N-terminal Pleckstrin Homology Research Articles

A Structural Network Analysis of Neuronal ArhGAP21/23 Interactors by Computational Modeling.

RhoGTPase-activating proteins (RhoGAPs) play multiple roles in neuronal development; however, details of their substrate recognition system remain elusive. ArhGAP21 and ArhGAP23 are RhoGAPs that contain N-terminal PDZ and pleckstrin homology domains. In the present study, the RhoGAP domain of these ArhGAPs was computationally modeled by template-based methods and the AlphaFold2 software program, and their intrinsic RhoGTPase recognition mechanism was analyzed from the domain structures using the protein docking programs HADDOCK and HDOCK. ArhGAP21 was predicted to preferentially catalyze Cdc42, RhoA, RhoB, RhoC, and RhoG and to downregulate RhoD and Tc10 activities. Regarding ArhGAP23, RhoA and Cdc42 were deduced to be its substrates, whereas RhoD downregulation was predicted to be less efficient. The PDZ domains of ArhGAP21/23 possess the FTLRXXXVY sequence, and similar globular folding consists of antiparalleled β-sheets and two α-helices that are conserved with PDZ domains of MAST-family proteins. A peptide docking analysis revealed the specific interaction of the ArhGAP23 PDZ domain with the PTEN C-terminus. The pleckstrin homology domain structure of ArhGAP23 was also predicted, and the functional selectivity for the interactors regulated by the folding and disordered domains in ArhGAP21 and ArhGAP23 was examined by an in silico analysis. An interaction analysis of these RhoGAPs revealed the existence of mammalian ArhGAP21/23-specific type I and type III Arf- and RhoGTPase-regulated signaling. Multiple recognition systems of RhoGTPase substrates and selective Arf-dependent localization of ArhGAP21/23 may form the basis of the functional core signaling necessary for synaptic homeostasis and axon/dendritic transport regulated by RhoGAP localization and activities.

Structural Insights Uncover the Specific Phosphoinositide Recognition by the PH1 Domain of Arap3.

Arap3, a dual GTPase-activating protein (GAP) for the small GTPases Arf6 and RhoA, plays key roles in regulating a wide range of biological processes, including cancer cell invasion and metastasis. It is known that Arap3 is a PI3K effector that can bind directly to PI(3,4,5)P3, and the PI(3,4,5)P3-mediated plasma membrane recruitment is crucial for its function. However, the molecular mechanism of how the protein recognizes PI(3,4,5)P3 remains unclear. Here, using liposome pull-down and surface plasmon resonance (SPR) analysis, we found that the N-terminal first pleckstrin homology (PH) domain (Arap3-PH1) can interact with PI(3,4,5)P3 and, with lower affinity, with PI(4,5)P2. To understand how Arap3-PH1 and phosphoinositide (PIP) lipids interact, we solved the crystal structure of the Arap3-PH1 in the apo form and complex with diC4-PI(3,4,5)P3. We also characterized the interactions of Arap3-PH1 with diC4-PI(3,4,5)P3 and diC4-PI(4,5)P2 in solution by nuclear magnetic resonance (NMR) spectroscopy. Furthermore, we found overexpression of Arap3 could inhibit breast cancer cell invasion in vitro, and the PIPs-binding ability of the PH1 domain is essential for this function.

Structure of the metastatic factor P-Rex1 reveals a two-layered autoinhibitory mechanism

P-Rex (PI(3,4,5)P3-dependent Rac exchanger) guanine nucleotide exchange factors potently activate Rho GTPases. P-Rex guanine nucleotide exchange factors are autoinhibited, synergistically activated by Gβγ and PI(3,4,5)P3 binding and dysregulated in cancer. Here, we use X-ray crystallography, cryogenic electron microscopy and crosslinking mass spectrometry to determine the structural basis of human P-Rex1 autoinhibition. P-Rex1 has a bipartite structure of N- and C-terminal modules connected by a C-terminal four-helix bundle that binds the N-terminal Pleckstrin homology (PH) domain. In the N-terminal module, the Dbl homology (DH) domain catalytic surface is occluded by the compact arrangement of the DH-PH-DEP1 domains. Structural analysis reveals a remarkable conformational transition to release autoinhibition, requiring a 126° opening of the DH domain hinge helix. The off-axis position of Gβγ and PI(3,4,5)P3 binding sites further suggests a counter-rotation of the P-Rex1 halves by 90° facilitates PH domain uncoupling from the four-helix bundle, releasing the autoinhibited DH domain to drive Rho GTPase signaling.

A RAC-alpha serine/threonine-protein kinase (CgAKT1) involved in the synthesis of CgIFNLP in oyster Crassostrea gigas

The RAC-alpha serine/threonine-protein kinase (AKT) is one of the most important protein kinases involved in many biological processes in eukaryotes. In the present study, a novel AKT homologue named CgAKT1 was identified from the Pacific oyster Crassostrea gigas. The open reading frame (ORF) of CgAKT1 cDNA was of 1482 bp encoding a peptide with 493 amino acid residues. There were classical domains in the predicted CgAKT1 protein, including an N-terminal pleckstrin homology domain, a central catalytic domain and a C-terminal hydrophobic domain. The mRNA transcripts of CgAKT1 were detected in all the examined tissues of C. gigas with higher level in gills (8.24-fold of that in mantle, p < 0.05) and haemocytes (3.62-fold of that in mantle, p < 0.05). After poly (I:C) stimulation, the mRNA expression of CgAKT1 decreased significantly in haemocytes from 3 h (0.44-fold of that in the control group, p < 0.001) to 24 h (0.20-fold of that in the control group, p < 0.001), and then increased significantly at 48 h (3.65-fold of that in the control group, p < 0.05). The expression level of CgAKT1 mRNA increased significantly at 6 h after rCgIFNLP stimulation, which was 3.60-fold of that in the control group (p < 0.001). The Alexa Fluor 488 positive signals of CgAKT1 protein were found to be distributed in the cytoplasm and cell membrane of haemocytes, while those in the cytoplasm became weaker after poly (I:C) stimulation. In CgAKT1-RNAi oysters, the mRNA expression of cyclic GMP-AMP synthase (CgcGAS) and TANK-binding kinase 1 (CgTBK1) did not change significantly, but the mRNA expression level of stimulator of interferon gene (CgSTING), interferon regulatory factor-1 (CgIRF-1), interferon regulatory factor-8 (CgIRF-8) and IFN-like protein (CgIFNLP) increased significantly, which was 1.40-fold, 1.53-fold, 1.72-fold and 1.99-fold of that in EGFP-RNAi oysters (p < 0.05), respectively. In CgIFNLP-RNAi oysters, the transcripts of CgAKT1 decreased significantly compared to those in EGFP-RNAi oysters (0.16-fold, p < 0.01). Moreover, the expression of p-CgTBK1, CgSTING and CgIFNLP at the protein level in the oysters treated with p-AKT1 activator (SC-79) was significantly suppressed after poly (I:C) stimulation. After the transfection of CgAKT1, the expression of p-cGAS protein in HEK293T cells increased significantly, while the cyclic GMP-AMP in the cells and the interferon (IFN-β) in the cell culture fluid decreased significantly compared with that in the control group. These results indicated that CgAKT1 might play a negative role in antiviral immunity of oyster by regulating the synthesis of CgIFNLP.

Simulations of Kindlin-2 PIP binding domains reveal protonation-dependent membrane binding modes

Kindlin-2, a member of the Kindlin family of peripheral membrane proteins, is important for integrin activation and stabilization of epidermal growth factor receptor. It associates with the cytoplasmic face of the plasma membrane via dedicated phosphatidylinositol phosphate binding domains located in the N-terminal F0 and Pleckstrin Homology domains. These domains have binding affinity for phosphatidylinositol 4,5-bisphosphate and, to a greater degree, phosphatidylinositol 3,4,5-trisphosphate. The biological significance of the differential binding of these phosphatidylinositol phosphates to Kindlin-2 and the mechanism by which they activate Kindlin-2 are not well understood. Recently, ssNMR identified the predominant protonation states of phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate near physiological pH in the presence of anionic lipids. Here, we perform atomistic simulation of the bound state of the Pleckstrin Homology and F0 domains of Kindlin-2 at membranes containing phosphatidylinositol 4,5-bisphosphate/phosphatidylinositol 3,4,5-trisphosphate with differing protonation states. This computational approach demonstrates that these two phosphatidylinositol phosphates differently modulate Kindlin-2 subdomain binding in a protonation-state-dependent manner. We speculate these variations in binding mode provide a mechanism for intracellular pH and Ca2+ influx to control the membrane binding behavior and activity of Kindlin-2.

Structural Insight on Functional Regulation of Human MINERVA Protein.

MINERVA (melanoma invasion by ERK), also known as FAM129B, is a member of the FAM129 protein family, which is only present in vertebrates. MINERVA is involved in key signaling pathways regulating cell survival, proliferation and apoptosis and found upregulated in many types of cancer promoting invasion. However, the exact function of the protein remains elusive. X-ray crystallographic methods were implemented to determine the crystal structure of MINERVAΔC, lacking C-terminal flexible region. Trypsin digestion was required before crystallization to obtain diffraction-quality crystals. While the N-terminal pleckstrin homology (PH) domain exhibits the typical fold of PH domains, lipid binding assay indicates specific affinity towards phosphatidic acid and inositol 3-phosphate. A helix-rich domain that constitutes the rest of the molecule demonstrates a novel L-shaped fold that encompasses the PH domain. The overall structure of MINERVAΔC with binding assays and cell-based experiments suggest plasma membrane association of MINERVA and its function seem to be tightly regulated by various motifs within the C-terminal flexible region. Elucidation of MINERVAΔC structure presents a novel fold for an α-helix bundle domain that would provide a binding platform for interacting partners.

The structural determinants of PH domain-mediated regulation of Akt revealed by segmental labeling.

Akt is a critical protein kinase that governs cancer cell growth and metabolism. Akt appears to be autoinhibited by an intramolecular interaction between its N-terminal pleckstrin homology (PH) domain and kinase domain, which is relieved by C-tail phosphorylation, but the precise molecular mechanisms remain elusive. Here, we use a combination of protein semisynthesis, NMR, and enzymological analysis to characterize structural features of the PH domain in its autoinhibited and activated states. We find that Akt autoinhibition depends on the length/flexibility of the PH-kinase linker. We identify a role for a dynamic short segment in the PH domain that appears to regulate autoinhibition and PDK1-catalyzed phosphorylation of Thr308 in the activation loop. We determine that Akt allosteric inhibitor MK2206 drives distinct PH domain structural changes compared to baseline autoinhibited Akt. These results highlight how the conformational plasticity of Akt governs the delicate control of its catalytic properties.

PI(4,5)P2-dependent regulation of exocytosis by amisyn, the vertebrate-specific competitor of synaptobrevin 2

The functions of nervous and neuroendocrine systems rely on fast and tightly regulated release of neurotransmitters stored in secretory vesicles through SNARE-mediated exocytosis. Few proteins, including tomosyn (STXBP5) and amisyn (STXBP6), were proposed to negatively regulate exocytosis. Little is known about amisyn, a 24-kDa brain-enriched protein with a SNARE motif. We report here that full-length amisyn forms a stable SNARE complex with syntaxin-1 and SNAP-25 through its C-terminal SNARE motif and competes with synaptobrevin-2/VAMP2 for the SNARE-complex assembly. Furthermore, amisyn contains an N-terminal pleckstrin homology domain that mediates its transient association with the plasma membrane of neurosecretory cells by binding to phospholipid PI(4,5)P2 However, unlike synaptrobrevin-2, the SNARE motif of amisyn is not sufficient to account for the role of amisyn in exocytosis: Both the pleckstrin homology domain and the SNARE motif are needed for its inhibitory function. Mechanistically, amisyn interferes with the priming of secretory vesicles and the sizes of releasable vesicle pools, but not vesicle fusion properties. Our biochemical and functional analyses of this vertebrate-specific protein unveil key aspects of negative regulation of exocytosis.

Characterization of phospholipid binding by the exocyst complex using lipid nanodiscs

Proper regulation of exocytosis is crucial for cell function. Post‐Golgi vesicles require precise localization and temporal specification to fuse at correct sites on the plasma membrane to secrete their cargo 1. Secretion is essential for cell survival and is involved in intercellular communication and cell migration 2. Multisubunit tethering complexes (MTCs) are protein assemblies that coordinate interactions required for membrane fusion. Exocyst is a hetero‐octameric MTC responsible for targeting secretory vesicles to the plasma membrane by recognizing SNAREs, small GTPases, and phospholipid 2. Two exocyst subunits, Sec3 and Exo70, have been shown to bind phosphatidylinositol 4,5 biphosphate (PI(4,5)P2). Sec3 contains an N‐terminal Pleckstrin homology (PH) domain and there is a positively charged patch in the C‐terminus of Exo70 3, 4. These two domains mediate interactions with PI(4,5)P2. However, these interactions have only been shown using recombinantly purified individual subunits or domains. Combining exocyst purified from cryo‐milled yeast lysate with lipid nanodiscs, we are able to study binding of the wildtype and mutant octameric complex to various phospholipids. This has allowed us to characterize differential contributions of Sec3 and Exo70 to PI(4,5)P2 binding. Further, nanodiscs allow us to investigate interactions of the exocyst complex with a wide range of phospholipids, demonstrating the specificity of exocyst‐PI(4,5)P2 binding. This research is crucial to understanding the mechanism by which exocytic machinery is targeted to sites of exocytosis.

Short AIP1 (ASK1-Interacting Protein-1) Isoform Localizes to the Mitochondria and Promotes Vascular Dysfunction.

Vascular endothelial cells (ECs) normally maintain vascular homeostasis and are regulated by proinflammatory cytokines and reactive oxygen species. A human genome-wide association study identified that AIP1 (ASK1 [apoptosis signal-regulating kinase 1]-interacting protein-1; also identified as DAB2IP) gene variants confer susceptibility to cardiovascular disease, but the underlying mechanism is unknown. Approach and Results: We detected a normal AIP1 form (named AIP1A) in the healthy aorta, but a shorter form of AIP1 (named AIP1B) was found in diseased aortae that contained atherosclerotic plaques and graft arteriosclerosis. AIP1B transcription in resting ECs was suppressed through epigenetic inhibition by RIF1 (Rap1 [ras-related protein 1]-interacting factor 1)/H3K9 (histone H3 lysine 9) methyltransferase-mediated H3K9 trimethylation, and this inhibition was released by proinflammatory cytokines. AIP1A, but not AIP1B, was downregulated by proteolytic degradation through a Smurf1 (SMAD [suppressor of mothers against decapentaplegic miscellaneous] ubiquitylation regulatory factor 1)-dependent pathway in ECs under inflammation. Therefore, AIP1B was the major form present during inflammatory conditions. AIP1B, which lacks the N-terminal pleckstrin homology domain of AIP1A, localized to the mitochondria and augmented TNFα (tumor necrosis factor alpha)-induced mitochondrial reactive oxygen species generation and EC activation. AIP1B-ECTG (EC-specific AIP1B transgenic) mice exhibited augmented reactive oxygen species production, EC activation, and neointima formation in vascular remodeling models. Our current study suggests that a shift from anti-inflammatory AIP1A to proinflammatory AIP1B during chronic inflammation plays a key role in inflammatory vascular diseases.

LIF-dependent survival of embryonic stem cells is regulated by a novel palmitoylated Gab1 signalling protein.

ABSTRACTThe cytokine leukaemia inhibitory factor (LIF) promotes self-renewal of mouse embryonic stem cells (ESCs) through activation of the transcription factor Stat3. However, the contribution of other ancillary pathways stimulated by LIF in ESCs, such as the MAPK and PI3K pathways, is less well understood. We show here that naive-type mouse ESCs express high levels of a novel effector of the MAPK and PI3K pathways. This effector is an isoform of the Gab1 (Grb2-associated binder protein 1) adaptor protein that lacks the N-terminal pleckstrin homology (PH) membrane-binding domain. Although not essential for rapid unrestricted growth of ESCs under optimal conditions, the novel Gab1 variant (Gab1β) is required for LIF-mediated cell survival under conditions of limited nutrient availability. This enhanced survival is absolutely dependent upon a latent palmitoylation site that targets Gab1β directly to ESC membranes. These results show that constitutive association of Gab1 with membranes through a novel mechanism promotes LIF-dependent survival of murine ESCs in nutrient-poor conditions.

Phosphorylation-Dependent Inhibition of Akt1.

Protein kinase B (Akt1) is a proto-oncogene that is overactive in most cancers. Akt1 activation requires phosphorylation at Thr308; phosphorylation at Ser473 further enhances catalytic activity. Akt1 activity is also regulated via interactions between the kinase domain and the N-terminal auto-inhibitory pleckstrin homology (PH) domain. As it was previously difficult to produce Akt1 in site-specific phosphorylated forms, the contribution of each activating phosphorylation site to auto-inhibition was unknown. Using a combination of genetic code expansion and in vivo enzymatic phosphorylation, we produced Akt1 variants containing programmed phosphorylation to probe the interplay between Akt1 phosphorylation status and the auto-inhibitory function of the PH domain. Deletion of the PH domain increased the enzyme activity for all three phosphorylated Akt1 variants. For the doubly phosphorylated enzyme, deletion of the PH domain relieved auto-inhibition by 295-fold. We next found that phosphorylation at Ser473 provided resistance to chemical inhibition by Akti-1/2 inhibitor VIII. The Akti-1/2 inhibitor was most effective against pAkt1T308 and showed four-fold decreased potency with Akt1 variants phosphorylated at Ser473. The data highlight the need to design more potent Akt1 inhibitors that are effective against the doubly phosphorylated and most pathogenic form of Akt1.

PI(4,5)P2 controls plasma membrane PI4P and PS levels via ORP5/8 recruitment to ER-PM contact sites.

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is a critically important regulatory lipid of the plasma membrane (PM); however, little is known about how cells regulate PM PI(4,5)P2 levels. Here, we show that the phosphatidylinositol 4-phosphate (PI4P)/phosphatidylserine (PS) transfer activity of the endoplasmic reticulum (ER)-resident ORP5 and ORP8 is regulated by both PM PI4P and PI(4,5)P2 Dynamic control of ORP5/8 recruitment to the PM occurs through interactions with the N-terminal Pleckstrin homology domains and adjacent basic residues of ORP5/8 with both PI4P and PI(4,5)P2 Although ORP5 activity requires normal levels of these inositides, ORP8 is called on only when PI(4,5)P2 levels are increased. Regulation of the ORP5/8 attachment to the PM by both phosphoinositides provides a powerful means to determine the relative flux of PI4P toward the ER for PS transport and Sac1-mediated dephosphorylation and PIP 5-kinase-mediated conversion to PI(4,5)P2 Using this rheostat, cells can maintain PI(4,5)P2 levels by adjusting the availability of PI4P in the PM.

Both the PH domain and N-terminal region of oxysterol-binding protein related protein 8S are required for localization to PM-ER contact sites

Oxysterol-binding protein-related proteins are implicated in the sensing and transporting lipids at the membrane contact sites. One of the members of the mammalian ORP family, ORP8, is thought to transport lipids through directly tethering both ER and PM membranes. Targeting to PM is thought to be mediated by N-terminal pleckstrin homology domain via binding to phosphoinositides. Sequence alignments and NMR structural determination revealed that the PH domain of ORP8 is atypical and contains an insertion of 20 amino acids in an unstructured loop region that may potentially block interactions with ligands. Using standard lipid-protein overlay assays or liposomal binding assays we could not detect binding of a recombinant version of the PH domain. Examination of a series of deletion constructs demonstrated that both the N-terminal polybasic region and the PH domain are required for proper targeting of the short splice variant ORP8S to the PM-ER contact site in Chinese hamster ovary cells.

DGAT1 from the arachidonic-acid-producing microalga Lobosphaera incisa shows late gene expression under nitrogen starvation and substrate promiscuity in a heterologous system

We report the identification and characterization of an acyl-CoA:diacylglycerol acyltransferase 1 (DGAT1)-encoding gene from the green oleaginous microalga Lobosphaera incisa (SAG 2468), a prolific photosynthetic producer of the n-6 very long chain polyunsaturated fatty acid (VLC-PUFA), arachidonic acid. The gene expression pattern of LiDGAT1 in L. incisa cells showed a weak increase in mRNA abundance in the course of nitrogen starvation under low light; however, LiDGAT1 expression was significantly upregulated with the progression of N-starvation under high light. Heterologous expression of LiDGAT1 in the neutral lipid-deficient mutant H1246 of Saccharomyces cerevisiae complemented the mutant phenotype and demonstrated an excelling TAG production compared to the yeast endogenous DGAT gene (DGA1). The TAG that formed in the LiDGAT1-expressing H1246 cells contained higher proportions of C16:0 and C18:0 fatty acids, suggesting that at least in a heterologous system, lacking PUFA biosynthesis, the enzyme seems to favor saturated over monounsaturated fatty acids. LiDGAT1 expression prompted an incorporation of several tested exogenous C18 PUFA and C20 VLC-PUFA into TAG. LiDGAT1-driven activity mediated the incorporation of either n-3 or n-6 VLC-PUFA, supplied as substrates for the TAG assembly; however, somewhat of a preference for 18:3n-3 over 20:4n-6 was demonstrated by lipidomics analysis. A structure-functional analysis of LiDGAT1 revealed that the N-terminal Pleckstrin homology (PH) domain is important but not essential for TAG generation in the yeast expression system. Deletion of the PH domain led to decreased TAG formation and ARA incorporation into TAG in yeast. Remarkably, we found the PH domain to be present in the DGAT1 of a number of chlorophytes, in a charophyceaen multicellular alga, in two diatoms and in the liverwort Marchantia polymorpha, but absent from those of red algae, higher plants and animals. Our findings indicate the promiscuity of LiDGAT1 for VLC-PUFA and suggest a specific role for this enzyme in the neutral lipid metabolism of L. incisa that needs to be further investigated by molecular engineering approaches.

The Tiam1 guanine nucleotide exchange factor is auto-inhibited by its pleckstrin homology coiled-coil extension domain

T-cell lymphoma invasion and metastasis 1 (Tiam1) is a Dbl-family guanine nucleotide exchange factor (GEF) that specifically activates the Rho-family GTPase Rac1 in response to upstream signals, thereby regulating cellular processes including cell adhesion and migration. Tiam1 contains multiple domains, including an N-terminal pleckstrin homology coiled-coiled extension (PHn-CC-Ex) and catalytic Dbl homology and C-terminal pleckstrin homology (DH-PHc) domain. Previous studies indicate that larger fragments of Tiam1, such as the region encompassing the N-terminal to C-terminal pleckstrin homology domains (PHn-PHc), are auto-inhibited. However, the domains in this region responsible for inhibition remain unknown. Here, we show that the PHn-CC-Ex domain inhibits Tiam1 GEF activity by directly interacting with the catalytic DH-PHc domain, preventing Rac1 binding and activation. Enzyme kinetics experiments suggested that Tiam1 is auto-inhibited through occlusion of the catalytic site rather than by allostery. Small angle X-ray scattering and ensemble modeling yielded models of the PHn-PHc fragment that indicate it is in equilibrium between "open" and "closed" conformational states. Finally, single-molecule experiments support a model in which conformational sampling between the open and closed states of Tiam1 contributes to Rac1 dissociation. Our results highlight the role of the PHn-CC-Ex domain in Tiam1 GEF regulation and suggest a combinatorial model for GEF inhibition and activation of the Rac1 signaling pathway.

Interaction between the PH and START domains of ceramide transfer protein competes with phosphatidylinositol 4-phosphate binding by the PH domain

De novo synthesis of the sphingolipid sphingomyelin requires non-vesicular transport of ceramide from the endoplasmic reticulum to the Golgi by the multidomain protein ceramide transfer protein (CERT). CERT's N-terminal pleckstrin homology (PH) domain targets it to the Golgi by binding to phosphatidylinositol 4-phosphate (PtdIns(4)P) in the Golgi membrane, whereas its C-terminal StAR-related lipid transfer domain (START) carries out ceramide transfer. Hyperphosphorylation of a serine-rich motif immediately after the PH domain decreases both PtdIns(4)P binding and ceramide transfer by CERT. This down-regulation requires both the PH and START domains, suggesting a possible inhibitory interaction between the two domains. In this study we show that isolated PH and START domains interact with each other. The crystal structure of a PH-START complex revealed that the START domain binds to the PH domain at the same site for PtdIns(4)P-binding, suggesting that the START domain competes with PtdIns(4)P for association with the PH domain. We further report that mutations disrupting the PH-START interaction increase both PtdIns(4)P-binding affinity and ceramide transfer activity of a CERT-serine-rich phosphorylation mimic. We also found that these mutations increase the Golgi localization of CERT inside the cell, consistent with enhanced PtdIns(4)P binding of the mutant. Collectively, our structural, biochemical, and cellular investigations provide important structural insight into the regulation of CERT function and localization.

An unpredicted aggregation-critical region of the actin-polymerizing protein TRIOBP-1/Tara, determined by elucidation of its domain structure

Aggregation of specific proteins in the brains of patients with chronic mental illness as a result of disruptions in proteostasis is an emerging theme in the study of schizophrenia in particular. Proteins including DISC1 (disrupted in schizophrenia 1) and dysbindin-1B are found in insoluble forms within brain homogenates from such patients. We recently identified TRIOBP-1 (Trio-binding protein 1, also known as Tara) to be another such protein through an epitope discovery and proteomics approach by comparing post-mortem brain material from schizophrenia patients and control individuals. We hypothesized that this was likely to occur as a result of a specific subcellular process and that it, therefore, should be possible to identify a region of the TRIOBP-1 protein that is essential for its aggregation to occur. Here, we probe the domain organization of TRIOBP-1, finding it to possess two distinct coiled-coil domains: the central and C-terminal domains. The central domain inhibits the depolymerization of F-actin and is also responsible for oligomerization of TRIOBP-1. Along with an N-terminal pleckstrin homology domain, the central domain affects neurite outgrowth. In neuroblastoma cells it was found that the aggregation propensity of TRIOBP-1 arises from its central domain, with a short "linker" region narrowed to within amino acids 324-348, between its first two coiled coils, as essential for the formation of TRIOBP-1 aggregates. TRIOBP-1 aggregation, therefore, appears to occur through one or more specific cellular mechanisms, which therefore have the potential to be of physiological relevance for the biological process underlying the development of chronic mental illness.

An Autoinhibitory Role for the Pleckstrin Homology Domain of Interleukin-2-Inducible Tyrosine Kinase and Its Interplay with Canonical Phospholipid Recognition.

Pleckstrin homology (PH) domains are well-known as phospholipid binding modules, yet evidence that PH domain function extends beyond lipid recognition is mounting. In this work, we characterize a protein binding function for the PH domain of interleukin-2-inducible tyrosine kinase (ITK), an immune cell specific signaling protein that belongs to the TEC family of nonreceptor tyrosine kinases. Its N-terminal PH domain is a well-characterized lipid binding module that localizes ITK to the membrane via phosphatidylinositol 3,4,5-trisphosphate (PIP3) binding. Using a combination of nuclear magnetic resonance spectroscopy and mutagenesis, we have mapped an autoregulatory protein interaction site on the ITK PH domain that makes direct contact with the catalytic kinase domain of ITK, inhibiting the phospho-transfer reaction. Moreover, we have elucidated an important interplay between lipid binding by the ITK PH domain and the stability of the autoinhibitory complex formed by full length ITK. The ITK activation loop in the kinase domain becomes accessible to phosphorylation to the exogenous kinase LCK upon binding of the ITK PH domain to PIP3. By clarifying the allosteric role of the ITK PH domain in controlling ITK function, we have expanded the functional repertoire of the PH domain generally and opened the door to alternative strategies to target this specific kinase in the context of immune cell signaling.

The carboxyl-terminal region of Dok-7 plays a key, but not essential, role in activation of muscle-specific receptor kinase MuSK and neuromuscular synapse formation.

As the synapse between a motor neuron and skeletal muscle, the neuromuscular junction (NMJ) is required for muscle contraction. The formation and maintenance of NMJs are controlled by the muscle-specific receptor kinase MuSK. Dok-7 is the essential cytoplasmic activator of MuSK, and indeed mice lacking Dok-7 form no NMJs. Moreover, DOK7 gene mutations underlie DOK7 myasthenia, an NMJ synaptopathy. Previously, we failed to detect MuSK activation in myotubes by Dok-7 mutated in the N-terminal pleckstrin homology (PH) or phosphotyrosine binding (PTB) domain or that lacked the C-terminal region (Dok-7-ΔC). Here, we found by quantitative analysis that Dok-7-ΔC marginally, but significantly, activated MuSK in myotubes, unlike the PH- or PTB-mutant. Purified, recombinant Dok-7-ΔC, but not other mutants, also showed marginal ability to activate MuSK's cytoplasmic portion, carrying the kinase domain. Consistently, forced expression of Dok-7-ΔC rescued Dok-7-deficient mice from neonatal lethality caused by the lack of NMJs, indicating restored MuSK activation and NMJ formation. However, these mice showed only marginal activation of MuSK and died by 3 weeks of age apparently due to an abnormally small number and size of NMJs. Thus, Dok-7's C-terminal region plays a key, but not fully essential, role in MuSK activation and NMJ formation.