PubMed2006

Structure of the autoinhibitory switch in formin mDia1.

Diaphanous-related formins (DRFs) regulate the nucleation and polymerization of unbranched actin filaments. The activity of DRFs is inhibited by an intramolecular interaction between their N-terminal regulatory region and a conserved C-terminal segment termed the Diaphanous autoinhibitory domain (DAD). Binding of GTP bound Rho to the mDia1 N terminus releases this autoinhibitory restraint. Here, we describe the crystal structure of the DAD segment of mDia1 in complex with the relevant N-terminal fragment, termed the DID domain. The structure reveals that the DAD segment forms an amphipathic helix that binds a conserved, concave surface on the DID domain. Comparison with the structure of the mDia1 N terminus bound to RhoC suggests that release of the autoinhibitory DAD interaction is accomplished largely by Rho-induced restructuring of the adjacent GTPase binding subdomain (GBD), but also by electrostatic repulsion and a small, direct steric occlusion of the DAD binding cleft by Rho itself.

Amino Acid Sequence↗

PubMed2005

Crystal structure of the FERM domain of focal adhesion kinase.

Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that localizes to focal adhesions in adherent cells. Through phosphorylation of proteins assembled at the cytoplasmic tails of integrins, FAK promotes signaling events that modulate cellular growth, survival, and migration. The amino-terminal region of FAK contains a region of sequence homology with band 4.1 and ezrin/radixin/moesin (ERM) proteins termed a FERM domain. FERM domains are found in a variety of signaling and cytoskeletal proteins and are thought to mediate intermolecular interactions with partner proteins and phospholipids at the plasma membrane and intramolecular regulatory interactions. Here we report two crystal structures of an NH2-terminal fragment of avian FAK containing the FERM domain and a portion of the regulatory linker that connects the FERM and kinase domains. The tertiary folds of the three subdomains (F1, F2, and F3) are similar to those of known FERM structures despite low sequence conservation. Differences in the sequence and relative orientation of the F3 subdomain alters the nature of the interdomain interface, and the phosphoinositide binding site found in ERM family FERM domains is not present in FAK. A putative protein interaction site on the F3 lobe is masked by the proximal region of the linker. Additionally, in one structure the adjacent Src SH3 and SH2 binding sites in the linker associate with the surfaces of the F3 and F1 lobes, respectively. These structural features suggest the possibility that protein interactions of the FAK FERM domain can be regulated by binding of Src kinases to the linker segment.

Amino Acid Sequence↗

PubMed2005

Crystal structure of the Jak3 kinase domain in complex with a staurosporine analog.

Jak (Janus kinase) family nonreceptor tyrosine kinases are central mediators of cytokine signaling. The Jak kinases exhibit distinct cytokine receptor association profiles and so transduce different signals. Jak3 expression is limited to the immune system, where it plays a key role in signal transduction from cytokine receptors containing the common gamma-chain, gammac. Patients unable to signal via gammac present with severe combined immunodeficiency (SCID). The finding that Jak3 mutations result in SCID has made it a target for development of lymphocyte-specific immunosuppressants. Here, we present the crystal structure of the Jak3 kinase domain in complex with staurosporine analog AFN941. The kinase domain is in the active conformation, with both activation loop tyrosine residues phosphorylated. The phosphate group on pTyr981 in the activation loop is in part coordinated by an arginine residue in the regulatory C-helix, suggesting a direct mechanism by which the active position of the C-helix is induced by phosphorylation of the activation loop. Such a direct coupling has not been previously observed in tyrosine kinases and may be unique to Jak kinases. The crystal structure provides a detailed view of the Jak3 active site and will facilitate computational and structure-directed approaches to development of Jak3-specific inhibitors.

Binding Sites↗

PubMed2005

EGFR mutation and resistance of non-small-cell lung cancer to gefitinib.

Mutations of the epidermal growth factor receptor (EGFR) gene have been identified in specimens from patients with non-small-cell lung cancer who have a response to anilinoquinazoline EGFR inhibitors. Despite the dramatic responses to such inhibitors, most patients ultimately have a relapse. The mechanism of the drug resistance is unknown. Here we report the case of a patient with EGFR-mutant, gefitinib-responsive, advanced non-small-cell lung cancer who had a relapse after two years of complete remission during treatment with gefitinib. The DNA sequence of the EGFR gene in his tumor biopsy specimen at relapse revealed the presence of a second point mutation, resulting in threonine-to-methionine amino acid change at position 790 of EGFR. Structural modeling and biochemical studies showed that this second mutation led to gefitinib resistance.

Aged↗

PubMed2005

Activating mutation in the tyrosine kinase JAK2 in polycythemia vera, essential thrombocythemia, and myeloid metaplasia with myelofibrosis.

Polycythemia vera (PV), essential thrombocythemia (ET), and myeloid metaplasia with myelofibrosis (MMM) are clonal disorders arising from hematopoietic progenitors. An internet-based protocol was used to collect clinical information and biological specimens from patients with these diseases. High-throughput DNA resequencing identified a recurrent somatic missense mutation JAK2V617F in granulocyte DNA samples of 121 of 164 PV patients, of which 41 had homozygous and 80 had heterozygous mutations. Molecular and cytogenetic analyses demonstrated that homozygous mutations were due to duplication of the mutant allele. JAK2V617F was also identified in granulocyte DNA samples from 37 of 115 ET and 16 of 46 MMM patients, but was not observed in 269 normal individuals. In vitro analysis demonstrated that JAK2V617F is a constitutively active tyrosine kinase.

Adult↗

PubMed2004

Structure and regulation of Src family kinases.

Src family kinases are prototypical modular signaling proteins. Their conserved domain organization includes a myristoylated N-terminal segment followed by SH3, SH2, and tyrosine kinase domains, and a short C-terminal tail. Structural dissection of Src kinases has elucidated the canonical mechanisms of phosphotyrosine recognition by the SH2 domain and proline-motif recognition by the SH3 domain. Crystallographic analysis of nearly intact Src kinases in the autoinhibited state has shown that these protein interaction motifs turn inward and lock the kinase in an inactive conformation via intramolecular interactions. The autoinhibited Src kinase structures reveal a mode of domain assembly used by other tyrosine kinases outside the Src family, including Abl and likely Tec family kinases. Furthermore, they illustrate the underlying regulatory principles that have proven to be general among diverse modular signaling proteins. Although there is considerable structural information available for the autoinhibited conformation of Src kinases, how they may assemble into active signaling complexes with substrates and regulators remains largely unexplored.

Animals↗

PubMed2004

Identifying and characterizing a novel activating mutation of the FLT3 tyrosine kinase in AML.

The FLT3 receptor is activated by juxtamembrane insertion mutations and by activation loop point mutations in patients with acute myeloid leukemia (AML). In a systematic tyrosine kinase gene exon resequencing study, 21 of 24 FLT3 exons were sequenced in samples from 53 patients with AML, 9 patients with acute lymphoblastic leukemia (ALL), and 3 patients with myelodysplasia samples. Three patients had novel point mutations at residue N841 that resulted in a change to isoleucine in 2 samples and to tyrosine in 1 sample. Introduction of FLT3-N841I cDNA into Ba/F3 cells led to interleukin-3 (IL-3)-independent proliferation, receptor phosphorylation, and constitutive activation of signal transducer and activator of transcription 5 (STAT5) and extracellular regulatory kinase (ERK), suggesting that the N841I mutation confers constitutive activity to the receptor. An FLT3 inhibitor (PKC412) inhibited the growth of Ba/F3-FLT3N841I cells (IC(50) 10 nM), but not of wild-type Ba/F3 cells cultured with IL-3. PKC412 also reduced tyrosine phosphorylation of the mutant receptor and inhibited STAT5 phosphorylation. Examination of the FLT3 autoinhibited structure showed that N841 is the key residue in a hydrogen-bonding network that likely stabilizes the activation loop. These results suggest that mutations at N841 represent a significant new activating mutation in patients with AML and that patients with such mutations may respond to small-molecule FLT3 inhibitors such as PKC412.

Adult↗

PubMed2004

EGFR mutations in lung cancer: correlation with clinical response to gefitinib therapy.

Receptor tyrosine kinase genes were sequenced in non-small cell lung cancer (NSCLC) and matched normal tissue. Somatic mutations of the epidermal growth factor receptor gene EGFR were found in 15of 58 unselected tumors from Japan and 1 of 61 from the United States. Treatment with the EGFR kinase inhibitor gefitinib (Iressa) causes tumor regression in some patients with NSCLC, more frequently in Japan. EGFR mutations were found in additional lung cancer samples from U.S. patients who responded to gefitinib therapy and in a lung adenocarcinoma cell line that was hypersensitive to growth inhibition by gefitinib, but not in gefitinib-insensitive tumors or cell lines. These results suggest that EGFR mutations may predict sensitivity to gefitinib.

Adenocarcinoma↗

PubMed2004

SAP increases FynT kinase activity and is required for phosphorylation of SLAM and Ly9.

The free Src homology 2 (SH2) domain protein SAP, encoded by the X-linked lymphoproliferative disease gene SH2D1A, controls signal transduction initiated by engagement of the SLAM-related receptors in T and NK cells. Here we demonstrate that SAP is required for phosphorylation of both SLAM and Ly9 in thymocytes and peripheral T cells. Furthermore, in vitro protein interaction studies and yeast two-hybrid analyses indicated that SAP binds directly to FynT and Lck. While SAP bound to both the SH3 domain and to the kinase domain of FynT, SAP bound solely to the kinase domain of Lck. The existence of a strong interaction between SAP and the SH3 domain of FynT prompted us to study the role of SAP in modulating the activity of FynT. In vitro addition of SAP to the autoinhibited form of FynT caused a large increase in FynT catalytic activity. By contrast, the SAP mutant R78E, which is unable to bind to the FynT SH3 domain, did not increase FynT activity and also displayed a reduced adaptor function upon transfection into T cells. Our results demonstrate that SAP is an adaptor that bridges SLAM and Ly9 with Src-like protein tyrosine kinases (PTKs), and has the ability to activate FynT.

Animals↗

PubMed2004

Crystal structures of a Formin Homology-2 domain reveal a tethered dimer architecture.

Formin proteins participate in a wide range of cytoskeletal processes in all eukaryotes. The defining feature of formins is a highly conserved approximately 400 residue region, the Formin Homology-2 (FH2) domain, which has recently been found to nucleate actin filaments. Here we report crystal structures of the S. cerevesiae Bni1p FH2 domain. The mostly alpha-helical FH2 domain forms a unique "tethered dimer" in which two elongated actin binding heads are tied together at either end by an unusual lasso and linker structure. Biochemical and crystallographic observations indicate that the dimer is stable but flexible, with flexibility between the two halves of the dimer conferred by the linker segments. Although each half of the dimer is competent to interact with filament ends, the intact dimer is required for actin nucleation and processive capping. The tethered dimer architecture may allow formins to stair-step on the barbed end of an elongating nascent filament.

Actins↗

PubMed2004

Small molecule blockade of transcriptional coactivation of the hypoxia-inducible factor pathway.

Homeostasis under hypoxic conditions is maintained through a coordinated transcriptional response mediated by the hypoxia-inducible factor (HIF) pathway and requires coactivation by the CBP and p300 transcriptional coactivators. Through a target-based high-throughput screen, we identified chetomin as a disrupter of HIF binding to p300. At a molecular level, chetomin disrupts the structure of the CH1 domain of p300 and precludes its interaction with HIF, thereby attenuating hypoxia-inducible transcription. Systemic administration of chetomin inhibited hypoxia-inducible transcription within tumors and inhibited tumor growth. These results demonstrate a therapeutic window for pharmacological attenuation of HIF activity and further establish the feasibility of disrupting a signal transduction pathway by targeting the function of a transcriptional coactivator with a small molecule.

Animals↗

PubMed2004

FERM domain interaction promotes FAK signaling.

From the results of deletion analyses, the FERM domain of FAK has been proposed to inhibit enzymatic activity and repress FAK signaling. We have identified a sequence in the FERM domain that is important for FAK signaling in vivo. Point mutations in this sequence had little effect upon catalytic activity in vitro. However, the mutant exhibits reduced tyrosine phosphorylation and dramatically reduced Src family kinase binding. Further, the abilities of the mutant to transduce biochemical signals and to promote cell migration were severely impaired. The results implicate a FERM domain interaction in cell adhesion-dependent activation of FAK and downstream signaling. We also show that the purified FERM domain of FAK interacts with full-length FAK in vitro, and mutation of this sequence disrupts the interaction. These findings are discussed in the context of models of FAK regulation by its FERM domain.

Animals↗

PubMed2003

A conserved mechanism for Bni1- and mDia1-induced actin assembly and dual regulation of Bni1 by Bud6 and profilin.

Formins have conserved roles in cell polarity and cytokinesis and directly nucleate actin filament assembly through their FH2 domain. Here, we define the active region of the yeast formin Bni1 FH2 domain and show that it dimerizes. Mutations that disrupt dimerization abolish actin assembly activity, suggesting that dimers are the active state of FH2 domains. The Bni1 FH2 domain protects growing barbed ends of actin filaments from vast excesses of capping protein, suggesting that the dimer maintains a persistent association during elongation. This is not a species-specific mechanism, as the activities of purified mammalian formin mDia1 are identical to those of Bni1. Further, mDia1 partially complements BNI1 function in vivo, and expression of a dominant active mDia1 construct in yeast causes similar phenotypes to dominant active Bni1 constructs. In addition, we purified the Bni1-interacting half of the cell polarity factor Bud6 and found that it binds specifically to actin monomers and, like profilin, promotes rapid nucleotide exchange on actin. Bud6 and profilin show additive stimulatory effects on Bni1 activity and have a synthetic lethal genetic interaction in vivo. From these results, we propose a model in which Bni1 FH2 dimers nucleate and processively cap the elongating barbed end of the actin filament, and Bud6 and profilin generate a local flux of ATP-actin monomers to promote actin assembly.

Actin Cytoskeleton↗

PubMed2003

Proteomics-based target identification: bengamides as a new class of methionine aminopeptidase inhibitors.

LAF389 is a synthetic analogue of bengamides, a class of marine natural products that produce inhibitory effects on tumor growth in vitro and in vivo. A proteomics-based approach has been used to identify signaling pathways affected by bengamides. LAF389 treatment of cells resulted in altered mobility of a subset of proteins on two-dimensional gel electrophoresis. Detailed analysis of one of the proteins, 14-3-3gamma, showed that bengamide treatment resulted in retention of the amino-terminal methionine, suggesting that bengamides directly or indirectly inhibited methionine aminopeptidases (MetAps). Both known MetAps are inhibited by LAF389. Short interfering RNA suppression of MetAp2 also altered amino-terminal processing of 14-3-3gamma. A high resolution structure of human MetAp2 co-crystallized with a bengamide shows that the compound binds in a manner that mimics peptide substrates. Additionally, the structure reveals that three key hydroxyl groups on the inhibitor coordinate the di-cobalt center in the enzyme active site.

14-3-3 Proteins↗

PubMed2003

A zinc clasp structure tethers Lck to T cell coreceptors CD4 and CD8.

The T cell coreceptors CD4 and CD8 both associate via their cytoplasmic tails with the N-terminus of the Src-family tyrosine kinase Lck. These interactions require zinc and are critical for T cell development and activation. We examined the folding and solution structures of ternary CD4-Lck-Zn2+ and CD8alpha-Lck-Zn2+ complexes. The coreceptor tails and the Lck N-terminus are unstructured in isolation but assemble in the presence of zinc to form compactly folded heterodimeric domains. The cofolded complexes have similar "zinc clasp" cores that are augmented by distinct structural elements. A dileucine motif required for clathrin-mediated endocytosis of CD4 is masked by Lck.

Amino Acid Motifs↗

PubMed2003

Homotetrameric structure of the SNAP-23 N-terminal coiled-coil domain.

SNARE proteins mediate intracellular membrane fusion by forming a coiled-coil complex to merge opposing membranes. A "fusion-active" neuronal SNARE complex is a parallel four-helix bundle containing two coiled-coil domains from SNAP-25 and one coiled-coil domain each from syntaxin-1a and VAMP-2. "Prefusion" assembly intermediate complexes can also form from these SNAREs. We studied the N-terminal coiled-coil domain of SNAP-23 (SNAP-23N), a non-neuronal homologue of SNAP-25, and its interaction with other coiled-coil domains. SNAP-23N can assemble spontaneously with the coiled-coil domains from SNAP-23C, syntaxin-4, and VAMP-3 to form a heterotetrameric complex. Unexpectedly, pure SNAP-23N crystallizes as a coiled-coil homotetrameric complex. The four helices have a parallel orientation and are symmetrical about the long axis. The complex is stabilized through the interaction of conserved hydrophobic residues comprising the a and d positions of the coiled-coil heptad repeats. In addition, a central, highly conserved glutamine residue (Gln-48) is buried within the interface by hydrogen bonding between glutamine side chains derived from adjacent subunits and to solvent molecules. A comparison of the SNAP-23N structure to other SNARE complex structures reveals how a simple coiled-coil motif can form diverse SNARE complexes.

Amino Acid Sequence↗

PubMed2003

Specificity in signaling by c-Yes.

c-Yes and c-Src are the two most closely related members of the Src family of nonreceptor tyrosine kinases. Although there is much evidence to support redundancy in signaling between these two kinases, there is also a growing body of evidence to indicate specificity in signaling. In this review, we summarize c-Yes, its potential functions and its ability to modulate signals that are distinct from c-Src.

Animals↗

PubMed2003

Assembling atomic resolution views of the immunological synapse.

Antigen-dependent T-cell activation is initiated by the organized interactions of a host of cell-surface receptors in the interface between the T cell and an antigen-presenting cell. Recent structural studies of many of the receptors that comprise this 'immunological synapse', including integrins, cadherins and co-stimulatory molecules, reveal in detail the molecular interactions that bridge the synapse. Additionally, structural analyses of the cytoplasmic tails of integrins and co-stimulatory receptors in complex with intracellular signaling proteins are providing insight into the mechanisms that couple receptor clustering on the cell surface with the initiation of cytoplasmic signaling.

Animals↗