N-terminal Proline-rich Region Research Articles

Isoforms of the Cytoskeletal LIM-Domain Protein Zyxin in the Early Embryogenesis of Xenopus laevis

Objective: The study of highly conserved mechanosensitive proteins, such as zyxin, is essential due to their role in shaping embryos of all animals during embryogenesis through coordinated morphogenetic processes and controlled cell differentiation. This study aims to identify endogenous zyxin isoforms in Xenopus laevis and investigate changes in their abundance and intracellular localization during embryogenesis. Methods: Endogenous proteins were primarily detected using specific antibodies. Polyclonal antibodies targeting the C-terminal region of zyxin containing the NES and three LIM domains (438–663 aa), as well as antibodies against the N-terminal proline-rich region of Zyxin (1–373 aa) crucial for interactions with actinin and cytoskeletal proteins, were employed. Western blotting with these antibodies was conducted on Xenopus laevis embryo cell samples after fractionation into nuclear and cytoplasmic fractions. Results and Discussion: The study revealed multiple isoforms of zyxin in Xenopus laevis, including a full-length modified protein (105 kDa), an unmodified form (70 kDa), and two truncated forms of 45 and 37 kDa. The number and subcellular distribution of the truncated forms were found to vary based on the developmental stage, with increased levels of the 45 and 37 kDa isoforms observed in the early stages. Conclusions: This work provides novel insights into changes in the abundance and localization of zyxin isoforms during embryonic development, shedding light on the dynamics of this mechanosensitive protein in the embryo.

Biophysical characterization of the DNA binding motif of human phospholipid scramblase 1.

Human phospholipid scramblase 1 (hPLSCR1) is a 37kDa multi-compartmental protein, which was initially identified as a Ca2+-dependent phospholipid translocator upon localizing to the plasma membrane. However, under certain physiological conditions, hPLSCR1 is localized to the nucleus where it interacts with the IP3R1 promoter (IP3R1P) and regulates its expression. In this study, the DNA binding properties of hPLSCR1 and ∆100-hPLSCR1 (N-terminal 100 amino acids deleted from hPLSCR1) were investigated by using a synthetic IP3R1P oligonucleotide and nonspecific scrambled-sequence oligonucleotides. Our results revealed that hPLSCR1 and ∆100-hPLSCR1 were bound to IP3R1P oligos in a 1:1 stoichiometry. In addition, ∆160-hPLSCR1 could not bind to IP3R1P oligonucleotide suggesting that the proposed DNA binding motif is the actual DNA binding motif. Specific binding of hPLSCR1 and ∆100-hPLSCR1 to IP3R1P oligos was demonstrated by fluorescence anisotropy assay. ITC analysis revealed that hPLSCR1 binds to IP3R1P oligos with Kd = 42.91 ± 0.23nM. Binding of IP3R1P oligos induces β-sheet formation in hPLSCR1 and increases the thermal stability of hPLSCR1 and ∆100-hPLSCR1. Binding of IP3R1P oligos to hPLSCR1 altered the B-form of the DNA, which was not observed with ∆100-hPLSCR1. Results from this study suggest that (i) ∆100-hPLSCR1 possesses a minimal DNA binding region and (ii) structural alterations of IP3R1P oligo by hPLSCR1 require proline-rich N-terminal region.

Impact of a conserved N-terminal proline-rich region of the α-subunit of CAAX-prenyltransferases on their enzyme properties

BackgroundThe CAAX-prenyltransferases farnesyltransferase (FTase) and geranylgeranyltransferase I (GGTase I) are heterodimers with a common α- (FTα) and unique β-subunits. Recently, α-subunits of species (e.g., human) that harbour an N-terminal proline-rich region (PRR) showed different dimerization behaviours than α-subunits without PRR (e.g., yeast). However, the specific function of the PRR has not been elucidated so far.MethodsTo determine whether the PRR is a conserved motif throughout eukaryotes, we performed phylogenetics. Elucidating the impact of the PRR on enzyme properties, we cloned human as well as rat PRR deficient FTα, expressed them heterologously and compared protein–protein interaction by pull-down as well as crosslinking experiments. Substrate binding, enzyme activity and sensitivity towards common FTase inhibitors of full length and PRR-deletion α-subunits and their physiological partners was determined by continuous fluorescence assays.ResultsThe PRR is highly conserved in mammals, with an exception for marsupials harbouring a poly-alanine region instead. The PRR shows similarities to canonical SH3-binding domains and to profilin-binding domains. Independent of the PRR, the α-subunits were able to dimerize with the different physiological β-subunits in in vitro as well as in yeast two-hybrid experiments. FTase and GGTase I with truncated FTα were active. The KM values for both substrates are in the single-digit µM range and show no significant differences between enzymes with full length and PRR deficient α-subunits within the species.ConclusionsOur data demonstrate that an N-terminal PRR of FTα is highly conserved in mammals. We could show that the activity and inhibitability is not influenced by the truncation of the N-terminal region. Nevertheless, this region shows common binding motifs for other proteins involved in cell-signalling, trafficking and phosphorylation, suggesting that this PRR might have other or additional functions in mammals. Our results provide new starting points due to the relevant but only partly understood role of FTα in eukaryotic FTase and GGTase I.83PbrUwLcXbuUPceRJ4UzrVideo

Artificial ER-Derived Vesicles as Synthetic Organelles for in Vivo Compartmentalization of Biochemical Pathways.

Compartmentalization in membrane-surrounded organelles has the potential to overcome obstacles associated with the engineering of metabolic pathways, such as unwanted side reactions, accumulation of toxic intermediates, drain of intermediates out of the cell, and long diffusion distances. Strategies utilizing natural organelles suffer from the presence of endogenous pathways. In our approach, we make use of endoplasmic reticulum-derived vesicles loaded with enzymes of a metabolic pathway ("metabolic vesicles"). They are generated by fusion of synthetic peptides containing the N-terminal proline-rich and self-assembling region of the maize storage protein gamma-Zein ("Zera") to the pathway enzymes. We have applied a strategy to integrate three enzymes of a cis,cis-muconic acid production pathway into those vesicles in yeast. Using fluorescence microscopy and cell fractionation techniques, we have proven the formation of metabolic vesicles and the incorporation of enzymes. Activities of the enzymes and functionality of the compartmentalized pathway were demonstrated in fermentation experiments.

Independent tubulin binding and polymerization by the proline-rich region of Tau is regulated by Tau's N-terminal domain

Tau is an intrinsically disordered, microtubule-associated protein that has a role in regulating microtubule dynamics. Despite intensive research, the molecular mechanisms of Tau-mediated microtubule polymerization are poorly understood. Here we used single-molecule fluorescence to investigate the role of Tau's N-terminal domain (NTD) and proline-rich region (PRR) in regulating interactions of Tau with soluble tubulin. We assayed both full-length Tau isoforms and truncated variants for their ability to bind soluble tubulin and stimulate microtubule polymerization. We found that Tau's PRR is an independent tubulin-binding domain that has tubulin polymerization capacity. In contrast to the relatively weak interactions with tubulin mediated by sites distributed throughout Tau's microtubule-binding region (MTBR), resulting in heterogeneous Tau: tubulin complexes, the PRR bound tubulin tightly and stoichiometrically. Moreover, we demonstrate that interactions between the PRR and MTBR are reduced by the NTD through a conserved conformational ensemble. On the basis of these results, we propose that Tau's PRR can serve as a core tubulin-binding domain, whereas the MTBR enhances polymerization capacity by increasing the local tubulin concentration. Moreover, the NTD appears to negatively regulate tubulin-binding interactions of both of these domains. The findings of our study draw attention to a central role of the PRR in Tau function and provide mechanistic insight into Tau-mediated polymerization of tubulin.

Litopenaeus vannamei stylicins are constitutively produced by hemocytes and intestinal cells and are differentially modulated upon infections

Stylicins are anionic antimicrobial host defense peptides (AAMPs) composed of a proline-rich N-terminal region and a C-terminal portion containing 13 conserved cysteine residues. Here, we have increased our knowledge about these unexplored crustacean AAMPs by the characterization of novel stylicin members in the most cultivated penaeid shrimp, Litopenaeus vannamei. We showed that the L. vannamei stylicin family is composed of two members (Lvan-Stylicin1 and Lvan-Stylicin2) encoded by different loci which vary in gene copy number. Unlike the other three gene-encoded antimicrobial peptide families from penaeid shrimp, the expression of Lvan-Stylicins is not restricted to hemocytes. Indeed, they are also produced by the columnar epithelial cells lining the midgut and its anterior caecum. Interestingly, Lvan-Stylicins are simultaneously transcribed at different transcriptional levels in a single shrimp and are differentially modulated in hemocytes after infections. While the expression of both genes showed to be responsive to damage-associated molecular patterns, only Lvan-Stylicin2 was induced after a Vibrio infection. Besides, Lvan-Stylicins also showed a distinct pattern of gene expression in the three portions of the midgut (anterior, middle and posterior) and during shrimp development. We provide here the first evidence of the diversity of the stylicin antimicrobial peptide family in terms of sequence and gene expression distribution and regulation.

Structural Basis of TRPV4 N Terminus Interaction with Syndapin/PACSIN1-3 and PIP2

Transient receptor potential (TRP) channels are polymodally regulated ion channels. TRPV4 (vanilloid 4) is sensitized by PIP2 and desensitized by Syndapin3/PACSIN3, which bind to the structurally uncharacterized TRPV4N terminus. We determined the nuclear magnetic resonance structure of the Syndapin3/PACSIN3 SH3 domain in complex with the TRPV4 N-terminal proline-rich region (PRR), which binds as a class I polyproline II (PPII) helix. This PPII conformation is broken by a conserved proline in a cis conformation. Beyond the PPII, we find that the proximal TRPV4N terminus is unstructured, a feature conserved across species thus explaining the difficulties in resolving it in previous structural studies. Syndapin/PACSIN SH3 domain binding leads to rigidification of both the PRR and the adjacent PIP2 binding site. We determined the affinities of the TRPV4N terminus for PACSIN1, 2, and 3 SH3 domains and PIP2 and deduce a hierarchical interaction network where Syndapin/PACSIN binding influences the PIP2 binding site but not vice versa.

Mapping and Engineering Functional Domains of the Assembly-Activating Protein of Adeno-associated Viruses.

Adeno-associated viruses (AAVs) encode a unique assembly-activating protein (AAP) within their genomes that is essential for capsid assembly. Studies to date have focused on establishing the role of AAP as a chaperone that mediates the stability, nucleolar transport, and assembly of AAV capsid proteins. Here, we map structure-function correlates of AAP using secondary structure analysis, followed by deletion and substitutional mutagenesis of specific domains, namely, the N-terminal hydrophobic region (HR), conserved core (CC), proline-rich region (PRR), threonine/serine-rich region (T/S), and basic region (BR). First, we establish that the centrally located PRR and T/S are flexible linker domains that can either be deleted completely or replaced by heterologous functional domains that enable ancillary functions such as fluorescent imaging or increased AAP stability. We also demonstrate that the C-terminal BR domains can be substituted with heterologous nuclear or nucleolar localization sequences that display various abilities to support AAV capsid assembly. Further, by replacing the BR domain with immunoglobulin (IgG) Fc domains, we assessed AAP complexation with AAV capsid subunits and demonstrate that the hydrophobic region (HR) and the conserved core (CC) in the AAP N terminus are the sole determinants for viral protein (VP) recognition. However, VP recognition alone is not sufficient for capsid assembly. Our study sheds light on the modular structure-function correlates of AAP and provides multiple approaches to engineer AAP that might prove useful toward understanding and controlling AAV capsid assembly.IMPORTANCE Adeno-associated viruses (AAVs) encode a unique assembly-activating protein (AAP) within their genomes that is essential for capsid assembly. Understanding how AAP acts as a chaperone for viral assembly could help improve efficiency and potentially control this process. Our studies reveal that AAP has a modular architecture, with each module playing a distinct role and can be engineered for carrying out new functions.

Identification and characterization of two arasin-like peptides in red swamp crayfish Procambarus clarkii

Antimicrobial peptides (AMPs) are small effectors in host defense by directly targeting microorganisms or by indirectly modulating immune responses. In the present study, two arasin like AMPs, named as Pc-arasin1 and Pc-arasin2, were identified in red swamp crayfish Procambarus clarkii with sequence similarity to the arasins found in Hyas araneus. Both Pc-arasins consisted of signal peptide, N-terminal proline-rich region and C-terminal region containing four conserved cysteine residues. The similarity of two Pc-arasins was 44.44%, and Pc-arasin2 contained several additional residues in the N-terminus. Multiple alignment of arasin family suggested the conservation of the C-terminus and the variation of the N-terminus of Pc-arasins. Both AMPs were found hemocytes-specific, and the expression could be induced the challenge of bacteria, espeacially by the pathogenic bacterium Aeromonas hydrophila. Knockdown of each Pc-arasin expression by double strand RNA would suppress the host immunity against A. hydrophila, and the commercially synthetic Pc-arasins could rescue the knockdown consequence. Both synthetic peptide showed broad antimicrobial activity towards 3 Gram-positive bacterium and 3 Gram-negative bacterium, and the minimal inhibitory concentrations varied from 6.25 μM to 50 μM. These results presented new data about the sequence, expression and function of arasin family, and emphasized the role of this family in host immune response against bacterial pathogens. The characterization of Pc-arasins also provided potential of therapeutic agent development for disease control in aquaculture based on these two newly identified AMPs.

Interacting protein partners of Arabidopsis RNA-binding protein AtRBP45b.

RNA binding proteins, important players in post-transcriptional gene regulation, usually exist in ribonuclear complexes. However, even in model systems like Arabidopsis characterisation of RBP associated proteins is limited. In this study, we investigated the interacting proteins of the Arabidopsis AtRBP45b, which is involved in stress signalling. In vivo localisation of AtRBP45b was conducted using 35S-GFP. FLAG-tagged AtRBP45b under control of the 35S promoter in the Atrbp45b-1 mutant background was used to pull down AtRBP45b interacting proteins. Yeast two-hybrid analysis, fluorescence energy resonance transfer assays were used to confirm the veracity of the AtRBP45b interacting proteins. In planta GFP-tagging indicated AtRBP45b is localised to the nucleus and the cytosol.AtRBP45b protein has a N-terminal proline-rich region and a C-terminal glutamine-rich domain that are usually involved in protein-protein interactions. Co-immunoprecipitation followed by mass spectrometry-based protein sequencing led to identification of 30 proteins that interacted with AtRBP45b. Using information from interactome databases (BIOGRID, INTACT and STRING), pull-down assays and localisation data, 12 putative interacting proteins were selected for yeast two-hybrid analysis. Cap-binding protein (CBP20, At5g44200) and polyA-binding protein (PAB8, At1g49760) were shown to interact with AtRBP45b. Based on its interacting partners we speculate that AtRBP45b may play an important role in RNA metabolism, especially in aspects related to mRNA stability and translation initiation during stress conditions in plants.

The topology, structure and PE interaction of LITAF underpin a Charcot-Marie-Tooth disease type 1C.

BackgroundMutations in Lipopolysaccharide-induced tumour necrosis factor-α factor (LITAF) cause the autosomal dominant inherited peripheral neuropathy, Charcot-Marie-Tooth disease type 1C (CMT1C). LITAF encodes a 17 kDa protein containing an N-terminal proline-rich region followed by an evolutionarily-conserved C-terminal ‘LITAF domain’, which contains all reported CMT1C-associated pathogenic mutations.ResultsHere, we report the first structural characterisation of LITAF using biochemical, cell biological, biophysical and NMR spectroscopic approaches. Our structural model demonstrates that LITAF is a monotopic zinc-binding membrane protein that embeds into intracellular membranes via a predicted hydrophobic, in-plane, helical anchor located within the LITAF domain. We show that specific residues within the LITAF domain interact with phosphoethanolamine (PE) head groups, and that the introduction of the V144M CMT1C-associated pathogenic mutation leads to protein aggregation in the presence of PE.ConclusionsIn addition to the structural characterisation of LITAF, these data lead us to propose that an aberrant LITAF-PE interaction on the surface of intracellular membranes contributes to the molecular pathogenesis that underlies this currently incurable disease.Electronic supplementary materialThe online version of this article (doi:10.1186/s12915-016-0332-8) contains supplementary material, which is available to authorized users.

Identification and expression analysis of a novel stylicin antimicrobial peptide from Kuruma shrimp (Marsupenaeus japonicus).

Antimicrobial peptides (AMPs) are important components of the innate immune system and function as the first line of defense against invading pathogens. In current study we identified, cloned and characterized a novel stylicin AMP from Kuruma shrimp Marsupenaeus japonicus (Mj-sty). The full-length cDNA of Mj-sty was 428 bp with an open reading frame of 315 bp that encoded 104 amino acids. The theoretical molecular mass of mature Mj-sty was 8.693 kDa with an isoelectric point (pI) of 4.79. A proline-rich N-terminal region and a C-terminal region contained 13 cysteine residues were identified. Genomic sequence analysis with respect to its cDNA showed that Mj-sty was organized into two exons interrupted by one intron. Tissue-specific expression revealed that Mj-sty was mainly transcribed in gills and hemocytes. Expression of Mj-sty in early developmental stages demonstrated that Mj-sty mRNA were present from fertilized eggs to post-larvae of 17 days (PL17), and the expression levels showed a significant variation in different developmental stages. After challenge of white spot syndrome virus (WSSV), the time-dependent expression pattern of Mj-sty in both gills and hepatopancrease showed down-regulation at the early hours of infection, subsequently up-regulation and down-regulation, and then up-regulation at the end hours to almost the half of the controls. The results indicate that Mj-sty is potentially involved in the ontogenesis and immune responses against WSSV.

The Regulatory T Cell Lineage Factor Foxp3 Regulates Gene Expression through Several Distinct Mechanisms Mostly Independent of Direct DNA Binding.

The lineage factor Foxp3 is essential for the development and maintenance of regulatory T cells, but little is known about the mechanisms involved. Here, we demonstrate that an N-terminal proline-rich interaction region is crucial for Foxp3’s function. Subdomains within this key region link Foxp3 to several independent mechanisms of transcriptional regulation. Our study suggests that Foxp3, even in the absence of its DNA-binding forkhead domain, acts as a bridge between DNA-binding interaction partners and proteins with effector function permitting it to regulate a large number of genes. We show that, in one such mechanism, Foxp3 recruits class I histone deacetylases to the promoters of target genes, counteracting activation-induced histone acetylation and thereby suppressing their expression.

Functional Significance of Cytochrome P450 1A2 Allelic Variants, P450 1A2*8, *15, and *16 (R456H, P42R, and R377Q).

P450 1A2 is responsible for the metabolism of clinically important drugs and the metabolic activation of environmental chemicals. Genetic variations of P450 1A2 can influence its ability to perform these functions, and thus, this study aimed to characterize the functional significance of three P450 1A2 allelic variants containing nonsynonymous single nucleotide polymorphisms (P450 1A2*8, R456H; *15, P42R; *16, R377Q). Variants containing these SNPs were constructed and the recombinant enzymes were expressed and purified in Escherichia coli. Only the P42R variant displayed the typical CO-binding spectrum indicating a P450 holoenzyme with an expression level of ∼ 170 nmol per liter culture, but no P450 spectra were observed for the two other variants. Western blot analysis revealed that the level of expression for the P42R variant was lower than that of the wild type, however the expression of variants R456H and R377Q was not detected. Enzyme kinetic analyses indicated that the P42R mutation in P450 1A2 resulted in significant changes in catalytic activities. The P42R variant displayed an increased catalytic turnover numbers (kcat) in both of methoxyresorufin O-demethylation and phenacetin O-deethylation. In the case of phenacetin O-deethylation analysis, the overall catalytic efficiency (kcat/Km) increased up to 2.5 fold with a slight increase of its Km value. This study indicated that the substitution P42R in the N-terminal proline-rich region of P450 contributed to the improvement of catalytic activity albeit the reduction of P450 structural stability or the decrease of substrate affinity. Characterization of these polymorphisms should be carefully examined in terms of the metabolism of many clinical drugs and environmental chemicals.

Grb10 is a dual regulator of receptor tyrosine kinase signaling

The adaptor protein Grb10 is a close homolog of Grb7 and Grb14. These proteins are characterized by an N-terminal proline-rich region, a Ras-GTPase binding domain, a PH domain, an SH2 domain and a BPS domain in between the PH and SH2 domains. Human Grb10 gene encodes three splice variants. These variants show differences in functionality. Grb10 associates with multiple proteins including tyrosine kinases in a tyrosine phosphorylation dependent or independent manner. Association with multiple proteins allows Grb10 to regulate different signaling pathways resulting in different biological consequences.

Probing the Interactions Between U24 from HHV-6A/7 and Fyn-SH3 or WW Domain Proteins

U24 is a type II tail-anchored putative membrane protein unique to the Roseolovirus family, including HHV-6 and HHV-7. It contains an N-terminal proline-rich region and is believed to function by disrupting the signalling pathway in order to ensure the virus' survival. HHV-6A is a neurovirulent virus as it is often found in multiple sclerosis (MS) patients. It has also been shown to directly induce demyelination, a typical MS symptom, in naive adult marmosets1. U24 from HHV-6A shares a seven residue identity with myelin basic protein (MBP), a protein responsible for the compaction of the myelin sheath in the CNS2. U24 from HHV-7, on the other hand, does not share this sequence identity with MBP, but this virus has also been implicated in MS, though indirectly3.In order to elucidate the exact role of U24 in MS, we have investigated the interaction of this protein with other partners such as the SH3 domain from Fyn tyrosine kinase and WW domain proteins. GST pull-downs, NMR titration data and molecular dynamics simulations will be presented. The differences in binding observed for U24 from HHV-6A and -7 will shed light into the hypothesis that U24 may function by mimicking MBP, as it has been previously shown that U24 and MBP can be phosphorylated in a similar manner4.

Coordinated activation of the Rac-GAP β2-chimaerin by an atypical proline-rich domain and diacylglycerol

Chimaerins, a family of GTPase activating proteins (GAPs) for the small G-protein Rac, have been implicated in development, neuritogenesis, and cancer. These Rac-GAPs are regulated by the lipid second messenger diacylglycerol (DAG) generated by tyrosine-kinases such as the epidermal growth factor receptor (EGFR). Here we identify an atypical Pro-rich motif in chimaerins that binds to the adaptor protein Nck1. Unlike most Nck1 partners, chimaerins bind to the third SH3 domain of Nck1. This association is mediated by electrostatic interactions of basic residues within the Pro-rich motif with acidic clusters in the SH3 domain. EGF promotes the binding of β2-chimaerin to Nck1 in the cell periphery in a DAG-dependent manner. Moreover, β2-chimaerin translocation to the plasma membrane and its peripheral association with Rac1 requires Nck1. Our studies underscore a coordinated mechanism for β2-chimaerin activation that involves lipid interactions via the C1 domain and protein-protein interactions via the N-terminal Pro-rich region.

Rapid screening of yeast mutants with reporters identifies new splicing phenotypes

Nuclear precursor mRNA splicing requires the stepwise assembly of a large complex, the spliceosome. Recent large-scale analyses, including purification of splicing complexes, high-throughput genetic screens and interactomic studies, have linked numerous factors to this dynamic process, including a well-defined core conserved from yeast to human. Intriguingly, despite extensive studies, no splicing defects were reported for some of the corresponding yeast mutants. To resolve this paradox, we screened a collection of viable yeast strains carrying mutations in splicing-related factors with a set of reporters including artificial constructs carrying competing splice sites. Previous analyses have indeed demonstrated that this strategy identifies yeast factors able to regulate alternative splicing and whose properties are conserved in human cells. The method, sensitive to subtle defects, revealed new splicing phenotypes for most analyzed factors such as the Urn1 protein. Interestingly, a mutant of PRP8 specifically lacking an N-terminal proline-rich region stimulated the splicing of a reporter containing competing branchpoint/3' splice site regions. Thus, using appropriate reporters, yeast can be used to quickly delineate the effect of various factors on splicing and identify those with the propensity to regulate alternative splicing events.

Frank-ter Haar Syndrome Protein Tks4 Regulates Epidermal Growth Factor-dependent Cell Migration

Mutations in the SH3PXD2B gene coding for the Tks4 protein are responsible for the autosomal recessive Frank-ter Haar syndrome. Tks4, a substrate of Src tyrosine kinase, is implicated in the regulation of podosome formation. Here, we report a novel role for Tks4 in the EGF signaling pathway. In EGF-treated cells, Tks4 is tyrosine-phosphorylated and associated with the activated EGF receptor. This association is not direct but requires the presence of Src tyrosine kinase. In addition, treatment of cells with LY294002, an inhibitor of PI 3-kinase, or mutations of the PX domain reduces tyrosine phosphorylation and membrane translocation of Tks4. Furthermore, a PX domain mutant (R43W) Tks4 carrying a reported point mutation in a Frank-ter Haar syndrome patient showed aberrant intracellular expression and reduced phosphoinositide binding. Finally, silencing of Tks4 was shown to markedly inhibit HeLa cell migration in a Boyden chamber assay in response to EGF or serum. Our results therefore reveal a new function for Tks4 in the regulation of growth factor-dependent cell migration.

Silencer of Death Domains (SODD) Inhibits Skeletal Muscle and Kidney Enriched Inositol 5-Phosphatase (SKIP) and Regulates Phosphoinositide 3-Kinase (PI3K)/Akt Signaling to the Actin Cytoskeleton

Phosphoinositide 3-kinase (PI3K) regulates cell polarity and migration by generating phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P(3)) at the leading edge of migrating cells. The serine-threonine protein kinase Akt binds to PI(3,4,5)P(3), resulting in its activation. Active Akt promotes spatially regulated actin cytoskeletal remodeling and thereby directed cell migration. The inositol polyphosphate 5-phosphatases (5-ptases) degrade PI(3,4,5)P(3) to form PI(3,4)P(2), which leads to diminished Akt activation. Several 5-ptases, including SKIP and SHIP2, inhibit actin cytoskeletal reorganization by opposing PI3K/Akt signaling. In this current study, we identify a molecular co-chaperone termed silencer of death domains (SODD/BAG4) that forms a complex with several 5-ptase family members, including SKIP, SHIP1, and SHIP2. The interaction between SODD and SKIP exerts an inhibitory effect on SKIP PI(3,4,5)P(3) 5-ptase catalytic activity and consequently enhances the recruitment of PI(3,4,5)P(3)-effectors to the plasma membrane. In contrast, SODD(-/-) mouse embryonic fibroblasts exhibit reduced Akt-Ser(473) and -Thr(308) phosphorylation following EGF stimulation, associated with increased SKIP PI(3,4,5)P(3)-5-ptase activity. SODD(-/-) mouse embryonic fibroblasts exhibit decreased EGF-stimulated F-actin stress fibers, lamellipodia, and focal adhesion complexity, a phenotype that is rescued by the expression of constitutively active Akt1. Furthermore, reduced cell migration was observed in SODD(-/-) macrophages, which express the three 5-ptases shown to interact with SODD (SKIP, SHIP1, and SHIP2). Therefore, this study identifies SODD as a novel regulator of PI3K/Akt signaling to the actin cytoskeleton.