anti-GST Antibody Research Articles

Molecular Recognition of the Tes LIM2–3 Domains by the Actin-related Protein Arp7A

Actin-related proteins (Arps) are a highly conserved family of proteins that have extensive sequence and structural similarity to actin. All characterized Arps are components of large multimeric complexes associated with chromatin or the cytoskeleton. In addition, the human genome encodes five conserved but largely uncharacterized “orphan” Arps, which appear to be mostly testis-specific. Here we show that Arp7A, which has 43% sequence identity with β-actin, forms a complex with the cytoskeletal proteins Tes and Mena in the subacrosomal layer of round spermatids. The N-terminal 65-residue extension to the actin-like fold of Arp7A interacts directly with Tes. The crystal structure of the 1–65Arp7A·LIM2–3Tes·EVH1Mena complex reveals that residues 28–49 of Arp7A contact the LIM2–3 domains of Tes. Two alanine residues from Arp7A that occupy equivalent apolar pockets in both LIM domains as well as an intervening GPAK linker that binds the LIM2–3 junction are critical for the Arp7A-Tes interaction. Equivalent occupied apolar pockets are also seen in the tandem LIM domain structures of LMO4 and Lhx3 bound to unrelated ligands. Our results indicate that apolar pocket interactions are a common feature of tandem LIM domain interactions, but ligand specificity is principally determined by the linker sequence.

Cloning and Expression of Spider Dragline Silk Protein Gene in <i>Escherichia coli</i> and Eukaryotic Cells

With the extreme tensile strength and toughness, spider silk has a high application value in a wide range of medical and industrial. However, the application of spider silk was limited because of its non-domestication. So, we tried to obtain the spider silk protein by genetic engineering. In this study, the dragline silk gene ( ASP ), a 837 bp fragment was cloned from the genome of spider ( Araneus ventricosus ) by Nested PCR. And then it was ligated into pGEX-6p-1 prokaryotic expression vector and pGFP-N2 eukaryotic expression vector, named as pASG and pASN, respectively. The fusion protein GST-ASP was successfully expressed in pASG and characterized by Western blotting with anti-GST antibodies after induced at 16℃ for 24 h in E. coli . The fusion proteins ASP-GFP was also successfully expressed in pASN by the detection of green fluorescence after transfected in insect sf9 cells for 8 h, which indicated that the ASP gene was correctly expressed in E. coli and eukaryotic cells, respectively. This study provides a beneficial attempt to develop the pathway for yielding spider silk protein by genetic engineering.

Cloning, recombinant expression and purification ofturbot (<I>Scophthalmus maximus</I>) mature IGF-I

Insulin-like growth factor-I (IGF-I) is a conserved peptide expressed ubiquitously,which shows highly homologous diverse effects on development,growth,and metabolism. With RT-PCR,the fragment encoding the turbot (Scophthalmus maximus) mature IGF-I peptide was amplified. It was predicted that the mature peptide was composed of 70 amino acids including 6 cysteines which may form 3 disulfide bonds. The target fragment was then successfully subcloned into the express vector pGEX-4T-1 and was highly expressed in E.coli BL21(DE3)plysS. The result of SDS-PAGE showed that the fusion protein expressed in the form of inclusion bodies with molecular weight of 34 ku and maximally amounted to 59 % of the whole protein in the E.coli cell 4 hours after being induced with IPTG. The western blotting indicated that recombinant protein had the antigenicity to anti-GST antibody. The inclusion bodies were dissolved in 6 mol/Lguanidine chloride followed by pulse renaturation in refolding buffer containing 0.5 mol/L L-Arginine,1.0 mol/L GSH and 0.2 mol/L GSSG. Then the renatured recombinant protein was purified by GSTrap FF affinity chromatography. The effect of purified GST-IGF-I on turbot kidney cells was analysed,and it indicated that the recombinant GST-IGF-I can stimulate the proliferation of the cells.

ETRAP (efficient trapping and purification) of target protein polyclonal antibodies from GST-protein immune sera1

Recombinant GST (glutathione transferase) proteins are widely used as immunogens to generate polyclonal antibodies. Advantages of using GST proteins include: commercially available cloning vectors, vast literature for protein expression in Escherichia coli, the ease of protein purification, immunogen can be used as an ELISA standard and GST can be removed in some systems. However, there are disadvantages: GST oligomerization, inclusion body formation and target protein insolubility after GST removal. Perhaps the most detrimental is the significant generation of anti-GST antibodies by the host animal. A two-column procedure using a glutathione-GST column and a glutathione-(GST-protein) column can yield affinity-purified anti-(GST-protein) polyclonal antibody. Several passes over the first column are often required, though, to completely extract the anti-GST antibodies from the immune sera. We reasoned that knowledge of the target protein linear epitope(s) would allow construction of a peptide affinity resin for a single-pass 'one and done' purification termed ETRAP (efficient trapping and purification). In the present paper, we describe our efforts and present data on rabbits and sheep immunized with GST proteins having target protein molecular masses of ~8, 21 and 33kDa. The titre and purity of the target antibodies using the ETRAP protocol were comparable to the more laborious multi-column purifications but with a considerable saving in time.

Screening serum markers of primary biliary cirrhosis with high-throughput protein chip encoded by the human genes

Objective To screen serum markers in patients with PBC by high-throughput protein chips encoded by the human genome. Methods High-throughput protein chips (contains a total of 38 400protein spots, including 17 718 human genes encoding proteins) were used to screen sera from 21 PBC patients, 20 disease control patients and 10 normal controls. Bioinformatics software was used to analyze information and statistical software was used to analyze the data to confirm the serum markers of PBC. Results The detection rate of protein spots using anti-GST antibody on the chip was 97. 6%, and the signal intensity correlation coefficient of double protein spots was 0. 98. Four serum markers( PDHA1, DBT,DLAT and HK1 )were screened by high-throughput protein chips between PBC group and the control group with a statistically significant. The positive rate of the four markers in the three groups was 66. 67% ( 14/21), 5.00% (1/20) and 0(0/10); 57. 14% ( 12/21 ), 5.00% (1/20) and 0(0/10); 52. 38% ( 11/21 ),0(0/20) and 0(0/10); 52. 38% ( 11/21 ), 0(0/20) and 0(0/10) respectively. All the four markers were different in the three groups with statistically significant (PDHA1 :x2 = 16. 79, P ＜0. 01 ;Fisher exact test,P=0. 000; DBT:x2 =12.86, P＜0. 01;Fisher exact test, P=0. 004; DLAT and HK1:Fisher exact test,P ＜0. 01 or 0. 05). Of those markers, antibodies to PDHA1, DBT and DLAT were the component of AMAM2 which had been used as the marker of PBC. Antibody to HK1 was identified as new marker of PBC,whose sensitivity to PBC was 52. 38% and specificity was 100. 00%. There were no serum marker were screened between the AMA-M2 positive and negative PBC patients. Only antibody to CENPB was identified to be significantly expressed between the ACA positive and negative PBC patients ( Fisher exact test, P =0. 000). Conclusions High-throughput protein chip encoded by the human gene is a technology for quick and comprehensive screening of new markers of PBC. Antibodies to HK1 could be used as new marker for PBC with highly sensitivity and specificity. No serum marker is found between the AMA-M2 positive and negative PBC patients whereas only antibody to CENPB is identified as marker between the ACA positive and negative PBC patients. Key words: Liver cirrhosis, biliary; Protein array analysis; Biological markers; Autoantibodies

Phospholipid Scramblase 1 Is Secreted by a Lipid Raft-dependent Pathway and Interacts with the Extracellular Matrix Protein 1 in the Dermal Epidermal Junction Zone of Human Skin

We examined the interaction of ECM1 (extracellular matrix protein 1) using yeast two-hybrid screening and identified the type II transmembrane protein, PLSCR1 (phospholipid scramblase 1), as a binding partner. This interaction was then confirmed by in vitro and in vivo co-immunoprecipitation experiments, and additional pull-down experiments with GST-tagged ECM1a fragments localized this interaction to occur within the tandem repeat region of ECM1a. Furthermore, immunohistochemical staining revealed a partial overlap of ECM1 and PLSCR1 in human skin at the basal epidermal cell layer. Moreover, in human skin equivalents, both proteins are expressed at the basal membrane in a dermal fibroblast-dependent manner. Next, immunogold electron microscopy of ultrathin human skin sections showed that ECM1 and PLSCR1 co-localize in the extracellular matrix, and using antibodies against ECM1 or PLSCR1 cross-linked to magnetic immunobeads, we were able to demonstrate PLSCR1-ECM1 interaction in human skin extracts. Furthermore, whereas ECM1 is secreted by the endoplasmic/Golgi-dependent pathway, PLSCR1 release from HaCaT keratinocytes occurs via a lipid raft-dependent mechanism, and is deposited in the extracellular matrix. In summary, we here demonstrate that PLSCR1 interacts with the tandem repeat region of ECM1a in the dermal epidermal junction zone of human skin and provide for the first time experimental evidence that PLSCR1 is secreted by an unconventional secretion pathway. These data suggest that PLSCR1 is a multifunctional protein that can function both inside and outside of the cell and together with ECM1 may play a regulatory role in human skin.

Proline Isomerase Pin1 Represses Terminal Differentiation and Myocyte Enhancer Factor 2C Function in Skeletal Muscle Cells

Reversible proline-directed phosphorylation at Ser/Thr-Pro motifs has an essential role in myogenesis, a multistep process strictly regulated by several signaling pathways that impinge on two families of myogenic effectors, the basic helix-loop-helix myogenic transcription factors and the MEF2 (myocyte enhancer factor 2) proteins. The question of how these signals are deciphered by the myogenic effectors remains largely unaddressed. In this study, we show that the peptidyl-prolyl isomerase Pin1, which catalyzes the isomerization of phosphorylated Ser/Thr-Pro peptide bonds to induce conformational changes of its target proteins, acts as an inhibitor of muscle differentiation because its knockdown in myoblasts promotes myotube formation. With the aim of clarifying the mechanism of Pin1 function in skeletal myogenesis, we investigated whether MEF2C, a critical regulator of the myogenic program that is the end point of several signaling pathways, might serve as a/the target for the inhibitory effects of Pin1 on muscle differentiation. We show that Pin1 interacts selectively with phosphorylated MEF2C in skeletal muscle cells, both in vitro and in vivo. The interaction with Pin1 requires two novel critical phospho-Ser/Thr-Pro motifs in MEF2C, Ser98 and Ser110, which are phosphorylated in vivo. Overexpression of Pin1 decreases MEF2C stability and activity and its ability to cooperate with MyoD to activate myogenic conversion. Collectively, these findings reveal a novel role for Pin1 as a regulator of muscle terminal differentiation and suggest that Pin1-mediated repression of MEF2C function could contribute to this function.

Inhibition of Osteoclast Bone Resorption by Disrupting Vacuolar H+-ATPase a3-B2 Subunit Interaction

Vacuolar H(+)-ATPases (V-ATPases) are highly expressed in ruffled borders of bone-resorbing osteoclasts, where they play a crucial role in skeletal remodeling. To discover protein-protein interactions with the a subunit in mammalian V-ATPases, a GAL4 activation domain fusion library was constructed from an in vitro osteoclast model, receptor activator of NF-κB ligand-differentiated RAW 264.7 cells. This library was screened with a bait construct consisting of a GAL4 binding domain fused to the N-terminal domain of V-ATPase a3 subunit (NTa3), the a subunit isoform that is highly expressed in osteoclasts (a1 and a2 are also expressed, to a lesser degree, whereas a4 is kidney-specific). One of the prey proteins identified was the V-ATPase B2 subunit, which is also highly expressed in osteoclasts (B1 is not expressed). Further characterization, using pulldown and solid-phase binding assays, revealed an interaction between NTa3 and the C-terminal domains of both B1 and B2 subunits. Dual B binding domains of equal affinity were observed in NTa, suggesting a possible model for interaction between these subunits in the V-ATPase complex. Furthermore, the a3-B2 interaction appeared to be moderately favored over a1, a2, and a4 interactions with B2, suggesting a mechanism for the specific subunit assembly of plasma membrane V-ATPase in osteoclasts. Solid-phase binding assays were subsequently used to screen a chemical library for inhibitors of the a3-B2 interaction. A small molecule benzohydrazide derivative was found to inhibit osteoclast resorption with an IC(50) of ∼1.2 μm on both synthetic hydroxyapatite surfaces and dentin slices, without significantly affecting RAW 264.7 cell viability or receptor activator of NF-κB ligand-mediated osteoclast differentiation. Further understanding of these interactions and inhibitors may contribute to the design of novel therapeutics for bone loss disorders, such as osteoporosis and rheumatoid arthritis.

Methionine Sulfoxide Reductase B1 (MsrB1) Recovers TRPM6 Channel Activity during Oxidative Stress

Mg(2+) is an essential ion for many cellular processes, including protein synthesis, nucleic acid stability, and numerous enzymatic reactions. Mg(2+) homeostasis in mammals depends on the equilibrium between intestinal absorption, renal excretion, and exchange with bone. The transient receptor potential melastatin type 6 (TRPM6) is an epithelial Mg(2+) channel, which is abundantly expressed in the luminal membrane of the renal and intestinal cells. It functions as the gatekeeper of transepithelial Mg(2+) transport. Remarkably, TRPM6 combines a Mg(2+)-permeable channel with an alpha-kinase domain. Here, by the Ras recruitment system, we identified methionine sulfoxide reductase B1 (MsrB1) as an interacting protein of the TRPM6 alpha-kinase domain. Importantly, MsrB1 and TRPM6 are both present in the renal Mg(2+)-transporting distal convoluted tubules. MsrB1 has no effect on TRPM6 channel activity in the normoxic conditions. However, hydrogen peroxide (H(2)O(2)) decreased TRPM6 channel activity. Co-expression of MsrB1 with TRPM6 attenuated the inhibitory effect of H(2)O(2) (TRPM6, 67 +/- 5% of control; TRPM6 + MsrB1, 81 +/- 5% of control). Cell surface biotinylation assays showed that H(2)O(2) treatment does not affect the expression of TRPM6 at the plasma membrane. Next, mutation of Met(1755) to Ala in TRPM6 reduced the inhibitory effect of H(2)O(2) on TRPM6 channel activity (TRPM6 M1755A: 84 +/- 10% of control), thereby mimicking the action of MsrB1. Thus, these data suggest that MsrB1 recovers TRPM6 channel activity by reducing the oxidation of Met(1755) and could, thereby, function as a modulator of TRPM6 during oxidative stress.

Use of SPR to Study the Interaction of G7-18NATE Peptide with the Grb7-SH2 Domain

Surface plasmon resonance (SPR) is a useful biosensor technique for the study of biomolecular interactions, with the potential for high-throughput screening of ligand interactions with drug targets. The key to its successful use, however, is in the appropriate design of the experiment, including the mode of immobilization to the biosensor chip. We report an investigation of the use of SPR for measuring the affinity of the G7-18NATE peptide ligand for its Grb7-SH2 domain target involved in the migratory and proliferative potential of cancer cells. Previous studies have shown that the cyclic non-phosphorylated peptide, G7-18NATE, inhibits Grb7 interactions with upstream binding partners and is able to inhibit both cell migration and proliferation of cancer cells. We report the synthesis of a biotinylated G7-18NATE covalently attached to a linker (G7-18NATE-ASASASK-Biotin) and compare its interaction with the Grb7-SH2 domain by SPR using three different immobilization strategies; immobilisation of the peptide via streptavidin, immobilization of glutathione S-transferase (GST)-Grb7-SH2 domain via anti-GST antibody, and immobilization of biotinylated Grb7-SH2 domain via streptavidin. This revealed that sensorgrams free from non-specific binding and displaying simple kinetics were most readily achieved by immobilising the protein rather than the peptide, in spite of the lower response associated with this method. KD values of ~300 μM were determined for both strategies at pH 7.4. This compared with a KD value of 4.4 μM at pH 6 demonstrating the importance of pH on this interaction. Overall, the immobilised protein systems are most suitable for future comparative screening efforts using SPR.

Use of a Single-Chain Antibody Library for Ovarian Cancer Biomarker Discovery

The discovery of novel early detection biomarkers of disease could offer one of the best approaches to decrease the morbidity and mortality of ovarian and other cancers. We report on the use of a single-chain variable fragment antibody library for screening ovarian serum to find novel biomarkers for the detection of cancer. We alternately panned the library with ovarian cancer and disease-free control sera to make a sublibrary of antibodies that bind proteins differentially expressed in cancer. This sublibrary was printed on antibody microarrays that were incubated with labeled serum from multiple sets of cancer patients and controls. The antibodies that performed best at discriminating disease status were selected, and their cognate antigens were identified using a functional protein microarray. Overexpression of some of these antigens was observed in cancer serum, tumor proximal fluid, and cancer tissue via dot blot and immunohistochemical staining. Thus, our use of recombinant antibody microarrays for unbiased discovery found targets for ovarian cancer detection in multiple sample sets, supporting their further study for disease diagnosis.

AKAP79/150 Interacts with AC8 and Regulates Ca2+-dependent cAMP Synthesis in Pancreatic and Neuronal Systems

Protein kinase A anchoring proteins (AKAPs) provide the backbone for targeted multimolecular signaling complexes that serve to localize the activities of cAMP. Evidence is accumulating of direct associations between AKAPs and specific adenylyl cyclase (AC) isoforms to facilitate the actions of protein kinase A on cAMP production. It happens that some of the AC isoforms (AC1 and AC5/6) that bind specific AKAPs are regulated by submicromolar shifts in intracellular Ca2+. However, whether AKAPs play a role in the control of AC activity by Ca2+ is unknown. Using a combination of co-immunoprecipitation and high resolution live cell imaging techniques, we reveal an association of the Ca2+-stimulable AC8 with AKAP79/150 that limits the sensitivity of AC8 to intracellular Ca2+ events. This functional interaction between AKAP79/150 and AC8 was observed in HEK293 cells overexpressing the two signaling molecules. Similar findings were made in pancreatic insulin-secreting cells and cultured hippocampal neurons that endogenously express AKAP79/150 and AC8, which suggests important physiological implications for this protein-protein interaction with respect to Ca2+-stimulated cAMP production.

Binding of p110 Retinoblastoma Protein Inhibits Nuclear Import of Simian Virus SV40 Large Tumor Antigen

Nuclear import of the simian virus 40 large tumor antigen (T-ag) is dependent on its nuclear localization signal (NLS) within amino acids 126-132 that is recognized by the importin alpha/beta1 heterodimer, as well as a protein kinase CK2 site at serine 112 upstream of the NLS, which enhances the interaction approximately 50-fold. Here we show for the first time that T-ag nuclear import is negatively regulated by N-terminal sequences (amino acids 102-110), which represent the binding site (BS) for the retinoblastoma (Rb) tumor suppressor protein (p110(Rb)). Quantitative confocal laser scanning microscopic analysis of the transport properties of T-ag constructs with or without Rb binding site mutations in living transfected cells or in a reconstituted nuclear transport system indicates that the presence of the RbBS significantly reduces nuclear accumulation of T-ag. A number of approaches, including the analysis of T-ag nuclear import in an isogenic cell pair with and without functional p110(Rb) implicate p110(Rb) binding as being responsible for the reduced nuclear accumulation, with the Ser(106) phosphorylation site within the RbBS appearing to enhance the inhibitory effect. Immunoprecipitation experiments confirmed association of T-ag and p110(Rb) and dependence thereof on negative charge at Ser(106). The involvement of p110(Rb) in modulating T-ag nuclear transport has implications for the regulation of nuclear import of other proteins from viruses of medical significance that interact with p110(Rb), and how this may relate to transformation.

Solution Structure of a Novel Cdc42 Binding Module of Bem1 and Its Interaction with Ste20 and Cdc42

Bem1 is a scaffold protein essential for the establishment of cell polarity in Saccharomyces cerevisiae. This work reports the solution structure of a Cdc42 binding module of Bem1 comprising the second SH3 domain (SH3b) and its C-terminal flanking region termed Cdc42 interacting (CI). First, the structure of Bem1 SH3b-CI was determined by NMR spectroscopy, which shows that Bem1 SH3b-CI is a structurally and functionally related domain that binds Cdc42. Next, the solution structure of Bem1 SH3b-CI in complex with the proline-rich region of p21-activated kinase Ste20 (Ste20 PRR) was determined. Finally, the interaction surface of Bem1 SH3b-CI with Cdc42 was identified based on chemical shift perturbation studies which reveals that Bem1 SH3b-CI interacts simultaneously with both Ste20 PRR and Cdc42 using the opposite surfaces. Thus, Bem1 can tether Cdc42 and Ste20 in close proximity so that Cdc42 can efficiently interact with Ste20 Cdc42 and Rac interactive binding (CRIB). Based on the present results together with the previous biochemical studies (Lamson, R. E., Winters, M. J., and Pryciak, P. M. (2002) Mol. Cell. Biol. 22, 2939-2951 and Winters, M. J., and Pryciak, P. M. (2005) Mol. Cell. Biol. 25, 2177-2190), a model was suggested that the autoinhibition of Ste20 kinase activity by CRIB is released through the Cdc42-CRIB interaction, which is mediated by Bem1, and Ste20 is subsequently activated, an initial step for the establishment of the cell polarity.

The 19-Amino Acid Insertion in the Tumor-associated Splice Isoform Rac1b Confers Specific Binding to p120 Catenin

The Rac1b splice isoform contains a 19-amino acid insertion not found in Rac1; this insertion leads to decreased GTPase activity and reduced affinity for GDP, resulting in the intracellular predominance of GTP-bound Rac1b. Here, using co-precipitation and proteomic methods, we find that Rac1b does not bind to many common regulators of Rho family GTPases but that it does display enhanced binding to SmgGDS, RACK1, and p120 catenin (p120(ctn)), proteins involved in cell-cell adhesion, motility, and transcriptional regulation. We use molecular modeling and structure analysis approaches to determine that the interaction between Rac1b and p120(ctn) is dependent upon protein regions that are predicted to be unstructured in the absence of molecular complex formation, suggesting that the interaction between these two proteins involves coupled folding and binding. We also find that directed cell movement initiated by Rac1b is dependent upon p120. These results define a distinct binding functionality of Rac1b and provide insight into how the distinct phenotypic program activated by this protein may be implemented through molecular recognition of effectors distinct from those of Rac1.

CaMKIIα interacts with M4 muscarinic receptors to control receptor and psychomotor function

Muscarinic acetylcholine receptors (mAChRs) are widely expressed in the mammalian brain and are essential for neuronal functions. These receptors are believed to be actively regulated by intracellular signals, although the underlying mechanisms are largely unknown. In this study, we show that Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) binds directly and selectively to one of five mAChR subtypes, M4 receptors (M4Rs), at their C-terminal regions of second intracellular loops. This binding relies on Ca(2+) activation of the kinase and leads to the phosphorylation of M4Rs at a specific threonine site (Thr145). Complementary in vivo studies in rat striatal neurons enriched with M4Rs confirm that rising Ca(2+) recruits CaMKIIalpha to M4Rs to potentiate receptor signalling, which controls behavioural sensitivity to dopamine stimulation in an activity-dependent manner. Our data identify a new model of protein-protein interactions. In a Ca(2+)-sensitive manner, CaMKIIalpha regulates M4R efficacy and controls the acetylcholine-dopamine balance in the basal ganglia and also the dynamics of movement.

Differential Effects of Sumoylation on Transcription and Alternative Splicing by Transcription Elongation Regulator 1 (TCERG1)

Modification of proteins by small ubiquitin-like modifier (SUMO) is emerging as an important control of transcription and RNA processing. The human factor TCERG1 (also known as CA150) participates in transcriptional elongation and alternative splicing of pre-mRNAs. Here, we report that SUMO family proteins modify TCERG1. Furthermore, TCERG1 binds to the E2 SUMO-conjugating enzyme Ubc9. Two lysines (Lys-503 and Lys-608) of TCERG1 are the major sumoylation sites. Sumoylation does not affect localization of TCERG1 to the splicing factor-rich nuclear speckles or the alternative splicing function of TCERG1. However, mutation of the SUMO acceptor lysine residues enhanced TCERG1 transcriptional activity, indicating that SUMO modification negatively regulates TCERG1 transcriptional activity. These results reveal a regulatory role for sumoylation in controlling the activity of a transcription factor that modulates RNA polymerase II elongation and mRNA alternative processing, which are discriminated differently by this post-translational modification.

Aph-1 Associates Directly with Full-length and C-terminal Fragments of γ-Secretase Substrates

γ-Secretase is a ubiquitous, multiprotein enzyme composed of presenilin, nicastrin, Aph-1, and Pen-2. It mediates the intramembrane proteolysis of many type 1 proteins, plays an essential role in numerous signaling pathways, and helps drive the pathogenesis of Alzheimer disease by excising the hydrophobic, aggregation-prone amyloid β-peptide from the β-amyloid precursor protein. A central unresolved question is how its many substrates bind and enter the γ-secretase complex. Here, we provide evidence that both the β-amyloid precursor protein holoprotein and its C-terminal fragments, the immediate substrates of γ-secretase, can associate with Aph-1 at overexpressed as well as endogenous protein levels. This association was observed using bi-directional co-immunoprecipitation in multiple systems and detergent conditions, and an β-amyloid precursor protein-Aph-1 complex was specifically isolated following in situ cross-linking in living cells. In addition, another endogenous canonical γ-substrate, Jagged, showed association of both its full-length and C-terminal fragment forms with Aph-1. We were also able to demonstrate that this interaction with substrates was conserved across the multiple isoforms of Aph-1 (β, αS, and αL), as they were all able to bind β-amyloid precursor protein with similar affinity. Finally, two highly conserved intramembrane histidines (His-171 and His-197) within Aph-1, which were recently shown to be important for γ-secretase activity, are required for efficient binding of substrates. Taken together, our data suggest a dominant role for Aph-1 in interacting with γ-secretase substrates prior to their processing by the proteolytic complex.

Regulation of FOXO1 by TAK1-Nemo-like Kinase Pathway

The FOXO family of forkhead transcription factors has a variety of important functions in stress response, metabolism, cell cycle, apoptosis, longevity, etc. The transcriptional activity and subcellular localization of FOXO are tightly regulated by post-translational modifications, including phosphorylation by various kinases. Here, we report that the transforming growth factor-beta-activated kinase (TAK1)-Nemo-like kinase (NLK) pathway negatively regulates FOXO1. We show that NLK binds and phosphorylates FOXO1 at Pro-directed Ser/Thr residues in the transactivation domain. The phosphorylation by TAK1-NLK pathway inhibits the transcriptional activity of FOXO1 and excludes FOXO1 from the nucleus, which is independent of phosphatidylinositol 3-kinase/Akt pathway. Consistently, knockdown of TAK1-NLK pathway dephosphorylates FOXO1 and decreases phospho-Ser-329 FOXO1 level. It also induces translocation of FOXO1 into the nucleus and leads to an increase in mRNA levels of FOXO target genes and poly(ADP-ribose) polymerase cleavage. In addition, we show the interaction between NLK and FOXO1 is evolutionarily conserved in Drosophila. Collectively, these findings provide the first evidence that TAK1-NLK pathway is a novel regulator of FOXO1.

Glycogen Synthase Kinase 3β Interaction Protein Functions as an A-kinase Anchoring Protein

A-kinase anchoring proteins (AKAPs) include a family of scaffolding proteins that target protein kinase A (PKA) and other signaling proteins to cellular compartments and thereby confine the activities of the associated proteins to distinct regions within cells. AKAPs bind PKA directly. The interaction is mediated by the dimerization and docking domain of regulatory subunits of PKA and the PKA-binding domain of AKAPs. Analysis of the interactions between the dimerization and docking domain and various PKA-binding domains yielded a generalized motif allowing the identification of AKAPs. Our bioinformatics and peptide array screening approaches based on this signature motif identified GSKIP (glycogen synthase kinase 3beta interaction protein) as an AKAP. GSKIP directly interacts with PKA and GSK3beta (glycogen synthase kinase 3beta). It is widely expressed and facilitates phosphorylation and thus inactivation of GSK3beta by PKA. GSKIP contains the evolutionarily conserved domain of unknown function 727. We show here that this domain of GSKIP and its vertebrate orthologues binds both PKA and GSK3beta and thereby provides a mechanism for the integration of PKA and GSK3beta signaling pathways.