N-terminal Myristoylation Research Articles

Enhanced SH3/Linker Interaction Overcomes Abl Kinase Activation by Gatekeeper and Myristic Acid Binding Pocket Mutations and Increases Sensitivity to Small Molecule Inhibitors*

Multidomain kinases such as c-Src and c-Abl are regulated by complex allosteric interactions involving their noncatalytic SH3 and SH2 domains. Here we show that enhancing natural allosteric control of kinase activity by SH3/linker engagement has long-range suppressive effects on the kinase activity of the c-Abl core. Surprisingly, enhanced SH3/linker interaction also dramatically sensitized the Bcr-Abl tyrosine kinase associated with chronic myelogenous leukemia to small molecule inhibitors that target either the active site or the myristic acid binding pocket in the kinase domain C-lobe. Dynamics analyses using hydrogen exchange mass spectrometry revealed a remarkable allosteric network linking the SH3 domain, the myristic acid binding pocket, and the active site of the c-Abl core, providing a structural basis for the biological observations. These results suggest a rational strategy for enhanced drug targeting of Bcr-Abl and other multidomain kinase systems that use multiple small molecules to exploit natural mechanisms of kinase control.

Posttranslational modifications and secretion efficiency of immunogenic hepatitis B virus L protein deletion variants

BackgroundSubviral particles of hepatitis B virus (HBV) composed of L protein deletion variants with the 48 N-terminal amino acids of preS joined to the N-terminus of S protein (1-48preS/S) induced broadly neutralizing antibodies after immunization of mice with a Semliki Forest virus vector. A practical limitation for use as vaccine is the suboptimal secretion of such particles. The role of the N-terminal preS myristoylation in the cellular retention of full-length L protein is described controversially in the literature and the relation of these data to the truncated L protein was unknown. Thus, we studied the effect of preS myristoylation signal suppression on 1-48preS/S secretion efficiency, glycosylation and subcellular distribution.FindingsThe findings are that 1-48preS/S is secreted, and that removal of the N-terminal myristoylation signal in its G2A variant reduced secretion slightly, but significantly. The glycosylation pattern of 1-48preS/S was not affected by the removal of the myristoylation signal (G2A mutant) but was different than natural L protein, whereby N4 of the preS and N3 of the S domain were ectopically glycosylated. This suggested cotranslational translocation of 1-48preS in contrast to natural L protein. The 1-48preS/S bearing a myristoylation signal was localized in a compact, perinuclear pattern with strong colocalization of preS and S epitopes, while the non-myristoylated mutants demonstrated a dispersed, granular cytoplasmic distribution with weaker colocalization.ConclusionsThe large deletion in 1-48preS/S in presence of the myristoylation site facilitated formation and secretion of protein particles with neutralizing preS1 epitopes at their surface and could be a useful feature for future hepatitis B vaccines.

HIV-1 Nef Impairs Heterotrimeric G-protein Signaling by Targeting Gαi2 for Degradation through Ubiquitination

The HIV Nef protein is an important pathogenic factor that modulates cell surface receptor trafficking and impairs cell motility, presumably by interfering at multiple steps with chemotactic receptor signaling. Here, we report that a dominant effect of Nef is to trigger AIP4 E3 ligase-mediated Gα(i2) ubiquitination, which leads to Gα(i2) endolysosomal sequestration and destruction. The loss of the Gα(i2) subunit was demonstrable in many cell types in the context of gene transfection, HIV infection, or Nef protein transduction. Nef directly interacts with Gα(i2) and ternary complexes containing AIP4, Nef, and Gα(i2) form. A substantial reversal of Gα(i2) loss and a partial recovery of impaired chemotaxis occurred following siRNA knockdown of AIP4 or NEDD4 or by inhibiting dynamin. The N-terminal myristoyl group, (62)EEEE(65) motif, and (72)PXXP(75) motif of Nef are critical for this effect to occur. Nef expression does not affect a Gq(i5) chimera where the five C-terminal residues of Gq are replaced with those of Gα(i2). Lysine at position 296 of Gα(i2) was identified as the critical determinant of Nef-induced degradation. By specifically degrading Gα(i2), Nef directly subverts leukocyte migration and homing. Impaired trafficking and homing of HIV Nef-expressing lymphocytes probably contributes to early immune dysfunction following HIV infection.

The X-linked Retinitis Pigmentosa protein RP2 facilitates traffic of cilia target proteins

Photoreceptors are specialized ciliated sensory neurons and aberrant traffic of proteins to the outer segment causes photoreceptor cell death. RP2 is a GTPase activating protein (GAP) for the small GTPase Arl3 and both proteins facilitate protein trafficking to primary cilia. We used GST-RP2 pull down from retinal lysates and identified the Gβ subunit of transducin (Gβ1) as a novel RP2 interacting protein. RP2 competes with Gγ1 for Gβ1 binding and does not interact with the Gβ:Gγ heterodimer. In SK-N-SH cells, overexpression of Gβ1 resulted in the cytoplasmic accumulation of the protein, whereas co-expression of Gβ1 with either RP2 or Gγ1 restored membrane association of Gβ1. Depletion of RP2 in ARPE19 cells by siRNA resulted in a shift of Gβ1 from the membrane to the cytosol, confirming that RP2 facilitates the membrane association of Gβ1. This shift in Gβ1 localization was rescued by Gγ1 overexpression. Membrane targeting of Gβ1 required RP2 N-terminal myristoylation and occurs via the co-factor C (TBCC) homology domain. The interaction was disrupted by the pathogenic RP2 mutation R118H, which blocks Arl3 GAP activity. Arl3-Q71L competed with Gβ1 for RP2 binding suggesting that RP2 GAP activity on Arl3 would release Gβ1. RP2 stimulated the association of Gβ1 with Rab11, an important GTPase for post-Golgi vesicle trafficking of photoreceptor proteins. Collectively our data support a role for RP2 in facilitating membrane association and traffic of Gβ1. Combined with other recent evidence, this suggests that RP2 may co-operate with Arl3 and its effectors in cilia associated trafficking of G proteins.

NMR structure of the calflagin Tb24 flagellar calcium binding protein of Trypanosoma brucei

Flagellar calcium binding proteins are expressed in a variety of trypanosomes and are potential drug targets for Chagas disease and African sleeping sickness. The flagellar calcium binding protein calflagin of Trypanosoma brucei (called Tb24) is a myristoylated and palmitoylated EF-hand protein that is targeted to the inner leaflet of the flagellar membrane. The Tb24 protein may also interact with proteins on the membrane surface that may be different from those bound to flagellar calcium binding proteins (FCaBPs) in T. cruzi. We report here the NMR structure of Tb24 that contains four EF-hand motifs bundled in a compact arrangement, similar to the overall fold of T. cruzi FCaBP (RMSD = 1.0 Å). A cluster of basic residues (K22, K25, K31, R36, and R38) located on a surface near the N-terminal myristoyl group may be important for membrane binding. Non-conserved residues on the surface of a hydrophobic groove formed by EF2 (P91, Q95, D103, and V108) and EF4 (C194, T198, K199, Q202, and V203) may serve as a target protein binding site and could have implications for membrane target recognition.

Identification and Characterization of an Extramitochondrial Human 3-Hydroxy-3-methylglutaryl-CoA Lyase

3-Hydroxy-3-methylglutaryl-CoA lyase-like protein (HMGCLL1) has been annotated in the Mammalian Genome Collection as a previously unidentified human HMG-CoA lyase (HMGCL). To test the validity of this annotation and evaluate the physiological role of the protein, plasmids were constructed for protein expression in Escherichia coli and Pichia pastoris. Protein expression in E. coli produced insoluble material. In contrast, active HMGCLL1 could be recovered upon expression in P. pastoris. Antibodies were prepared against a unique peptide sequence found in the N terminus of the protein. In immunodetection experiments, the antibodies discriminated between HMGCLL1 and mitochondrial HMGCL. Purified enzyme was characterized and demonstrated to cleave HMG-CoA to acetoacetate and acetyl-CoA with catalytic and affinity properties comparable with human mitochondrial HMGCL. The deduced HMGCLL1 sequence contains an N-terminal myristoylation motif; the putative modification site was eliminated by construction of a G2A HMGCLL1. Modification of both proteins was attempted using human N-myristoyltransferase and [(3)H]myristoyl-CoA. Wild-type protein was clearly modified, whereas G2A protein was not labeled. Myristoylation of HMGCLL1 affects its cellular localization. Upon transfection of appropriate expression plasmids into COS1 cells, immunofluorescence detection indicates that G2A HMGCLL1 exhibits a diffuse pattern, suggesting a cytosolic location. In contrast, wild-type HMGCLL1 exhibits a punctate as well as a perinuclear immunostaining pattern, indicating myristoylation dependent association with nonmitochondrial membrane compartments. In control experiments with the HMGCL expression plasmid, protein is localized in the mitochondria, as anticipated. The available results for COS1 cell expression, as well as endogenous expression in U87 cells, indicate that HMGCLL1 is an extramitochondrial hydroxymethylglutaryl-CoA lyase.

Repression of Transcription by WT1-BASP1 Requires the Myristoylation of BASP1 and the PIP2-Dependent Recruitment of Histone Deacetylase

The Wilms' tumor 1 protein WT1 is a transcriptional regulator that is involved in cell growth and differentiation. The transcriptional corepressor BASP1 interacts with WT1 and converts WT1 from a transcriptional activator to a repressor. Here, we demonstrate that the N-terminal myristoylation of BASP1 is required in order to elicit transcriptional repression at WT1 target genes. We show that myristoylated BASP1 binds to nuclear PIP2, which leads to the recruitment of PIP2 to the promoter regions of WT1-dependent target genes. BASP1's myristoylation and association with PIP2 are required for the interaction of BASP1 with HDAC1, which mediates the recruitment of HDAC1 to the promoter and elicits transcriptional repression. Our findings uncover a role for myristoylation in transcription, as well as a critical function for PIP2 in gene-specific transcriptional repression through the recruitment of histone deacetylase.

GTP-Binding-Defective ARL4D Alters Mitochondrial Morphology and Membrane Potential

ARL4D, ARL4A, and ARL4C are closely related members of the ADP-ribosylation factor/ARF-like protein (ARF/ARL) family of GTPases. All three ARL4 proteins contain nuclear localization signals (NLSs) at their C-termini and are primarily found at the plasma membrane, but they are also present in the nucleus and cytoplasm. ARF function and localization depends on their controlled binding and hydrolysis of GTP. Here we show that GTP-binding-defective ARL4D is targeted to the mitochondria, where it affects mitochondrial morphology and function. We found that a portion of endogenous ARL4D and the GTP-binding-defective ARL4D mutant ARL4D(T35N) reside in the mitochondria. The N-terminal myristoylation of ARL4D(T35N) was required for its localization to mitochondria. The localization of ARL4D(T35N) to the mitochondria reduced the mitochondrial membrane potential (ΔΨm) and caused mitochondrial fragmentation. Furthermore, the C-terminal NLS region of ARL4D(T35N) was required for its effect on the mitochondria. This study is the first to demonstrate that the dysfunctional GTP-binding-defective ARL4D is targeted to mitochondria, where it subsequently alters mitochondrial morphology and membrane potential.

Widespread Occurrence of N-Terminal Acylation in Animal Globins and Possible Origin of Respiratory Globins from a Membrane-Bound Ancestor

Proteins of the (hemo-)globin superfamily have been identified in many different animals but also occur in plants, fungi, and bacteria. Globins are renowned for their ability to store and to transport oxygen, but additional globin functions such as sensing, signaling, and detoxification have been proposed. Recently, we found that the zebrafish globin X protein is myristoylated and palmitoylated at its N-terminus. The addition of fatty acids results in an association with the cellular membranes, suggesting a previously unrecognized globin function. In this study, we show that N-terminal acylation likely occurs in globin proteins from a broad range of phyla. An N-terminal myristoylation site was identified in 90 nonredundant globins from Chlorophyta, Heterokontophyta, Cnidaria, Mollusca, Arthropoda, Nematoda, Echinodermata, Hemichordata, and Chordata (including Cephalochordata), of which 66 proteins carry an additional palmitoylation site. Bayesian phylogenetic analyses identified five major globin families, which may mirror the ancient globin diversity of the Metazoa. Globin X-like proteins form two related clades, which diverged before the radiation of the Eumetazoa. Vertebrate hemoglobin (Hb), myoglobin, cytoglobin, globin E, and globin Y form a strongly supported common clade, which is the sister group of a clade consisting of invertebrate Hbs and relatives. The N-terminally acylated globins do not form a single monophyletic group but are distributed to four distinct clades. This pattern may be either explained by multiple introduction of an N-terminal acylation site into distinct globin lineages or by the origin of animal respiratory globins from a membrane-bound ancestor. Similarly, respiratory globins were not monophyletic. This suggests that respiratory globins might have emerged independently several times and that the early metazoan globins might have been associated with a membrane and carried out a function that was related to lipid protection or signaling.

Myristoylation Exerts Direct and Allosteric Effects on Gα Conformation and Dynamics in Solution

Coupling of heterotrimeric G proteins to activated G protein-coupled receptors results in nucleotide exchange on the Gα subunit, which in turn decreases its affinity for both Gβγ and activated receptors. N-Terminal myristoylation of Gα subunits aids in membrane localization of inactive G proteins. Despite the presence of the covalently attached myristoyl group, Gα proteins are highly soluble after GTP binding. This study investigated factors facilitating the solubility of the activated, myristoylated protein. In doing so, we also identified myristoylation-dependent differences in regions of Gα known to play important roles in interactions with receptors, effectors, and nucleotide binding. Amide hydrogen-deuterium exchange and site-directed fluorescence of activated proteins revealed a solvent-protected amino terminus that was enhanced by myristoylation. Furthermore, fluorescence quenching confirmed that the myristoylated amino terminus is in proximity to the Switch II region in the activated protein. Myristoylation also stabilized the interaction between the guanine ring and the base of the α5 helix that contacts the bound nucleotide. The allosteric effects of myristoylation on protein structure, function, and localization indicate that the myristoylated amino terminus of Gα(i) functions as a myristoyl switch, with implications for myristoylation in the stabilization of nucleotide binding and in the spatial regulation of G protein signaling.

Acylation-dependent and-independent membrane targeting and distinct functions of small myristoylated proteins (SMPs) in Leishmania major

Trypanosomatid parasites express a number of mono- and diacylated proteins that are targeted to distinct regions of the plasma membrane including the cell body, the flagellum and the flagellar pocket. The extent to which the acylation status and other protein motifs regulate the targeting and/or retention of these proteins to the distinct membrane domains is poorly defined. We have previously described a family of small myristoylated proteins (SMPs) that are either monoacylated (myristoylated) or diacylated (myristoylated and palmitoylated) and targeted to distinct plasma membrane domains. Diacylated SMP-1 is a major constituent of the flagellar membrane, whereas monoacylated SMP-2 resides in the flagellar pocket in Leishmania major. Here, we show that a third SMP family member, monoacylated SMP-4, localizes predominantly to the pellicular membrane. Density gradient centrifugation of detergent-insoluble membranes indicated that SMP-4 was associated with detergent-insoluble domains but was not tightly associated with the subpellicular cytoskeleton. Based on the localisation of truncated SMP proteins, we conclude that the flagellum targeting of SMP-1 is primarily dependent on the dual-acylation motif. In contrast, the localisation of SMP-4 to the cell body membrane is dependent on N-terminal myristoylation and a C-terminal peptide subdomain with a predicted α-helical structure. Strikingly, a SMP-1 chimera containing the SMP-4 C-terminal extension was selectively trafficked to the distal tip of the flagellum and failed to complement the loss of native SMP-1 in a Δsmp1/2 double knockout strain. Collectively, these results suggest that dual acylation is sufficient to target some SMP proteins to the flagellum, while the unique C-terminal extensions of these proteins may confer additional membrane targeting signals that are important for both localisation and SMP function.

Plant plasma membrane-bound staphylococcal-like DNases as a novel class of eukaryotic nucleases

BackgroundThe activity of degradative nucleases responsible for genomic DNA digestion has been observed in all kingdoms of life. It is believed that the main function of DNA degradation occurring during plant programmed cell death is redistribution of nucleic acid derived products such as nitrogen, phosphorus and nucleotide bases. Plant degradative nucleases that have been studied so far belong mainly to the S1-type family and were identified in cellular compartments containing nucleic acids or in the organelles where they are stored before final application. However, the explanation of how degraded DNA components are exported from the dying cells for further reutilization remains open.ResultsBioinformatic and experimental data presented in this paper indicate that two Arabidopsis staphylococcal-like nucleases, named CAN1 and CAN2, are anchored to the cell membrane via N-terminal myristoylation and palmitoylation modifications. Both proteins possess a unique hybrid structure in their catalytic domain consisting of staphylococcal nuclease-like and tRNA synthetase anticodon binding-like motifs. They are neutral, Ca2+-dependent nucleaces showing a different specificity toward the ssDNA, dsDNA and RNA substrates. A study of microarray experiments and endogenous nuclease activity revealed that expression of CAN1 gene correlates with different forms of programmed cell death, while the CAN2 gene is constitutively expressed.ConclusionsIn this paper we present evidence showing that two plant staphylococcal-like nucleases belong to a new, as yet unidentified class of eukaryotic nucleases, characterized by unique plasma membrane localization. The identification of this class of nucleases indicates that plant cells possess additional, so far uncharacterized, mechanisms responsible for DNA and RNA degradation. The potential functions of these nucleases in relation to their unique intracellular location are discussed.

Molecular structure and target recognition of neuronal calcium sensor proteins

Neuronal calcium sensor (NCS) proteins, a sub-branch of the EF-hand superfamily, are expressed in the brain and retina where they transduce calcium signals and are genetically linked to degenerative diseases. The amino acid sequences of NCS proteins are highly conserved but their physiological functions are quite distinct. Retinal recoverin and guanylate cyclase activating proteins (GCAPs) both serve as calcium sensors in retinal rod cells, neuronal frequenin (NCS1) modulates synaptic activity and neuronal secretion, K+ channel interacting proteins (KChIPs) regulate ion channels to control neuronal excitability, and DREAM (KChIP3) is a transcriptional repressor that regulates neuronal gene expression. Here we review the molecular structures of myristoylated forms of NCS1, recoverin, and GCAP1 that all look very different, suggesting that the sequestered myristoyl group helps to refold these highly homologous proteins into very different structures. The molecular structure of NCS target complexes have been solved for recoverin bound to rhodopsin kinase (RK), NCS-1 bound to phosphatidylinositol 4-kinase, and KChIP1 bound to A-type K+ channels. We propose that N-terminal myristoylation is critical for shaping each NCS family member into a different structure, which upon Ca2+-induced extrusion of the myristoyl group exposes a unique set of previously masked residues that interact with a particular physiological target.

Myristic Acid Increases Dihydroceramide Δ4‐Desaturase 1 (DES1) Activity in Cultured Rat Hepatocytes

Dihydroceramide Δ4-desaturase 1 (DES1) catalyzes the last step of the de novo ceramide biosynthesis, which consists of the introduction of a trans Δ4-double bond in the carbon chain of the dihydroceramide. It was previously observed that myristic acid binds DES1 through N-myristoylation. This N-terminal modification significantly increased the activity of the recombinant DES1 in COS-7 cells and targeted part of the enzyme initially present in the endoplasmic reticulum to the mitochondrial outer membrane, leading to an increase in ceramide levels. Since these results were obtained in a recombinant COS-7 cell model with high expression of rat DES1, the purpose of the present study was to investigate if the native DES1 enzyme was really upregulated by its N-myristoylation in cultured rat hepatocytes. We first showed that DES1 was the main dihydroceramide desaturase isoform expressed in rat hepatocytes. In this model, the wild-type myristoylable recombinant form of rat DES1 was found in both the endoplasmic reticulum and the mitochondria whereas the mutated non-myristoylable recombinant form (N-terminal glycine replaced by an alanine) was almost exclusively localized in the endoplasmic reticulum, which evidenced the importance of the myristoylation. Then, we showed that compared to other fatty acids, myristic acid was the only one to increase native DES1 activity, in both total cell lysates and mitochondrial fractions. The myristic acid-associated increase in DES1 activity was not linked to elevated mRNA or protein expression but more likely to its N-terminal myristoylation. Finally, the myristic acid-associated increase in DES1 activity slightly enhanced the number of apoptotic cells.

N-Terminally Myristoylated Feline Foamy Virus Gag Allows Env-Independent Budding of Sub-Viral Particles

Foamy viruses (FVs) are distinct retroviruses classified as Spumaretrovirinae in contrast to the other retroviruses, the Orthoretrovirinae. As a unique feature of FVs, Gag is not sufficient for sub-viral particle (SVP) release. In primate and feline FVs (PFV and FFV), particle budding completely depends on the cognate FV Env glycoproteins. It was recently shown that an artificially added N-terminal Gag myristoylation signal (myr-signal) overcomes this restriction in PFV inducing an Orthoretrovirus-like budding phenotype. Here we show that engineered, heterologous N-terminal myr-signals also induce budding of the distantly related FFV Gag. The budding efficiency depends on the myr-signal and its location relative to the N-terminus of Gag. When the first nine amino acid residues of FFV Gag were replaced by known myr-signals, the budding efficiency as determined by the detection of extracellular SVPs was low. In contrast, adding myr-signals to the intact N-terminus of FFV Gag resulted in a more efficient SVP release. Importantly, budding of myr-Gag proteins was sensitive towards inhibition of cellular N-myristoyltransferases. As expected, the addition or insertion of myr-signals that allowed Env-independent budding of FFV SVPs also retargeted Gag to plasma membrane-proximal sites and other intracellular membrane compartments. The data confirm that membrane-targeted FV Gag has the capacity of SVP formation.

Trafficking of Platelet-Activating Factor Acetylhydrolase Type II in Response to Oxidative Stress

Platelet-activating factor acetylhydrolase type II (PAFAH-II) is an intracellular phospholipase A(2) enzyme that hydrolyzes platelet-activating factor and oxidatively fragmented phospholipids. This N-terminally myristoylated protein becomes associated with cytoplasm-facing cell membranes under oxidative stress. The structural requirements for binding of PAFAH-II to membranes in response to oxidative stress are unknown. To begin elucidating the mechanism of trafficking and stress response, we constructed a homology model of PAFAH-II. From the predicted membrane orientation of PAFAH-II, the N-terminal myristoyl group and a hydrophobic patch are hypothesized to be involved in membrane binding. Localization studies of human PAFAH-II in HEK293 cells indicated that an unmyristoylated mutant remained cytoplasmic under stressed and unstressed conditions. The myristoylated wild-type enzyme was partially localized to the cytoplasmic membranes prior to stress and became more localized to these membranes upon stress. A triple mutation of three hydrophobic patch residues of the membrane binding region likewise did not localize to membranes following stress. These results indicate that both the myristoyl group and the hydrophobic patch are essential for proper trafficking of the enzyme to the membranes following oxidative stress. Additionally, colocalization studies using organelle-specific proteins demonstrate that PAFAH-II is transported to the membranes of both the endoplasmic reticulum and Golgi apparatus.

Neurospora crassa homologue of Neuronal Calcium Sensor-1 has a role in growth, calcium stress tolerance, and ultraviolet survival

NCU04379 gene encodes a conserved Ca(2+) and/or calmodulin binding protein that possesses a consensus signal for N-terminal myristoylation and four EF-hands, characteristics of Neuronal Calcium Sensor-1proteins. The NCU04379.2 knockout mutant shows slow growth rate, increased sensitivity to calcium and ultraviolet (UV) irradiation, and a wild-type fragment carrying NCU04379 gene complements the mutant. Therefore, NCU04379 gene has a role in growth, calcium stress tolerance, and UV survival. Crosses homozygous for ΔNCU04379.2 mutant strains were fully fertile; however, we found evidence for involvement of Ca(2+)/calmodulin-dependent protein kinase encoding genes NCU02283 and NCU09123 in sexual development.

Expression and purification of myristoylated matrix protein of Mason-Pfizer monkey virus for NMR and MS measurements

Matrix proteins play multiple roles both in early and late stages of the viral replication cycle. Their N-terminal myristoylation is important for interaction with the host cell membrane during virus budding. We used Escherichia coli, carrying N-myristoyltransferase gene, for the expression of the myristoylated His-tagged matrix protein of Mason-Pfizer monkey virus. An efficient, single-step purification procedure eliminating all contaminating proteins including, importantly, the non-myristoylated matrix protein was designed. The comparison of NMR spectra of matrix protein with its myristoylated form revealed substantial structural changes induced by this fatty acid modification.

Discovery and Characterization of a Cell-Permeable, Small-Molecule c-Abl Kinase Activator that Binds to the Myristoyl Binding Site

c-Abl kinase activity is regulated by a unique mechanism involving the formation of an autoinhibited conformation in which the N-terminal myristoyl group binds intramolecularly to the myristoyl binding site on the kinase domain and induces the bending of the αI helix that creates a docking surface for the SH2 domain. Here, we report a small-molecule c-Abl activator, DPH, that displays potent enzymatic and cellular activity in stimulating c-Abl activation. Structural analyses indicate that DPH binds to the myristoyl binding site and prevents the formation of the bent conformation of the αI helix through steric hindrance, a mode of action distinct from the previously identified allosteric c-Abl inhibitor, GNF-2, that also binds to the myristoyl binding site. DPH represents the first cell-permeable, small-molecule tool compound for c-Abl activation.

HID-1 is a peripheral membrane protein primarily associated with the medial- and trans- Golgi apparatus

Caenorhabditis elegans hid-1 gene was first identified in a screen for mutants with a high-temperature-induced dauer formation (Hid) phenotype. Despite the fact that the hid-1 gene encodes a novel protein (HID-1) which is highly conserved from Caenorhabditis elegans to mammals, the domain structure, subcellular localization, and exact function of HID-1 remain unknown. Previous studies and various bioinformatic softwares predicted that HID-1 contained many transmembrane domains but no known functional domain. In this study, we revealed that mammalian HID-1 localized to the medial- and trans- Golgi apparatus as well as the cytosol, and the localization was sensitive to brefeldin A treatment. Next, we demonstrated that HID-1 was a peripheral membrane protein and dynamically shuttled between the Golgi apparatus and the cytosol. Finally, we verified that a conserved N-terminal myristoylation site was required for HID-1 binding to the Golgi apparatus. We propose that HID-1 is probably involved in the intracellular trafficking within the Golgi region.