Direct Phosphorylation of SRC Homology 3 Domains by Tyrosine Kinase Receptors Disassembles Ligand-Induced Signaling Networks.

Phosphotyrosine (pTyr) signaling has evolved into a key cell-to-cell communication system. Activated receptor tyrosine kinases (RTKs) initiate several pTyr-dependent signaling networks by creating the docking sites required for the assembly of protein complexes. However, the mechanisms leading to network disassembly and its consequence on signal transduction remain essentially unknown. We show that activated RTKs terminate downstream signaling via the direct phosphorylation of an evolutionarily conserved Tyr present in most SRC homology (SH) 3 domains, which are often part of key hub proteins for RTK-dependent signaling. We demonstrate that the direct EPHA4 RTK phosphorylation of adaptor protein NCK SH3s at these sites results in the collapse of signaling networks and abrogates their function. We also reveal that this negative regulation mechanism is shared by other RTKs. Our findings uncover a conserved mechanism through which RTKs rapidly and reversibly terminate downstream signaling while remaining in a catalytically active state on the plasma membrane.

Exploring the short linear motif-mediated protein-protein interactions of CrkL through ProP-PD.

Adaptor proteins play a pivotal role in cellular signaling mediating a multitude of protein-protein interaction critical for cellular homeostasis. Dysregulation of these interactions has been linked to the onset of various cancer pathologies and exploited by viral pathogens during host cell takeover. CrkL is an adaptor protein composed of an N-terminal SH2 domain followed by two SH3 domains that mediate interactions with diverse partners through the recognition of specific binding motifs. In this study, we employed proteomic peptide-phage display (ProP-PD) to comprehensively explore the short linear motif (SLiM)-based interactions of CrkL. Furthermore, we scrutinized how the binding affinity for selected peptides was influenced in the context of the full-length CrkL versus the isolated N-SH3 domain. Importantly, our results provided insights into SLiM-binding sites within previously reported interactors, as well as revealing novel human and viral ligands, expanding our understanding of the interactions mediated by CrkL and highlighting the significance of SLiM-based interactions in mediating adaptor protein function, with implications for cancer and viral pathologies.

Three archetypical classes of macromolecular regulators of protein liquid-liquid phase separation.

Membraneless organelles, corresponding to the droplet phase upon liquid-liquid phase separation (LLPS) of protein or protein-RNA mixtures, mediate myriad cellular functions. Cells use a variety of biochemical signals such as expression level and posttranslational modification to regulate droplet formation and dissolution, but the physical basis of the regulatory mechanisms remains ill-defined and quantitative assessment of the effects is largely lacking. Our computational study predicted that the strength of attraction by droplet-forming proteins dictates whether and how macromolecular regulators promote or suppress LLPS. We experimentally tested this prediction, using the pentamers of SH3 domains and proline-rich motifs (SH35 and PRM5) as droplet-forming proteins. Determination of the changes in phase boundary and the partition coefficients in the droplet phase over a wide range of regulator concentrations yielded both a quantitative measure and a mechanistic understanding of the regulatory effects. Three archetypical classes of regulatory effects were observed. Ficoll 70 at high concentrations indirectly promoted SH35-PRM5 LLPS, by taking up volume in the bulk phase and thereby displacing SH35 and PRM5 into the droplet phase. Lysozyme had a moderate partition coefficient and suppressed LLPS by substituting weaker attraction with SH35 for the stronger SH35-PRM5 attraction in the droplet phase. By forming even stronger attraction with PRM5, heparin at low concentrations partitioned heavily into the droplet phase and promoted LLPS. These characteristics were recapitulated by computational results of patchy particle models, validating the identification of the 3 classes of macromolecular regulators as volume-exclusion promotors, weak-attraction suppressors, and strong-attraction promotors.

The Nebulin Family LIM and SH3 Proteins Regulate Postsynaptic Development and Function.

Neuronal dendrites have specialized actin-rich structures called dendritic spines that receive and integrate most excitatory synaptic inputs. The stabilization of dendrites and spines during neuronal maturation is essential for proper neural circuit formation. Changes in dendritic morphology and stability are largely mediated by regulation of the actin cytoskeleton; however, the underlying mechanisms remain to be fully elucidated. Here, we present evidence that the nebulin family members LASP1 and LASP2 play an important role in the postsynaptic development of rat hippocampal neurons from both sexes. We find that both LASP1 and LASP2 are enriched in dendritic spines, and their knockdown impairs spine development and synapse formation. Furthermore, LASP2 exerts a distinct role in dendritic arbor and dendritic spine stabilization. Importantly, the actin-binding N-terminal LIM domain and nebulin repeats of LASP2 are required for spine stability and dendritic arbor complexity. These findings identify LASP1 and LASP2 as novel regulators of neuronal circuitry.SIGNIFICANCE STATEMENT Proper regulation of the actin cytoskeleton is essential for the structural stability of dendrites and dendritic spines. Consequently, the malformation of dendritic structures accompanies numerous neurologic disorders, such as schizophrenia and autism. Nebulin family members are best known for their role in regulating the stabilization and function of actin thin filaments in muscle. The two smallest family members, LASP1 and LASP2, are more structurally diverse and are expressed in a broader array of tissues. While both LASP1 and LASP2 are highly expressed in the brain, little is currently known about their function in the nervous system. In this study, we demonstrate the first evidence that LASP1 and LASP2 are involved in the formation and long-term maintenance of dendrites and dendritic spines.

FID-Net: A versatile deep neural network architecture for NMR spectral reconstruction and virtual decoupling.

In recent years, the transformative potential of deep neural networks (DNNs) for analysing and interpreting NMR data has clearly been recognised. However, most applications of DNNs in NMR to date either struggle to outperform existing methodologies or are limited in scope to a narrow range of data that closely resemble the data that the network was trained on. These limitations have prevented a widescale uptake of DNNs in NMR. Addressing this, we introduce FID-Net, a deep neural network architecture inspired by WaveNet, for performing analyses on time domain NMR data. We first demonstrate the effectiveness of this architecture in reconstructing non-uniformly sampled (NUS) biomolecular NMR spectra. It is shown that a single network is able to reconstruct a diverse range of 2D NUS spectra that have been obtained with arbitrary sampling schedules, with a range of sweep widths, and a variety of other acquisition parameters. The performance of the trained FID-Net in this case exceeds or matches existing methods currently used for the reconstruction of NUS NMR spectra. Secondly, we present a network based on the FID-Net architecture that can efficiently virtually decouple 13Cα-13Cß couplings in HNCA protein NMR spectra in a single shot analysis, while at the same time leaving glycine residues unmodulated. The ability for these DNNs to work effectively in a wide range of scenarios, without retraining, paves the way for their widespread usage in analysing NMR data.

A helical lock and key model of polyproline II conformation with SH3.

MOTIVATION: More than half of the human proteome contains the proline-rich motif, PxxP. This motif has a high propensity for adopting a left-handed polyproline II (PPII) helix and can potentially bind SH3 domains. SH3 domains are generally grouped into two classes, based on whether the PPII binds in a positive (N-to-C terminal) or negative (C-to-N terminal) orientation. Since the discovery of this structural motif, over six decades ago, a systematic understanding of its binding remains poor and the consensus amino acid sequence that binds SH3 domains is still ill defined. RESULTS: Here, we show that the PPII interaction with SH3 domains is governed by the helix backbone and its prolines, and their rotation angle around the PPII helical axis. Based on a geometric analysis of 131 experimentally solved SH3 domains in complex with PPIIs, we observed a rotary translation along the helical screw axis, and separated them by 120° into three categories we name α (0-120°), ß (120-240°) and γ (240-360°). Furthermore, we found that PPII helices are distinguished by a shifting PxxP motif preceded by positively charged residues which act as a structural reading frame and dictates the organization of SH3 domains; however, there is no one single consensus motif for all classified PPIIs. Our results demonstrate a remarkable apparatus of a lock with a rotating and translating key with no known equivalent machinery in molecular biology. We anticipate our model to be a starting point for deciphering the PPII code, which can unlock an exponential growth in our understanding of the relationship between protein structure and function. AVAILABILITY AND IMPLEMENTATION: We have implemented the proposed methods in the R software environment and in an R package freely available at https://github.com/Grantlab/bio3d. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

A disordered encounter complex is central to the yeast Abp1p SH3 domain binding pathway.

Protein-protein interactions are involved in a wide range of cellular processes. These interactions often involve intrinsically disordered proteins (IDPs) and protein binding domains. However, the details of IDP binding pathways are hard to characterize using experimental approaches, which can rarely capture intermediate states present at low populations. SH3 domains are common protein interaction domains that typically bind proline-rich disordered segments and are involved in cell signaling, regulation, and assembly. We hypothesized, given the flexibility of SH3 binding peptides, that their binding pathways include multiple steps important for function. Molecular dynamics simulations were used to characterize the steps of binding between the yeast Abp1p SH3 domain (AbpSH3) and a proline-rich IDP, ArkA. Before binding, the N-terminal segment 1 of ArkA is pre-structured and adopts a polyproline II helix, while segment 2 of ArkA (C-terminal) adopts a 310 helix, but is far less structured than segment 1. As segment 2 interacts with AbpSH3, it becomes more structured, but retains flexibility even in the fully engaged state. Binding simulations reveal that ArkA enters a flexible encounter complex before forming the fully engaged bound complex. In the encounter complex, transient nonspecific hydrophobic and long-range electrostatic contacts form between ArkA and the binding surface of SH3. The encounter complex ensemble includes conformations with segment 1 in both the forward and reverse orientation, suggesting that segment 2 may play a role in stabilizing the correct binding orientation. While the encounter complex forms quickly, the slow step of binding is the transition from the disordered encounter ensemble to the fully engaged state. In this transition, ArkA makes specific contacts with AbpSH3 and buries more hydrophobic surface. Simulating the binding between ApbSH3 and ArkA provides insight into the role of encounter complex intermediates and nonnative hydrophobic interactions for other SH3 domains and IDPs in general.

Drebrin-like (Dbnl) Controls Neuronal Migration via Regulating N-Cadherin Expression in the Developing Cerebral Cortex.

The actin cytoskeleton is crucial for neuronal migration in the mammalian developing cerebral cortex. The adaptor protein Drebrin-like (Dbnl) plays important roles in reorganization of the actin cytoskeleton, dendrite formation, and endocytosis by interacting with F-actin, cobl, and dynamin. Although Dbnl is known to be expressed in the brain, the functions of this molecule during brain development are largely unknown. In this study, to examine the roles of Dbnl in the developing cerebral cortex, we conducted experiments using mice of both sexes with knockdown of Dbnl, effected by in utero electroporation, in the migrating neurons of the embryonic cortex. Time-lapse imaging of the Dbnl-knockdown neurons revealed that the presence of Dbnl is a prerequisite for appropriate formation of processes in the multipolar neurons in the multipolar cell accumulation zone or the deep part of the subventricular zone, and for neuronal polarization and entry into the cortical plate. We found that Dbnl knockdown decreased the amount of N-cadherin protein expressed on the plasma membrane of the cortical neurons. The defect in neuronal migration caused by Dbnl knockdown was rescued by moderate overexpression of N-cadherin and αN-catenin or by transfection of the phospho-mimic form (Y337E, Y347E), but not the phospho-resistant form (Y337F, Y347F), of Dbnl. These results suggest that Dbnl controls neuronal migration, neuronal multipolar morphology, and cell polarity in the developing cerebral cortex via regulating N-cadherin expression.SIGNIFICANCE STATEMENT Disruption of neuronal migration can cause neuronal disorders, such as lissencephaly and subcortical band heterotopia. During cerebral cortical development, the actin cytoskeleton plays a key role in neuronal migration; however, the mechanisms of regulation of neuronal migration by the actin cytoskeleton still remain unclear. Herein, we report that the novel protein Dbnl, an actin-binding protein, controls multiple events during neuronal migration in the developing mouse cerebral cortex. We also showed that this regulation is mediated by phosphorylation of Dbnl at tyrosine residues 337 and 347 and αN-catenin/N-cadherin, suggesting that the Dbnl-αN-catenin/N-cadherin pathway is important for neuronal migration in the developing cortex.

MC159 of Molluscum Contagiosum Virus Suppresses Autophagy by Recruiting Cellular SH3BP4 via an SH3 Domain-Mediated Interaction.

MC159 is a viral FLIP (FLICE inhibitory protein) encoded by the molluscum contagiosum virus (MCV) enabling MCV to evade antiviral immunity and to establish persistent infections in humans. Here, we show that MC159 contains a functional SH3 binding motif, which mediates avid and selective binding to SH3BP4, a signaling protein known to regulate endocytic trafficking and suppress cellular autophagy. The capacity to bind SH3BP4 was dispensable for regulation of NF-κB-mediated transcription and suppression of proapoptotic caspase activation but contributed to inhibition of amino acid starvation-induced autophagy by MC159. These results provide new insights into the cellular functions of MC159 and reveal SH3BP4 as a novel host cell factor targeted by a viral immune evasion protein.IMPORTANCE After the eradication of smallpox, molluscum contagiosum virus (MCV) is the only poxvirus restricted to infecting humans. MCV infection is common and causes benign skin lesions that usually resolve spontaneously but may persist for years and grow large, especially in immunocompromised individuals. While not life threatening, MCV infections pose a significant global health burden. No vaccine or specific anti-MCV therapy is available. MCV encodes several proteins that enable it to evade antiviral immunity, a notable example of which is the MC159 protein. In this study, we describe a novel mechanism of action for MC159 involving hijacking of a host cell protein called SH3BP4 to suppress autophagy, a cellular recycling mechanism important for antiviral immunity. This study contributes to our understanding of the host cell interactions of MCV and the molecular function of MC159.

The Ca2+ channel subunit CaV ß2a-subunit down-regulates voltage-activated ion current densities by disrupting actin-dependent traffic in chromaffin cells.

ß-Subunits of the Ca2+ channel have been conventionally regarded as auxiliary subunits that regulate the expression and activity of the pore-forming α1 subunit. However, they comprise protein-protein interaction domains, such as a SRC homology 3 domain (SH3) domain, which make them potential signaling molecules. Here we evaluated the role of the ß2a subunit of the Ca2+ channels (CaV ß2a) and its SH3 domain (ß2a-SH3) in late stages of channel trafficking in bovine adrenal chromaffin cells. Cultured bovine adrenal chromaffin cells were injected with CaV ß2a or ß2a-SH3 under different conditions, in order to acutely interfere with endogenous associations of these proteins. As assayed by whole-cell patch clamp recordings, Ca2+ currents were reduced by CaV ß2a in the presence of exogenous α1-interaction domain. ß2a-SH3, but not its dimerization-deficient mutant, also reduced Ca2+ currents. Na+ currents were also diminished following ß2a-SH3 injection. Furthermore, ß2a-SH3 was still able to reduce Ca2+ currents when dynamin-2 function was disrupted, but not when SNARE-dependent exocytosis or actin polymerization was inhibited. Together with the additional finding that both CaV ß2a and ß2a-SH3 diminished the incorporation of new actin monomers to cortical actin filaments, ß2a-SH3 emerges as a signaling module that might down-regulate forward trafficking of ion channels by modulating actin dynamics.

Identification of a new type of haematopoietic progenitor kinase-interacting protein (HIP-55) in Aedes aegypti mosquito haemocytes and its involvement in immunity-like functions in mosquito: a molecular study.

In this study, we characterize the HIP-55 protein in the mosquito Aedes aegypti for the first time. HIP-55 is a 55-kDa HPK1-interacting protein that is also called SH3P7. HIP-55 constitutively binds HPK1 'via' an HPK1 proline-rich motif 2(PR2) through its C-terminal SH3 domain. HIP-55 critically interacts with ZAP-70, and this interaction was induced by TCR signalling. ZAP-70 phosphorylated HIP-55 at Tyr-334 and Tyr-344 in vitro and in vivo. In our previous findings, AaZAP gene expression strongly proved that AaZAP-70 was involved in immunity-like functions in mosquito. Northern blot analysis of HIP-55 mRNA expression confirmed that it is only expressed in the abdomen and haemocyte tissues; this prediction correlates 100% and a polyclonal antibody also confirmed its localization in haemocytes and the abdomen. We prepared extracts to show the cytoplasmic expression (CE) of this protein. Previous results had proven that this protein is secreted from the cytoplasm; thus, we confirmed here that the protein is a cytoplasmic adaptor protein in mosquitoes and mammalian systems. Furthermore, our polyclonal antibody against HIP-55 also demonstrated that this protein is found in haemocytes and abdomen tissues, which assumes that the protein may be involved in phagocytic-like functions. RNAi (siRNA) silencing studies were used to degrade mosquito HIP-55; however, silencing only slightly affected the HIP-55 sequence and the gene transcriptional level. To characterize this protein, we cloned 609 bp from the 1.6-kb full-length cDNA using a pET28 vector for polyclonal antibody production. Graphical abstract.

Crystal Structures and Thermodynamic Analysis Reveal Distinct Mechanisms of CD28 Phosphopeptide Binding to the Src Homology 2 (SH2) Domains of Three Adaptor Proteins.

Full activation of T cells and differentiation into effector T cells are essential for many immune responses and require co-stimulatory signaling via the CD28 receptor. Extracellular ligand binding to CD28 recruits protein-tyrosine kinases to its cytoplasmic tail, which contains a YMNM motif. Following phosphorylation of the tyrosine, the proteins growth factor receptor-bound protein 2 (Grb2), Grb2-related adaptor downstream of Shc (Gads), and p85 subunit of phosphoinositide 3-kinase may bind to pYMNM (where pY is phosphotyrosine) via their Src homology 2 (SH2) domains, leading to downstream signaling to distinct immune pathways. These three adaptor proteins bind to the same site on CD28 with variable affinity, and all are important for CD28-mediated co-stimulatory function. However, the mechanism of how these proteins recognize and compete for CD28 is unclear. To visualize their interactions with CD28, we have determined the crystal structures of Gads SH2 and two p85 SH2 domains in complex with a CD28-derived phosphopeptide. The high resolution structures obtained revealed that, whereas the CD28 phosphopeptide bound to Gads SH2 is in a bent conformation similar to that when bound to Grb2 SH2, it adopts a more extended conformation when bound to the N- and C-terminal SH2 domains of p85. These differences observed in the peptide-protein interactions correlated well with the affinity and other thermodynamic parameters for each interaction determined by isothermal titration calorimetry. The detailed insight into these interactions reported here may inform the development of compounds that specifically inhibit the association of CD28 with these adaptor proteins to suppress excessive T cell responses, such as in allergies and autoimmune diseases.

Transforming growth factor-ß1 impairs lymph node homing of dendritic cells by downregulating C-type lectin receptor-2 expression.

Trafficking of dendritic cells (DCs) from peripheral tissues to draining lymph nodes is a prerequisite for induction of adaptive immunity. An immunosuppressive cytokine transforming growth factor (TGF)-ß1, however, inhibits migration of DCs by downregulating the expression of chemokine receptor CCR-7. Whether TGF-ß1 engages any other receptor to mediate this inhibitory effect is currently unknown. In this article, we report that TGF-ß1 attenuated the lymph node homing ability of mouse DCs by reducing C-type lectin receptor-2 (CLEC-2) expression. Notably, TGF-ß1 inhibited CLEC-2 expression in DCs via c-Src. DCs silenced for c-Src were resistant to TGF-ß1-induced inhibition of CLEC-2 expression. Furthermore, silencing of c-Src substantially improved the lymph node homing capacity of TGF-ß1-treated DCs by restoring CLEC-2 expression. These results document a critical role for c-Src and CLEC-2 in TGF-ß1-mediated impairment of DC migration and define a previously unknown mechanism by which TGF-ß1 attenuates the lymph node homing ability of DCs.

Graf1 Controls the Growth of Human Parainfluenza Virus Type 2 through Inactivation of RhoA Signaling.

UNLABELLED: Rho GTPases are involved in a variety of cellular activities and are regulated by guanine nucleotide exchange factors and GTPase-activating proteins (GAPs). We found that the activation of Rho GTPases by lysophosphatidic acid promotes the growth of human parainfluenza virus type 2 (hPIV-2). Furthermore, hPIV-2 infection causes activation of RhoA, a Rho GTPase. We hypothesized that Graf1 (also known as ARHGAP26), a GAP, regulates hPIV-2 growth by controlling RhoA signaling. Immunofluorescence analysis showed that hPIV-2 infection altered Graf1 localization from a homogenous distribution within the cytoplasm to granules. Graf1 colocalized with hPIV-2 P, NP, and L proteins. Graf1 interacts with P and V proteins via their N-terminal common region, and the C-terminal Src homology 3 domain-containing region of Graf1 is important for these interactions. In HEK293 cells constitutively expressing Graf1, hPIV-2 growth was inhibited, and RhoA activation was not observed during hPIV-2 infection. In contrast, Graf1 knockdown restored hPIV-2 growth and RhoA activation. Overexpression of hPIV-2 P and V proteins enhanced hPIV-2-induced RhoA activation. These results collectively suggested that hPIV-2 P and V proteins enhanced hPIV-2 growth by binding to Graf1 and that Graf1 inhibits hPIV-2 growth through RhoA inactivation. IMPORTANCE: Robust growth of hPIV-2 requires Rho activation. hPIV-2 infection causes RhoA activation, which is suppressed by Graf1. Graf1 colocalizes with viral RNP (vRNP) in hPIV-2-infected cells. We found that Graf1 interacts with hPIV-2 P and V proteins. We also identified regions in these proteins which are important for this interaction. hPIV-2 P and V proteins enhanced the hPIV-2 growth via binding to Graf1, while Graf1 inhibited hPIV-2 growth through RhoA inactivation.

Non-canonical dynamic mechanisms of interaction between the p66Shc protein and Met receptor.

Met receptor tyrosine kinase (RTK) is known to bind to the three distinct protein isoforms encoded by the ShcA (Shc) gene. Structure-function studies have unveiled critical roles for p52Shc-dependent signalling pathways in Met-regulated biological functions. The molecular basis of the interaction between the Met and p52Shc proteins is well-defined, but not for the longest protein isoform, p66Shc. In the present study, co-immunoprecipitation assays were performed in human embryonic kidney 293 (HEK293) cells, transiently co-transfected with Met and p66Shc mutants, in order to define the molecular determinants involved in mediating Met-p66Shc interaction. Our results show that p66Shc interacts constitutively with the receptor Met, and the Grb2 (growth factor receptor-bound protein-2) and Gab1 (Grb2-associated binder-1) adaptor proteins. Although its phosphotyrosine-binding domain (PTB) and Src homology 2 (SH2) domains co-ordinate p66Shc binding to non-activated Met receptor, these phosphotyrosine-binding modules, and its collagen homology domain 2 (CH2) region, exert negative constraints. In contrast, p66Shc interaction with the activated Met depends mainly on the integrity of its PTB domain, and to a lesser extent of its SH2 domain. Even though not required for the recruitment of p66Shc, tyrosine phosphorylation of p66Shc by activated Met enhances these interactions by mechanisms not reliant on the integrity of the Met multisubstrate-binding site. In turn, this increases phosphotyrosine-dependent p66Shc-Grb2-Gab1 complex formation away from the receptor, while blocking Grb2 and Gab1 recruitment to activated Met. In conclusion, we identify, for the first time, a novel non-canonical dynamic mode of interaction between Met and the p66 protein isoform of Shc and its effects on rewiring binding effector complexes according to the activation state of the receptor.

Prolyl isomerization as a molecular memory in the allosteric regulation of the signal adapter protein c-CrkII.

c-CrkII is a central signal adapter protein. A domain opening/closing reaction between its N- and C-terminal Src homology 3 domains (SH3N and SH3C, respectively) controls signal propagation from upstream tyrosine kinases to downstream targets. In chicken but not in human c-CrkII, opening/closing is coupled with cis/trans isomerization at Pro-238 in SH3C. Here, we used advanced double-mixing experiments and kinetic simulations to uncover dynamic domain interactions in c-CrkII and to elucidate how they are linked with cis/trans isomerization and how this regulates substrate binding to SH3N. Pro-238 trans → cis isomerization is not a simple on/off switch but converts chicken c-CrkII from a high affinity to a low affinity form. We present a double-box model that describes c-CrkII as an allosteric system consisting of an open, high affinity R state and a closed, low affinity T state. Coupling of the T-R transition with an intrinsically slow prolyl isomerization provides c-CrkII with a kinetic memory and possibly functions as a molecular attenuator during signal transduction.

The SH3 domain distinguishes the role of I-BAR proteins IRTKS and MIM in chemotactic response to serum.

The family of inverse BAR (I-BAR) domain proteins participates in a range of cellular processes associated with membrane dynamics and consists of five distinct members. Three of the I-BAR proteins, including insulin receptor tyrosine kinase substrate (IRTKS), contain an SH3 domain near their C-termini. Yet, the function of the SH3 domain of IRTKS remains uncharacterized. Here we report that in contrast to MIM, which is a prototype of I-BAR proteins and does not contain an SH3 domain, IRTKS promoted serum-induced cell migration along with enhanced phosphorylation of mitogen activated kinases Erk1/2 and p38, and activation of small GTPases Rac1 and Cdc42. In addition, cells overexpressing IRTKS exhibited an increased polarity characterized by elongated cytoplasm and extensive lamellipodia at leading edges. However, a mutant with deletion of the SH3 domain attenuated both cellular motility and p38 phosphorylation but had little effect on Erk1/2 phosphorylation. Also, a chimeric mutant in which the N-terminal portion of MIM is fused with the C-terminal IRTKS, including the SH3 domain, was able to promote chemotactic response to serum and cellular polarity. In contrast, a chimeric mutant in which the N-terminal IRTKS is fused with the C-terminal MIM failed to do so. Furthermore, treatment of cells with SB203580, a selective inhibitor of p38, also neutralized the effect of IRTKS on cell migration. These data indicate that the SH3 domain distinguishes the function of IRTKS in promoting cell migration and inducing signal transduction from those of SH3-less I-BAR proteins.

Creation of an apoptin-derived peptide that interacts with SH3 domains and inhibits glioma cell migration and invasion.

Glioblastoma multiforme (GBM) is an aggressive tumor of the central nervous system characterized by high rates of recurrence, morbidity, and mortality. This study investigated the antitumor effects of an apoptin-derived peptide (ADP) on glioma cells and explored the underlying mechanisms. The U251, U87, and C6 glioma cell lines were used in the present study, and the expression of p-Akt, Akt, and MMP-9 was determined through Western blotting, quantitative real-time PCR, and hematoxylin and eosin (HE) staining. Tumor growth was evaluated by magnetic resonance imaging, and cell viability was assessed through an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide MTT assay. Glioma cell metastasis was evaluated using transwell migration, invasion, and scratch-wound assays. An ADP was designed and synthesized based on the results of a domain-based analysis of the structure of apoptin. The ADP inhibited glioma cell viability, invasion and migration, and treatment with the synthesized ADP led to downregulation of p-Akt and MMP-9 and inhibited MMP-9 translation. The ADP also inhibited glioma invasion and migration in vivo, and HE staining showed decreases in the satellite-like invasion of cell masses and apoptotic cell populations after treatment with the ADP. Our findings demonstrate that treatment with an ADP can suppress glioma cell migration and invasion via the PI3K/Akt/MMP-9 signaling pathway and provide a new platform for the development of drugs for treating glioma.

The accessory factor Nef links HIV-1 to Tec/Btk kinases in an Src homology 3 domain-dependent manner.

The HIV-1 Nef virulence factor interacts with multiple host cell-signaling proteins. Nef binds to the Src homology 3 domains of Src family kinases, resulting in kinase activation important for viral infectivity, replication, and MHC-I down-regulation. Itk and other Tec family kinases are also present in HIV target cells, and Itk has been linked to HIV-1 infectivity and replication. However, the molecular mechanism linking Itk to HIV-1 is unknown. In this study, we explored the interaction of Nef with Tec family kinases using a cell-based bimolecular fluorescence complementation assay. In this approach, interaction of Nef with a partner kinase juxtaposes nonfluorescent YFP fragments fused to the C terminus of each protein, resulting in YFP complementation and a bright fluorescent signal. Using bimolecular fluorescence complementation, we observed that Nef interacts with the Tec family members Bmx, Btk, and Itk but not Tec or Txk. Interaction with Nef occurs through the kinase Src homology 3 domains and localizes to the plasma membrane. Allelic variants of Nef from all major HIV-1 subtypes interacted strongly with Itk in this assay, demonstrating the highly conserved nature of this interaction. A selective small molecule inhibitor of Itk kinase activity (BMS-509744) potently blocked wild-type HIV-1 infectivity and replication, but not that of a Nef-defective mutant. Nef induced constitutive Itk activation in transfected cells that was sensitive to inhibitor treatment. Taken together, these results provide the first evidence that Nef interacts with cytoplasmic tyrosine kinases of the Tec family and suggest that Nef provides a mechanistic link between HIV-1 and Itk signaling in the viral life cycle.