L-JNKI reduced rhTRAIL-induced cell death but enhanced cell death induced by selective activation of DR4 or DR5. This difference was unrelated to receptor internalisation or differential activation of c-Jun, but activation of different JNK isoforms. Our data demonstrate that JNK1, but not JNK2 is activated by rhTRAIL in the examined colon cancer cell lines. Although rhTRAIL activated both the long and short isoforms of JNK1, selective activation of DR4 or DR5 led to predominant activation of the short JNK1 isoforms (JNK1α1 and/or JNK1β1). Knockdown of JNK1α1 by shRNA enhanced apoptosis induced by TRAIL, agonistic DR4 or DR5 antibodies. On the other hand, knockdown of the long JNK1 isoforms (JNK1α2 and JNK1β2) had the opposite effect; it reduced TRAIL-induced cell death. These data indicate that the short JNK1 isoforms transmit an antiapoptotic signal, whereas the long isoforms (JNK1α2 or JNK1β2) act in a proapoptotic manner.Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis on binding to its receptors, death receptor 4 and 5 . TRAIL can also activate c-Jun N-terminal kinase (JNK) through the adaptor molecules, TNF receptor-associated factor 2 (TRAF2) and receptor-interacting protein (RIP). The role of JNK in TRAIL-induced tumour cell apoptosis is unclear. In this study, we demonstrate that JNK is activated by TRAIL in colon cancer cells. Inhibition of JNK with  Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) is a member of the TNF ligand superfamily . TRAIL iBinding of homotrimeric TRAIL to DR4 and DR5 induces receptor trimerisation and activation leading to recruitment of adaptor proteins and formation of the death-inducing signalling complex (DISC). Procaspase-8 and/or -10 are recruited to the DISC, leading to their oligomerisation and activation. Active caspase-8/-10 can activate the executioner caspases  and/or initiate the mitochondrial apoptotic pathway by cleaving the BH3-only protein Bid.Generally, caspase activation is the main outcome following activation of DR4/5 by TRAIL. However, TRAIL, via adaptor molecules such as TNF receptor-associated factor 2 (TRAF2), receptor-interacting protein (RIP), and the mitogen-activated protein kinase kinases (MKK)-4 and -7, can also activate the c-Jun N-terminal kinase (JNK) pathway . JNK1α1, JNK1β1, JNK2α1 and JNK2β1 isoforms all have a molecular weight of 46 kDa, whereas JNK1α2, JNK1β2, JNK2α2 and JNK2β2 isoforms are larger due to an extended C-terminus and their molecular weight is 54 kDa. JNK3 has two isoforms, JNK3α1 (46 kDa) and JNK3α2 (54 kDa). The α- and β-isoforms correspond to two alternative stretches of sequences in the kinase subdomains IX and X , agonistic anti-DR4 or anti-DR5 antibodies . To inhibit JNK activation, L-JNKI , a cell-permeable, 21-amino-acid peptide inhibitor of activated JNK was added 30 min before treatment with TRAIL or agonistic antibodies. UV treatment was done at 25 J m−2 for 3 min as a positive control. All reagents were from Sigma-Aldrich  unless otherwise stated.Colo205 cells were obtained from American Tissue Culture Collection . HCT15 and HCA7 cells were a kind gift from Professor L Egan . Colo205 and HCT15 cells were maintained in RPMI-1640 medium and HCA7 in DMEM medium, both media supplemented with 10% fetal bovine serum (FBS), 2 mCell viability was monitored by 2--2, 5-diphenyl tetrazolium bromide (MTT) assay as described before  for 15 min on ice in the dark. A wash step in calcium buffer was carried out before acquisition on a FacsCalibur flow cytometer .Cell death was monitored by labelling phosphatidyl serine on the surface of apoptotic cells with Annexin-V-FITC. Following treatment, cells were collected by gentle trypsinisation and incubated for 10 min at 37°C to allow membrane recovery after trypsinisation, pelleted by centrifugation at 350 × M Tris/HCl pH 8.0, 200 mM sodium chloride (NaCl), 5 mM EDTA, 10% glycerol, 1 mM dithiothreitol (DTT), 1 mM phenylmethylsulphonyl fluoride (PMSF), 5 μg ml−1 aprotinin, 2.5 μg ml−1 leupeptin, 1 mM sodium orthovanadate (Na2VO3) and 1 mM sodium fluoride (NaF). Cellular proteins (30 μg) were separated by electrophoresis on 10% SDS polyacrylamide gel and transferred onto nitrocellulose membranes. Blots were incubated with rabbit monoclonal antibodies to total JNK, phospho (p)-JNK , JNK1  or mouse monoclonal antibodies to p-JNK (1 : 500), Caspase-8  and JNK2 . For detection, the appropriate horseradish peroxidase-conjugated goat secondary antibodies were used. Protein bands were visualised with SuperSignal West Pico Chemiluminescent Substrate  on X-ray film .Cells were lysed in a buffer containing 1% Triton X-100, 100 mμg protein) were incubated overnight at 4°C with GST-c-Jun fusion protein beads. After washing, the beads were resuspended in kinase buffer containing ATP and kinase reaction was allow to proceed for 30 min at 30°C. Reactions were stopped by the addition of polyacrylamide gel electrophoresis (PAGE) sample loading buffer. Proteins were separated by electrophoresis on a 10% PAGE gel, transferred on PVDF membrane and incubated with phospho-c-Jun (Ser63) antibody. Finally, blots were subjected to enhanced chemiluminescence and kinase activity determined by densitometric analysis.JNK activity was measured using a specific kit  following the manufacturer's instructions and using GST fusion peptide as the specific substrate for JNK. In brief, cell lysates  for 40 min. After two wash steps with PBS/BSA, anti-mouse IgG-FITC (Sigma) secondary antibody was added for 30 min. All incubations were carried out on ice. Negative controls contained isotype control antibody. Cells were analysed by FacsCalibur flow cytometer (Becton Dickinson).5 Colo205 cells were incubated at 4 or 37°C in the presence of 50 ng ml−1 FITC-conjugated TRAIL or agonistic DR4/5 antibody cross-linked with FITC-labelled anti-mouse antibody for 30 min. Samples were rapidly chilled on ice to inhibit endocytosis and cells were collected by a brief centrifugation at 4°C. After washing twice in prechilled wash buffer , cell surface-bound ligand/antibody was removed by resuspension in prechilled acid wash solution  for 5 min on ice. Cells were subsequently washed three times in wash buffer and resuspended in cold PBS containing 2% (w/v) FBS before immediate quantification of ligand internalisation using a FacsCalibur flow cytometer (Becton Dickinson).To measure cellular uptake of receptor bound TRAIL and agonistic DR4/5 antibody, 2 × 10M sodium phosphate, 137 mM NaCl, 25 mM sodium-β-glycerol phosphate, 2 mM sodium pyrophosphate, 2 mM EDTA, 1% Triton X-100, 10% glycerol, 1 mM DTT, 1 mM PMSF, 5 μg ml−1 aprotinin, 2.5 μg ml−1 leupeptin, 1 mM Na2VO3 and 1 mM NaF with sonication. To cross-link mouse monoclonal JNK1 (BD Pharmigen) and p-JNK  antibodies to protein G-sepharose beads (Sigma), 2 μg antibody was incubated with 30 μl of beads for 1 h at 4°C, washed twice with PLB, resuspended in 0.2 M sodium borate pH 9.0 containing 20 mM dimethylpimelidate (DMP) and incubated for 45 min at room temperature. The reaction was stopped by washing the beads once with 0.2 M ethanolamine/HCl pH 8.0 and incubating at room temperature for 2 h. The beads were washed twice in 200 mM glycine and 200 mM NaCl pH 2.5 followed by a wash in 500 mM Tris/HCl pH 8.0. Protein (300 μg) was incubated for 2 h at 4°C with cross-linked JNK1 or p-JNK  antibodies bound to protein G-Sepharose. After two rounds of washes with PLB, the beads were resuspended in 30 μl of 1X Laemmli buffer. The supernatant was loaded on a 10% SDS–PAGE acrylamide gel and transferred to nitrocellulose membranes. Membranes containing the JNK1 immunoprecipitates were incubated with mouse monoclonal JNK1 (BD Pharmigen) and rabbit monoclonal p-JNK  antibodies to identify which JNK1 isoforms were activated. Membranes of the p-JNK immunoprecipitates were probed with JNK2 (Santa Cruz) and rabbit monoclonal total p-JNK  antibodies to identify the JNK2 isoforms activated. Detection and visualisation was carried out as described above.Cells were lysed in phosphate lysis buffer (PLB) containing 20 m6) were pelleted and resuspended in transfection solution V  containing 2.5 μg of mammalian JNK1α1 siRNA and the negative control expression plasmid, pKD-JNK1α1/SAPK1c-v1  and pKD-NegCon-v1 , or siRNA against JNK1α2/β2 and GFP as negative control by nucleofection using program T13 as per manufacturer's protocol (Amaxa). GFP plasmid (2.5 μg) was also transfected to analyse transfection efficiencies. Twenty-four hours after transfection, cells were resuspended in RPMI-1640 and seeded for Annexin V assays, RNA isolation or protein sampling. Sequence of JNK1α2/β2 siRNA1: sense–UUAGGUGCAGCAGUGAUCAtt; JNK1α2/β2 siRNA2: sense–UAGGUGCAGCAGUGAUCAAtt; GFP sense–GGCUACGUCCAGGAGCGCACCtt.Cells   using 20 U/25 μl reaction avian myeloblastosis virus reverse transcriptase. cDNAs for genes of interest were amplified during 32 cycles of 30 s denaturation at 94°C, 30 s annealing at 56°C, and 60 s extension at 72°C, with the following primers: JNK1α1 reverse – TCA CTG CTG CAC CTG TGC TAA AGG, forward – TGC CAC AAA ATC CTC TTT CCA GGA; JNK1 reverse – TCT TGG TTC TCT CCT CCA AGT C, forward – GTC AGG CAA GGG ATT TGT TAT; JNK1β1 reverse – ACT GCT GCA CCT GTG CTA AAG GAG, forward – AGG TGG TGT TTT GTT CCC AGG TAC, GAPDH was used as a loading control; GAPDH reverse – TCC ACC ACC CTG TTG CTG; forward – ACC ACA GTC CAT GCC ATC. The binding sites of the isoform specific primers in relation to the different JNK splice variants are depicted in Total RNA was isolated from cells using the GenElute Mammalian Total RNA Extraction kit (Sigma). Reverse transcription was carried out with 2 t-test, with a significance of P<0.05. Error bars are shown as standard error of mean (s.e.m.). All statistical analysis was carried on Graphpad Prism 4 .Differences in Annexin V staining between the treatment groups were analysed used a non-paired Student's −1 of rhTRAIL sufficient to decrease cell viability by 61.8±2.3%  resulted in phosphorylation of JNK in all three cell lines (M) induced almost equal levels of apoptosis in HCT15 cells  measured at 5 h after treatment.To examine whether the JNK pathway was activated during TRAIL-induced colon cancer cell death, phosphorylation of JNK and its target, c-Jun were assessed by western blot analysis following treatment with rhTRAIL. rhTRAIL , however enhanced agonistic DR4- (P=0.0002) and DR5 antibody-induced cell death . On the other hand (as was observed in Colo205 cells), inhibition of JNK reduced rhTRAIL-induced apoptosis , whereas the long isoform  was only phosphorylated after rhTRAIL treatment  whereas rhTRAIL led to phosphorylation of both the short and long JNK1 isoforms (ie JNK1α2 and/or JNK1β2).To assess which JNK2 isoforms were phosphorylated following selective receptor activation or rhTRAIL treatment, immunoprecipitation with JNK2-specific antibody was attempted, but was unsuccessful due to technical difficulties . As an alternative strategy, phospho-JNK  was immunoprecipitated, electrophoresed by SDS–PAGE and probed with total JNK2 antibody (this identified whether the short or long JNK2 isoforms were phosphorylated). Total phospho-JNK levels of the same blots were also determined to quantify the levels of phospho-JNK2 isoforms, in a similar manner as for the JNK1 isoforms . The levα1 and/or JNK1β1) may play an antiapoptotic role on selective activation of DR4 or DR5. In an effort to dissect its role, Colo205 cells were transiently transfected with an shRNA expression plasmid to JNK1α1. RT–PCR demonstrated knockdown of JNK1α1 following transient transfection of JNK1α1 shRNA, without affecting total JNK1 and JNK1β1 mRNA levels confirming isotype-specific knockdown  compared to cells transfected with a scrambled shRNA expressing plasmid  for 3 h and induction of apoptosis was measured .The only region that differs in the long JNK1 isoforms from the short JNK1 isoforms is a 5-nucleotide sequence and thus this was the only region targetable by siRNA. We designed two siRNAs against this region with selectivity for the antibody . JNK1α2/measured . KnockdoL-JNKI. L-JNKI was chosen over the widely used SP600125  and the difference in cell death seen following JNK inhibition may be related to the different JNK1 isoforms, with the short isoforms of JNK1 (JNK1α1 and JNK1β1) transmitting an antiapoptotic signal and the long isoforms of JNK1 (JNK1α2 or JNK1β2) mediating a proapoptotic signal. As a reason why activation of individual TRAIL receptors by agonistic antibodies activates different JNK isoforms from rhTRAIL, it is likely that the agonistic DR4- and DR5-specific antibodies trigger different receptor clustering, or intracellular conformational changes from those induced by rhTRAIL. It is also possible that on rhTRAIL treatment, higher-order heteromeric receptor complexes (receptosomes) including both DR4 and DR5 are formed where the interaction between the death domains of the various receptor trimers allows for the recruitment of more and/or different adaptor proteins. It has been shown that different agonists of DR4 and DR5 (agonistic antibodies and recombinant ligand) trigger different conformational changes in the receptors resulting in differences in adaptor protein recruitment, such as FADD. This can lead to activation of different downstream signal transduction and kinase pathways  is very difficult. Even 30–50% of the remaining protein may be sufficient to transmit a signal. Selective targeting one JNK isoform is even more complicated and thus our method probably underestimated the real potency of JNK isoforms in modulating TRAIL-mediated apoptosis.To elucidate the roles of the different JNK1 isoforms, JNK1α1 and JNK1β1 have been previously identified by α1 led to the induction of a number of antiapoptotic/prosurvival genes including proliferin (mitogen-regulated protein), Ack protein tyrosine kinase and serum and glucocorticoid regulated kinase (sgk) and repression of proapoptotic proteins, such as insulin-like growth factor binding protein-4 (IGFBP-4) and suppressor of cytokine signalling-3 (SOCS-3) as a strong indication that JNK1α1 indeed triggers antiapoptotic signalling (Genes regulated by JNK1vs selective activation of DR4 or DR5 result in the activation of different JNK isoforms. Moreover, the short isoform of JNK1, JNK1α1, generates an antiapoptotic signal whereas the long isoforms of JNK1 trigger proapoptotic signalling. Our findings shed light on the apparently contradictory results surrounding the role of JNK signalling in TRAIL-induced apoptosis and also suggest a way forward to target cancer cells for sensitisation to killing by inhibition of short isoforms of JNK1.In conclusion, we show for the first time that combined activation of all TRAIL receptors


The Crk adaptor proteins (Crk and CrkL) constitute an integral part of a network of essential signal transduction pathways in humans and other organisms that act as major convergence points in tyrosine kinase signaling. Crk proteins integrate signals from a wide variety of sources, including growth factors, extracellular matrix molecules, bacterial pathogens, and apoptotic cells. Mounting evidence indicates that dysregulation of Crk proteins is associated with human diseases, including cancer and susceptibility to pathogen infections. Recent structural work has identified new and unusual insights into the regulation of Crk proteins, providing a rationale for how Crk can sense diverse signals and produce a myriad of biological responses. There are over 110 SH2 domains and 300 SH3 domains in the human genome, making this general signaling strategy widely utilized in metazoan cells to transmit intracellular signals. As the name adaptor implies, these molecules physically bridge tyrosine phosphorylated proteins to various intracellular signaling pathways  and Src Homology 3 (SH3) domains separated by flexible linker sequences that act as building blocks to assemble multiprotein complexes. SH2 domains are structurally conserved protein domains of ~100 amino acids contained within the Src oncogene and other signaling proteins that bind tyrosine phosphorylated proteins in the context of short peptide sequences and localize SH2 domains to tyrosine phosphorylated proteins. SH3 domains are structurally conserved domains of ~60 amino acids that bind a consensus sequence of Xs Figure . An imprs Figure . The Crkchicken tumor Virus 10) [CT10 regulator of a tyrosine kinase.The Crk gene product was identified approximately 20 years ago in the form of a transforming gene (Gag-Crk) encoded in the genome of a defective avian sarcoma retrovirus called CT10 (irus 10) . The mosirus 10) ,5. Howevv-crk or gag-crk, Reichman, et al. reported the isolation of the cellular homolog of Crk by screening a chicken brain library using a portion of the v-crk probe devoid of the retrovirus-derived gag sequences [crk (c-crk) revealed a molecule with similar structural organization to v-Crk, but containing a ~50 amino acid proline-rich linker and an additional C-terminal SH3 domain (SH3C)  Figure . Matsudagenicity . Hence, The Crk and CrkL proteins have central roles in an astonishingly vast number of biological processes, ranging from cell proliferation, cell adhesion and migration, phagocytic and endocytic pathways for apoptotic cells and parasitic organisms respectively, apoptosis, and regulation of gene expression . Until tOver the past two decades, over 40 cellular proteins have been identified that bind to the SH2 and SH3N domains, and for this reason, it is clearly daunting to assign one specific cellular function to Crk . Indeed,Another important aspect of Crk signaling that requires definition is to assess which signaling pathways depend exclusively on Crk or CrkL versus those signaling pathways whereby Crk and CrkL can compensate for each other. Despite considerable homology between Crk II and CrkL in their SH2 and SH3 domains, development of knockout mouse models clearly show these gene products have distinct, non-overlapping roles, during embryonic development as both Crk knockout and CrkL knockout mice die perinatally with different developmental defects. Moreover, in Crk II (-/-) fibroblasts, CrkL proteins were not overexpressed in a compensatory manner, and vice versa of CrkII expression in CrkL (-/-) cells, again suggesting little molecular crosstalk at the level of expression in embryonic cells . InteresHowever, in contrast to the aforementioned observations showing independent roles for CrkL and Crk II during development, a number of recent studies suggest that Crk II and CrkL are co-expressed in somatic cells and can compensate to each in cellular signaling. An example of such redundancy comes from several independent studies dissecting the Reelin signaling pathway in cortical pyramidal cells of the hippocampus. Reelin, a secreted glycoprotein that binds to receptors on hippocampal neurons such as ApoER2, VLDLR, and α3β1 integrin, controls cell positioning during adult neurogenesis via Src-dependent tyrosine phosphorylation of the scaffold protein Dab1 . Upon reMuch of the early emphasis on Crk biology focused on the mechanisms of increased cellular tyrosine phosphorylation and models for v-Crk transformation. Although it is still reasonable to consider that v-Crk binds to and "hijacks" a cellular tyrosine kinase, no model in which a specific tyrosine kinase is activated has provided a unifying molecular mechanism for v-Crk and Crk I transformation. However, a number of studies indicate that expression of v-Crk in Src (-/-) or Fyn (-/-) fibroblasts significantly reduces Crk-inducible tyrosine phosphorylation , and sucCrk associated substrate), the most ubiquitously expressed member, was in fact discovered as a result of its elevated phosphotyrosine levels observed in CT10-tranformed cells [With respect to the substrates of tyrosine phosphorylation and Crk transformation, a great deal of attention has been placed on the p130Cas/HEF1/Efs proteins, a family of non-enzymatic docking proteins that contain multiple interacting domains and serve as important anchoring points for protein-protein interactions ,91. p130ed cells ,19,92. Ted cells ,94. Receed cells . Moreoveed cells . Overexped cells ,97. Indeed cells . Althouged cells . Since min utero from congestive heart failure, edema, and hemorrhaging due to breakdown in the regulation of vascular permeability [Mice with homozygous null mutations of p130Cas exhibit marked growth retardation with poorly developed heart and vasculature and die eability . Isolateeability . In fibreability ,102. SpeAt the molecular level, p130Cas has multiple protein-protein interaction domains including an N-terminal SH3 domain that binds FAK and Pyk2, an interior "substrate domain" characterized by 15 YxxP motifs, a C-terminal Src binding domain, followed by a highly conserved C-terminal region that binds to the Nsp family of proteins   Figure . Based oThe biological role of p130Cas phosphorylation is also intriguing from the vantage point that a vast variety of extracellular matrix molecules, growth factors, hormones, and metabolic intermediates that induce tyrosine phosphorylation of p130Cas and subsequent coupling to Crk Table . These mIn vivo, both of these kinases have been proposed to contribute to p130Cas phosphorylation, possibly to permit different mechanisms and different input signals to converge on assembling Crk linkages [Once extended, the central region of p130Cas is a substrate for activated tyrosine kinases, and p130Cas itself has binding sites for FAK  and Pyk2linkages . To gainlinkages  developelinkages . In appalinkages .Analogous to the theme described above for p130Cas, other scaffolding molecules that are acted upon by extrinsic signals ultimately interact with Crk, thereby launching alternative versions of a common signaling paradigm. Numerous stimuli, ranging from growth factors and cytokines to ECM, apoptotic cells, microbial products, immune complexes, and endogenous metabolic products activate trans-membrane receptors that inducibly phosphorylate proteins that, in turn, engage the Crk SH2 domain  was isolated in a screen for Crk SH3N-binding partners and was the first protein identified that bound to the SH3 domain of Crk [C3G  of each ommatidium fails to form. Subsequent studies indicated that SOS was homologous to the yeast exchange factor for Ras, CDC25, and then identified as a central conduit to link receptor tyrosine kinases to Ras following growth factor stimulation in metazoan cells [SOS was first identified in  pathway . When sean cells . In mamman cells .Both SOS1 and SOS2 possess repetitive proline motifs that conform to consensus Crk SH3 binding motifs and direct stable association between Crk and SOS. A functional significance for this interaction is evident from co-expression studies showing that dominant negative Ras protein suppresses v-Crk-induced cell transformation in CEFs . MoreoveDownstream of Crk; DOCK1) was originally cloned by far-Western blotting, with Crk SH3N domain as a "bait" molecule [DOCK180  or vascular-endothelial growth factor receptor (VEGFR), Crk/DOCK180/ELMO/Rac interactions mediate JNK activation, leading to dorsal closure [In addition to the ability of Crk to activate JNK through C3G and R-Ras noted above, Crk II can also interact with JNK directly through a proline-rich sequence in JNK and the SH3N domain of Crk . Similar closure .Abl is a non-receptor tyrosine kinase first identified as the cellular homolog of the v-Abl oncogene product of the Abelson murine leukemia virus, and has multiple roles in cell growth, transformation, cell stress, apoptosis, and remodeling of the actin cytoskeleton. Arg (Abl-related gene) is over 70% homologous to Abl, and both proteins contain four tandem proline-rich motifs located just C-terminal to the kinase domain ,158 thatIn vitro, addition of the Crk SH3N domain can reverse the inhibition of Abl activity by F-actin, further suggesting that Crk transactivation may occur locally in the focal adhesions [In general, the interaction between Abl and Crk is suggested to be bi-directional, meaning that Crk can both transactivate Abl and Abl can transinhibit Crk II and CrkL. Transactivation of Abl by Crk II was first reported by Shishido and Hanafusa based on observations that co-expresion of Crk and Abl increased the net tyrosine phosphorylation of cellular proteins in Abl-expressing cells Figure . Subsequdhesions .Subsequent to the binding and transactivation of Abl by Crk, Crk Y221 is itself an excellent substrate for Abl, and binding of Crk to Abl reflects a potent downmodulatory signal . HoweverTyrosine phosphorylation of Crk on Y221 is also not of inconsequence with respect to signal transduction pathways initiated by integrins and growth factors that led to p130Cas phosphorylation. Klemke and colleagues were the first to demonstrate that Abl inducible Crk Y221 phosphorylation acted as a molecular switch to dissociate a p130Cas/Crk/DOCK180 ternary complex, in order to block motility and invasive behavior of human pancreatic cancer cells  Figure . More reFinally, a very interesting study by Peterson and Long identified an important functional role in inhibitory signaling downstream of the human killer cell Ig-like receptor (KIR) that inhibits Natural Killer (NK) cell activation and cytotoxicity . Using aRecent studies also suggest that pY221Crk is not necessarily a biologically inert species. Interesting work by Vuori and colleagues showed that pY221 Crk was required for recruiting activated Rac1 to the membrane fraction, and Rac1 re-localization was prevented by expression of the Y221F Crk mutant . Most inSince the discovery of v-Crk and c-Crk II, it is clear that the C-terminal region exerts negative regulation on Crk signaling and transformation. Whereas models for protein interactions mediated by the SH2 and SH3N have been well documented to explain how Crk functions as an adaptor protein ,168,174,C. elegans to mammals, there are notable differences in the Crk SH3C compared to canonical SH3 domains that bind PxxP motifs. First, although the amino acids that comprise the structural elements of the hydrophobic core are highly conserved, the amino acids that comprise the surface of the SH3C, in particular, where the PxxPxK is expected to bind, are highly divergent [NMR studies on various Crk proteins that contain the SH3C domain and SH3 linker have begun to offer explanations as to the nature of its regulatory aspects. Like the Y221YAQP motif, the SH3C contributes to the negative regulation of Crk II and CrkL by intramolecular domain crosstalk. Although the Crk SH3C is highly conserved from ivergent ,176. Theivergent . Howeverivergent . This suivergent .cis-trans isomerization at the proline from the PFY motif at the boundary of the linker-SH3C, a region previously identified as an important negative regulatory region in chicken Crk [cis and trans forms appear to have entirely different regulatory functions: the cis isomer is locked in a configuration that tethers the SH3N, preventing ligand binding, while the trans configuration appears extended and flexible  may be a specific inhibitor of Crk pathways in vivo. Another interesting aspect of this regulatory mechanism involving the PFY motif in chicken Crk is that F239, adjacent to P238, was absolutely required to achieve cis-trans isomerization, and mutation of F239 to Ile or Val abrogated this regulatory mechanism. This is most intriguing, given the fact that chicken is the only species of Crk with the PFY motif. This motif diverges to PIY in human and PVY in mouse   Figure . InteresC. elegans using classical worm genetics [C. elegans elegantly identified genes that mediated the manifestations of apoptosis (Ced-3/Caspases and Ced-4/APAF-1) and in the suppression of apoptosis (Ced-9/Bcl-2) [C. elegans model, a second related, if not overlapping, phenotype was noted in the Ced-2/Crk deficient worms that involved defects in the migration of cells into the distal tip [Drosophila metamorphosis, elegantly demonstrated that Drosophila Crk (Dcrk) cooperates with DOCK180  and ELMO for border cell migration in oogenesis and hemocyte migration during embryogenesis, and further mapped this pathway downstream of PVR, the fly equivalent molecule of PDGF Receptor and VEGF-Receptor [A significant advance towards a molecular understanding of Crk biology was revealed following a screen for genes involved in cell-death pathways in genetics . Earlier9/Bcl-2) ,184. Upo9/Bcl-2) . In addistal tip . DefectsReceptor . In addiYersinia pseudotuberculosis infection into human epithelial cells activated the p130Cas-Crk-DOCK180-Rac1 pathway via binding of the bacterial invasin to the β1 integrin receptor [Shigella flexneri infection into NIH 3T3 cells using a curious pathway involving Abl-Arg-mediated Crk Y221 phosphorylation, whereby Crk Y221F mutation blocked S. Flexneri infection [Pseudomonas aeruginosa infection [Helicobacter pylori infection [Pseudomonas aeruginosa internalization [H. pylori, Crk associates with tyrosine phosphorylated CagA, and the CagA/Crk complex regulates Erk signaling and Rac1-mediated cytoskeletal assemblages [H. pylori-associated gastric cancer, CagA-SHP2 signaling is known to play a central role, the functional significance of the CagA/Crk complex for cellular infection should be evaluated. Finally, exoenzyme T (ExoT) of P. aeruginosa ADP-ribosylates Crk I and Crk II at specific residues in the SH2 domain (Arg20) that impairs binding of Crk to p130Cas [In recent years, a growing and unexpected body of evidence suggests that Crk may contribute to bacterial pathogenesis by influencing entry into cells or by serving as targets of bacterial toxins that disrupt essential cellular function through Crk-dependent mechanisms. A role for Crk in bacterial uptake pathways was first noted by Weidow and colleagues, who demonstrated that receptor ,186. Thinfection . In verynfection  and Helinfection , Crk applization . In the emblages . Althoug p130Cas . Althoug p130Cas .A number of studies have suggested that Crk may play an important role in human cancers. Immunohistochemical analysis of human cancers showed the over-expression of Crk in adenocarcinomas of lung, breast, and stomach, and also sarcomas, followed by the demonstration of increased levels of Crk mRNA in more aggressive phenotypes of lung adenocarcinoma ,190-194.Based on these observations, several groups have begun to investigate the effects of Crk knockdown on the phenotypic behavior of human tumor cells. Simultaneous downregulation of both Crk I and Crk II by siRNA demonstrated the essential role of Crk in the malignant features of human ovarian cancer cells , synoviaThe above studies suggest that Crk may have a central role in cell motility and metastasis in highly aggressive motile cancer cells. There are a plethora of studies showing that p130Cas-HEF and the FAK-Src circuitry is involved in tumorigenesis, and Crk may indeed be an attractive target due to its central integrative downstream role in signaling by these molecules. Recent studies now suggest that DOCK180 and/or ELMO may be oncogenes, or have oncogenic potential, and Crk may be an attractive drug target in tumor cells that use Rac1 activation to acquire motile or metastatic potential. DOCK180, for example, is overexpressed at the borders, but not at the centers, of human malignant glioma, and together with ELMO1, it regulates Rac activity to enhance tumor invasion . AlthougAnother area of clinical investigation is the role of tyrosine phosphorylation of CrkL and Crk II in CML -201. ThiXenopus by activating caspases [In recent years, both Crk II and CrkL have been implicated in nuclear signal transduction events. For CrkL, elegant studies have shown that CrkL binds tyrosine phosphorylated Stat5 in Bcr-Abl expressing cells, and in a variety of cytokine-stimulated hematopoietic cells ,203. In caspases . Depleticaspases . Evidenccaspases . Recent caspases . These sThe Crk gene was identified about 20 years ago, and the characterization of Crk and its mode of cell signaling has arguably revolutionized the way we think about intracellular signal transduction. Despite significant advances and an enormous wealth of new information, the field is now faced with the challenging problem of how these complex signaling networks are controlled in time and space to produce biological responses. Efforts will need to be redirected from the relatively simple task of identifying specific effector proteins to an understanding of when and where these complexes are formed in cells. Development of FRET technology that reports specific protein-protein interactions, as well as the generation of a battery of phosphospecific antiseras against specific post-translational epitopes, should help provide a better understanding of Crk biology. This exciting field can expect substantial progress as the new observations continue. One thing is certain: the field of Crk biology will continue to grow over the next 20 years.The authors declare that they have no competing interests.RB wrote the background and significance and the sections on the biological significance of Crk. CK and FI wrote parts of the sections on Crk structure. ST wrote the sections on Crk and Cancers. All authors read and approved the final manuscript.A rotational view of the Crk II structure is shown in the supplemental data. The organization of the SH2 and SH3 domains are indicated in order to illustrate negative regulation.Click here for filePhenotype of Crki expressing cells. When Crk is downmodulated by siRNA, cells exhibit decreased motility and invasion.Click here for filePhenotype of Crki expressing cells. When Crk is downmodulated by siRNA, cells exhibit decreased motility and invasion.Click here for file


Since their discovery a little more than a decade ago, the docking proteins of the Gab/DOS family have emerged as important signalling elements in metazoans. Gab/DOS proteins integrate and amplify signals from a wide variety of sources including growth factor, cytokine and antigen receptors as well as cell adhesion molecules. They also contribute to signal diversification by channelling the information from activated receptors into signalling pathways with distinct biological functions. Recent approaches in protein biochemistry and systems biology have revealed that Gab proteins are subject to complex regulation by feed-forward and feedback phosphorylation events as well as protein-protein interactions. Thus, Gab/DOS docking proteins are at the centre of entire signalling subsystems and fulfil an important if not essential role in many physiological processes. Furthermore, aberrant signalling by Gab proteins has been increasingly linked to human diseases from various forms of neoplasia to Alzheimer's disease.In this review, we provide a detailed overview of the structure, effector functions, regulation and evolution of the Gab/DOS family. We also summarize recent findings implicating Gab proteins, in particular the Gab2 isoform, in leukaemia, solid tumours and other human diseases. Scaffgnalling ,5. Indeef action -7. The df action -10. Sincf action ,12.Drosophila and Caenorhabditis contain only one Gab gene [The Gab proteins are large scaffold or docking proteins of 50 to 100 kDa found in metazoans ,12. FuncGab gene ,7,20-22.via a second mechanism involving the small adaptor protein Grb2, and this represents the only mode of receptor interaction for Gab2 [knock-in mice expressing a Grb2 binding-deficient Gab1 mutant [Docking proteins of the Gab family use several different mechanisms to regulate their subcellular localization. Firstly, the PH domain confers recruitment of Gab proteins to plasma membrane patches enriched in specific phosphatidyl-inositol-phosphates (PIPs) -29. In afor Gab2 . The sigfor Gab2  and by t1 mutant . This in1 mutant ,33,35-40Ciona and the sea anemone Nematostella [via its N-terminal SH3 domain, while it uses its C-terminal SH3 domain to bind to two SH3 binding motifs within Gab proteins [Vertebrate Gab proteins possess at least two regions that are potentially involved in the recruitment of Grb2 or other proteins containing SH3 domains such as Mona/Gads  [Two recruitment motifs can be distinguished in Gab/Dos proteins, a "typical recruitment" motif and an "atypical" Grb2 binding site . The typ binding  and consno acid) . Howeveret al. (2002) have addressed the first issue for DOS by showing that mutation of either of the two Grb2 binding sites impairs R7 photoreceptor cell development in a moderate manner, while simultaneous mutation abrogates R7 development completely [et al. (2003) have shown that the atypical Grb2 binding site plays a dominant role in the Gads/Grb2-mediated recruitment of Gab2 to the LAT signalling complex in the lipid rafts of T lymphocytes [Since mutation/deletion experiments have clearly demonstrated the importance of the Gab2/Grb2 interaction for the activation of the various effector pathways controlled by this docking protein ,40, impompletely . Furtherphocytes . A finalphocytes ,39,49,50phocytes .via Gab1/p85 interaction in the signalling complex [A more complex indirect recruitment mechanism is utilized by Gab1 in FGF receptor signalling ,52. Here complex . The dif3,4,5P3 or PIP3 in short), phosphatidylinositol-3,4-bisphosphate  as well as phosphatidylinositol-4,5-bisphosphate  [3,4,5P3, which is only found within the plasma membrane, and less to PI3,4P2 and PI4,5P2 [via the EGF-, VEGF- or B cell antigen receptors (BCR) [Several PH domains are able to recognize specific phosphoinositides such as phosphatidylinositol-3,4,5-trisphosphate  ,56. Inte PI4,5P2 -25,40,57rs (BCR) ,26,27,58rs (BCR) ,25. In trs (BCR)  and is rrs (BCR) . In concet al. (2008) reiterates the importance of the PH domain by showing that it is required for the EGF-triggered recruitment of Gab1 to the plasma membrane in the presence of low doses of EGF, while the recruitment of this docking protein by high doses of EGF relies on Grb2 [The PH domain mediated recruitment of Gab proteins provides the opportunity to modulate their membrane residency by PI3K and lipid phosphatases in a very dynamic manner without the need to disrupt large signalosomes such as the EGFR/Grb2/Gab complex that are potentially stabilised by various direct and indirect protein-protein interactions. A recent study by Sampaio  on Grb2 . The dep on Grb2 .via the Grb2 binding sites, as indicated by various lines of evidence. Firstly, the tyrosine phosphorylation of Gab1 is drastically reduced in mouse embryonic fibroblasts (MEFs) lacking Grb2 or expressing a functionally impaired Grb2 protein in which its SH2 domain has been rendered non-functional by the E89K knock-in mutation [knock-in mice that express a Gab1 mutant lacking the Grb2 binding sites display an embryonic lethal phenotype and defects in liver, placenta and craniofacial development [3 hydrolysing phosphatases such as SHIP and PTEN [If the PH domain were to play such an important role under low growth factor stimulation, one would expect that the membrane recruitment mechanisms reliant on protein-protein interactions such as the c-MET/Gab1 and Grb2/Gab interactions would be largely dispensable. In the following, we review several lines of evidence from various experimental settings indicating that the PH domain alone cannot confer long-term plasma-membrane residency or ensure adequate physiological Gab signalling. For example, the MBD plays an important role in Gab1 recruitment under certain circumstances . A stronmutation . The oppmutation ,59,61,62mutation . Most imelopment . This fielopment ,40. Thisand PTEN ,65. This3 levels due to the loss of PTEN expression or PIK3CA mutations, respectively [et al. showed that the EPO-induced tyrosine phosphorylation of Gab2 is much more reliant on PI3K activity than that of Gab1 [Overall, it appears that the relative roles played by these alternative recruitment mechanisms are context-dependent. The reports reviewed in this section invite for detailed future studies that not only take the amount and timing of the extra-cellular stimulus into account, but also consider the lineage and transformation status of the cell lines. Indeed, the Gab/Grb2 interaction might be more relevant in primary tissues or immortalized cell lines such as BMMCs and MCF-10A, than in particular tumour cell lines often used in signalling studies, e.g. Jurkat or MCF-7 cells, which display elevated PIPectively -68. Last of Gab1 .bona fide signal transducers, Gab proteins not only possess structural motifs for their receptor recruitment, but also contain features that are involved in the transduction, localization and amplification of receptor-derived signals . These mice display impaired development of muscles and the placenta [Probably the best-characterized effector arm of Gab proteins is mediated e domain ,97. Manye domain ,98. Thuse domain ,98-101. e domain  as well e domain ,103. Thee domain ,104, cele domain  and acine domain . These s elegans ,105. Recplacenta , an orgaplacenta .Ras-GAP on the activated receptors for PDGF and EGF [Ras-GAP is recruited via its SH2 domain to phosphorylated Y317 on Gab1, which is dephosphorylated by SHP2. Like Gab1, DOS is also de-phosphorylated by the SHP2 orthologue Corkscrew (CSW) resulting in enhanced Ras activation [Taken together, these findings demonstrate that SHP2 is an important positive modulator of ERK activation. However, why the recruitment of SHP2 by Gab proteins is required for full activation of ERK signalling downstream of receptor tyrosine kinases is still not fully understood, but several mechanisms may contribute. Firstly, SHP2 de-phosphorylates binding sites for p120 and EGF ,108 and  and EGF  and thertivation . Secondltivation  and paxitivation , leadingtivation .knock-in mice expressing a Gab1 protein defective in p85 recruitment (Gab1Δp85) demonstrated that the interaction between Gab1 and PI3K downstream of the EGFR is important in embryonic development for eyelid closure and for keratinocyte migration [knock-out is embryonic lethal [via direct p85 binding sites and via Gab proteins, for example c-Kit and the NGF receptor [via co-receptors and via Gab2 [via a Shc/Grb2/Gab2 complex and other cytokine receptors lacking direct PI3K binding sites might use the same pathway [et al. (2000) [Through the recruitment of PI3K to activated receptors, Gab proteins contribute to the initiation of signalling pathways promoting cellular growth, survival, migration and proliferation . The genigration . Neverthc lethal ,111, Gabc lethal -114. Howc lethal . The rolreceptor ,115. A rreceptor . Similarvia Gab2 ,117. Thu pathway . Lastly,. (2000) .et al. (2006) found that PLCγ2 not only interacts with Gab2, but also enhances its interaction with the receptor RANK and its tyrosine phosphorylation, suggesting that PLCγ2 plays a scaffolding or recruitment role in the RANK/Gab2 relationship [Association of Gab proteins and PLCγ 1/2 most likely reflects a direct interaction involving tyrosine-phosphorylated residues on the docking protein and the SH2 domains of the PLCγ isoform. For example, HGF induces tyrosine phosphorylation of Gab1 at Y307, Y373 and Y407, which in turn recruit PLCγ1, a critical event for MET-induced branching morphogenesis of MDCK cells . Gab2 altionship . The phytionship , indicatvia Grb2. The latter mechanism has been demonstrated for Gab2 signalling complexes from EGF-stimulated mammary epithelial cells and from Fcε RI- or stem cell factor (SCF)-stimulated mast cells [et al. (2001) identified Shc in Gab2 complexes from M-CSF stimulated cells, but failed to purify Gab2 using GST fusion proteins bearing either the SH2 or PTB domain of Shc [via Shc [Another prominent component of immuno-purified Gab signalling complexes are the Shc adaptor proteins. In many cases, however, it is still unclear as to whether Shc interacts directly with Gab proteins or is recruited st cells ,49,102. n of Shc . These dn of Shc  predictsn of Shc . Consequn of Shc  or the Sn of Shc ,125? Indn of Shc ,125. Simn of Shc , a humann of Shc  and in Mn of Shc . Althougn of Shc , several[via Shc ,125. Thevia their SH3 domains e.g. guanine nucleotide exchange factors for Rac and Rap-GTPases. Thereby, they potentially regulate cellular motility, adhesion and morphology [et al. have recently demonstrated that HGF/c-MET signalling in human synovial sarcoma cell lines induces sustained tyrosine phosphorylation of Y307 on Gab1, which serves as a recruitment site for both Crk and PLCγ [Listeria monocytogenes, implicating the Gab1/Crk complex in promotion of cytoskeletal rearrangements required for pathogen internalization [Crk proteins constitute another group of Gab interaction partners. These adaptor proteins consist of one N-terminal SH2 domain followed by one (CrkI) or two (CrkII) SH3 domains ,128. As rphology ,129. Intand PLCγ . The recand PLCγ . Furtherand PLCγ ,131,132.lization .et al. (2003) isolated a novel GTPase activating protein (GAP) for Rho-GTPases, which was named GC-GAP [in vitro GTPase stimulating activity towards RhoA, Rac1 and Cdc42 and towards Rac1 and Cdc42 upon ectopic expression in HEK293T cells. RNAi-mediated suppression of GC-GAP was correlated with reduced proliferation of C6 astroglioma cells. Although the original identification of the Gab2/GC-GAP interaction in the Y2H screen suggests a direct interaction between both proteins, it should be noted that GC-GAP also interacts in mammalian cells with the N-terminal SH3 domain of Crk [In a Y2H screen conducted with a portion of Gab2 (aa 120-587) as a bait, Zhao d GC-GAP . This inn of Crk .et al. (2009) has identified the Ser/Thr-kinase and Rac/Cdc42 effector PAK4 as a specific interaction partner of the Gab1 isoform [via its GEF-interacting domain to a region in Gab1 located between the PH domain and the first of the three Crk binding sites (aa 116-234). Interestingly, the authors could show that ectopically co-expressed Gab1 and PAK4 cooperate in HGF-induced epithelial cell scattering and invasiveness and that PAK4 knockdown or deletion of the PAK4 recruitment region impaired these biological responses.A recent study by Paliouras  isoform . PAK4 bimycosis fungoides, tyrosine-phosphorylated Gab2 interacted with SHP2 and STAT5a in a IL-2- regulated fashion [et al. (2004) uncovered a complex interplay between Gab2, SHP2 and STAT5 in IL-2 stimulated T cells [via the phosphorylated tyrosine residues Y614 and Y643. Interestingly, activation of the ERK pathway was blunted, as expected by other studies, by the Y614F mutation and slightly increased by the Gab2S623A mutant. In contrast, IL-2-induced STAT5 activation was enhanced by the SHP2 binding mutant Gab2Y614F and inhibited by Gab2S623A. These data indicate a potential role of STAT5, its interaction partners or its upstream kinases as SHP2 substrates.Although the molecular details remain ill-defined, the Gab2 isoform is increasingly implicated in JAK/STAT signalling. An early study demonstrated that in CD4-positive T cells derived from the rare human neoplasia  fashion . Later,  T cells . Here, Sgab2 gene is one of the top candidates on the modifier locus located on chromosome 7 that modulates the engraftment of hematopoietic stem cells (HSC) during steady-state haematopoiesis, a process dependent on intact cytokine signalling [via the Ras/ERK and PI-3K/AKT pathways [via a direct interaction or via a mutual binding partner such as p85. Clearly, further work is required to characterize mechanisms underpinning the interplay between Gab2 and STAT5, and to determine how STAT5 antagonizes Shp2 recruitment to this docking protein.Additional observations support the concept of a functional cooperation between STAT5 and Gab2. First, the murine gnalling . Second,pathways ,135. In pathways . This suet al. (2007) have demonstrated that murine and human Gab2 orthologues, but not Gab1, contains a canonical STAT3 binding motif (YXXQ). Using a Y194F substitution mutant, the authors could demonstrate that this site is indeed required for the recruitment of STAT3 and the efficient Friend erythroleukemia virus-mediated transformation of murine hematopoietic progenitors [In addition to the STAT5/Gab2 relationship, Ni genitors . It remaMonosiga brevicollis, which represents an outgroup to metazoans, and the basal metazoan Trichoplax adhaerens and the starlet sea anemone Nematostella vectensis. Whilst there is no evidence for bona fide Gab/DOS proteins in either the Monosiga nor Trichoplax genomes (our own observations), we and the Feller group identified a Gab-like protein in the Nematostella genome . However, it should be noted that, whilst there are also tyrosine residues in NeGab that align with those involved in the recruitment of SHP2 to mammalian Gab proteins, the surrounding residues are not conserved and therefore do not constitute a bona fide recognition motif for both SH2 domains of mammalian SHP2 [Recent genome analyses have revealed that the emergence of PTK signalling networks precedes the advent of true multi-cellularity and that these expand dramatically at the base of the animal kingdom Fig. 1636529; ). This c636529; .1636529; . It shou1636529; . Importatostella  and dataian SHP2 ). These ian SHP2 , the SH2Ciona and Branchiostoma possess only one Gab-like protein [GAB4 gene, for which entries at both the genomic DNA and transcript level have been recorded for humans and chimpanzees. The human GAB4 gene is located on chromosome 22q11.1 and its nucleotide sequence is most related to Gab2  could show that both Gab1 and Gab2 play an important role in the postnatal maintenance of cardiac function [Using cardiomyocyte-specific Gab1/Gab2 double-deficient mice, Nakaoka function . Neureguknock-out.Gab2 plays an important albeit not essential role in the development of various haematopoietic lineages , except Gab2-deficient mice also display an osteopetrotic phenotype that is explained by the role of Gab2 as a key regulator of RANK signalling . Bone hoet al. (1999) reported that ectopic expression of Gab2 in PC12 cells increased NGF-independent neuronal differentiation and survival via PI3K- and MEK-dependent pathways [All three mammalian Gab proteins are expressed in neuronal tissues ,29,155, So far, studies have not identified any specific roles for Gab3. Genetically-modified mice deficient for Gab3 are healthy and viable and despite the strong up-regulation of this protein during macrophage development, no obvious phenotype was identified in Gab3-deficient macrophages .Gab proteins fulfill critical roles in the communication between various receptor classes and several signalling pathways involved in the control of proliferation, cell death, migration and differentiation. Consequently, their expression, subcellular localisation and signalling competent state must be strictly regulated. Although our knowledge about these processes is still very limited, it is becoming apparent that several layers of negative regulation are applied to Gab docking proteins, which we will now discuss.Firstly, as the PH domain plays an important role in membrane recruitment, Gab signalling is influenced by the balance between the activities of PI3K and lipid phosphatases such as PTEN or SHIP1/2. As discussed above, the latter proteins are recruited into Gab signalosomes . SimilarSecondly, tyrosine-phosphorylated Gab docking proteins recruit SHP2 and it is therefore highly likely that the phosphorylation of certain tyrosine residues and their associated downstream signalling events are directly regulated by this protein tyrosine phosphatase (PTP). Indeed, studies on both DOS and Gab1 have shown that they are dephosphorylated by CSW and SHP2, respectively ,21,159. GAB gene promoters remains poorly-characterized, one study has shown that transcription of the GAB2 gene is induced by the transcription factor E2F [A third regulatory layer is the control of the expression level of Gab proteins by various mechanisms acting at the transcriptional and post-transcriptional levels. Although the regulation of ctor E2F . Furtherctor E2F  and studctor E2F ,145,164.Another study has demonstrated that Gab2 is subject to ubiquitin-mediated degradation in FcεRI-stimulated RBL-2H3 basophilic leukaemia cells . HoweverA fourth and emerging mode of negative regulation of docking proteins is mediated by Ser/Thr-phosphorylation, which is often correlated with their reduced tyrosine phosphorylation and/or changes in subcellular localisation  identified S623 as the site of action of this ERK-mediated feedback loop and provided evidence that the Gab2/SHP2 interaction is enhanced by a S623A mutation.Gab2 is also regulated by ERK-mediated negative feedback phosphorylation, as identified by Arnaud phocytes . Previouphocytes ,180, howTaken together, a series of studies over the last decade has shown that the tyrosine phosphorylation and signalling potential of docking proteins such as those of Gab, IRS, FRS and SLP families is counteracted by their Ser/Thr-phosphorylation, which usually represents the endpoint of feedback loops from cytoplasmic signalling cascades ,177,181.cis/trans peptidyl-prolyl-isomerisation  and the neuro-cardio-facious cutaneous syndromes (NCFC) are human pathologies caused by aberrant Ras/ERK signalling. The NCFC syndromes comprise neurofibromatosis (NF) and the Noonan (NS), Costello (CS), LEOPARD (LS) and cardio-facious-cutaneous (CFC) syndromes, which are correlated with autosomal-dominant germ-line mutations within either the core components  or modulators of the Ras/ERK pathway . The resulting mutant proteins display aberrant activities and consequently disturb the overall fine-tuning of the Ras/ERK pathway and to a certain degree the Ras/PI3K pathway ,193. As reported -199. Thereported . Howeverproteins ,201. Nevrylation .wt. Taken together, these studies identify Gab proteins as important "accomplices" of NCFC-associated SHP2 mutants and suggest that a better knowledge of Gab signalling will contribute to an improved understanding and treatment of these syndromes. Furthermore, the close relationship between SHP2 and Gab proteins and the important role of Gab proteins as modulators of Ras signalling also raise the question as to whether the Gab genes themselves are awaiting their identification as novel "NCFC" alleles.While NS patients carry mostly gain-of-function mutations in SHP2, this phosphatase often contains dominant-negative mutations in LS patients ,203. IntMET amplification in lung cancer cells, which have become resistant towards the EGFR inhibitor gefitinib [GAB1 gene in human breast and lung cancers, albeit at very low frequencies . HoweveGAB2 locus on 11q13-14 [GAB2 promoter, and aberrant estrogen receptor (ER) signalling [et al. applied a bi-cistronic retroviral expression system to adjust the Gab2 expression in MCF-10A cells to levels observed in human breast cancer cell lines and analysed the intracellular signalling events in these cells [In contrast to Gab1, Gab2 is developing a strong track record as an oncoprotein in its own right in various solid tumours. Firstly, Gab2 is frequently over-expressed in human breast cancer cell lines and primary tumours and becomes tyrosine phosphorylated in these cells in response to EGF, insulin and bFGF stimulation . This in11q13-14 , a regio11q13-14 ,206, whignalling . Indeed,gnalling . These ognalling  and genegnalling . In the se cells . In monose cells . Very sise cells . The effse cells . Importase cells .GAB2 gene has been also recently reported for ovarian [et al. have shown that, similar to breast cancers and other neoplasias, the GAB2 gene is amplified and/or over-expressed in 11% and 50% of human metastatic melanomas, respectively [et al. (2009) demonstrated that GAB2 amplification is associated with melanoma arising from sun-protected sites and often occurs independently from oncogenic NRAS or BRAF mutations or amplification of the KIT gene [Amplification and/or over-expression of the human  ovarian  and gast ovarian  and acut ovarian , althoug ovarian ,212. Firectively . MoreoveKIT gene . In contKIT gene .wt, but not Gab2ΔSHP2, with the RTK Neu  resulted in an invasive growth phenotype of MCF-10A cells in 3D culture [We have previously reported that MCF-10A cells expressing very high levels of Gab2 generate large disorganized structures in 3D culture with defective luminal clearance , a pheno culture . Importaet al. [via increased E2F activity. However, Ke et al. reported that loss of Gab2 in mice had only a modest effect on the initiation and growth rate of mammary tumours induced by a constitutively active neu transgene (Neu2-5) or a signalling-compromised version, NeuYD, which can only recruit Shc proteins [knock-out strategies, with one strain expressing low amounts of a N-terminally truncated Gab2 protein [neu transgene in Drosophila [The studies of Bentires-Alj et al.  were comet al. . Here, tproteins . There aproteins . Firstly protein . Secondlosophila .et al. (2008) investigated the biological consequences of the co-expression of Gab2 and Src proteins in the aforementioned MCF-10A model [Δp85, significantly enhanced acinar disruption induced by the hyper-active v-Src and c-SrcY527F mutants [The cooperation of Gab2 with oncoproteins in solid tumours is not restricted to oncogenic RTKs such as ErbB2 and v-Sea. For example, the non-receptor tyrosine kinase c-Src is often aberrantly expressed or activated in human breast cancers, sometimes as a consequence of dys-regulated ErbB2 activity ,219. As 0A model . This st mutants . This phBRAFV600E oncogene in melanoma. The V600E mutation is a very frequent and early-arising event in the nevi-melanoma progression series but, by itself, induces only a transient enhancement of proliferation followed by cell cycle arrest with hallmarks of cellular senescence [BRAFV600E cooperates with the loss of PTEN in the induction of metastatic melanomas [BRAFV600E requires increased levels of PI3K activity to drive malignant melanomas. Thus, Gab2 might even cooperate with oncogenes that are not directly associated with the Gab2 signalosome.Lastly, as Gab2 is an important amplifier of PI3K signalling, it is tempting to speculate that Gab2 overexpression might cooperate with the nescence . Indeed,elanomas , which uIn summary, a series of studies conducted in various experimental settings have now demonstrated that Gab2 is not only an important interaction partner of oncoproteins involved in the transformation of hematopoietic cells, but also of those playing a well-described role in solid tumours. It appears likely that more co-operating oncogenes for Gab2 in solid tumours will be found in the not too distant future. One of these candidates might be again SHP2, which is mutated at low frequency in AML and several solid cancer types and commonly overexpressed in breast cancer, where it is involved in regulating epithelial/mesenchymal transition -224. StuHelicobacter pylori is translocated into gastric epithelium cells of the host where it interacts with Grb2, becomes tyrosine phosphorylated and recruits effectors such as SHP2 and Crk to enhance Ras/ERK signalling and cellular transformation [via molecular mimicry of these eukaryotic docking proteins. Given the association between CagA and development of gastric carcinoma [Helicobacter infection and that the gastric epithelium is susceptible towards transformation by aberrant SHP2 activity. Interestingly, this concept of molecular mimicry is now strongly supported by recent experiments in Drosophila demonstrating that a cagA transgene can rescue larval viability and photoreceptor development in mutant animals lacking DOS [The CagA protein of the gastric pathogen ormation -228. Howarcinoma , an intearcinoma  triggersking DOS . Furtheret al. (2007) identified certain GAB2 alleles as modifiers of disease susceptibility in carriers of the APOEε4 allele that is strongly associated with late-onset AD [et al. (2007) could demonstrate that siRNA-mediated reduction of Gab2 expression in neuroglioma cells results in increased Tau protein phosphorylation at Ser 262, a residue, which is hyper-phosphorylated in AD and has been implicated in neurofibrillary tangle formation. As this residue is targeted by GSK-3 and this kinase is inhibited by AKT-mediated phosphorylation [In addition to the various neoplastic diseases, Gab2 is also increasingly implicated in Alzheimer's disease (AD). Reiman onset AD . Althougonset AD -233, theonset AD ,235 and onset AD ,237. It rylation , this fivia phosphorylation events. They orchestrate multiple protein-protein and -lipid interactions and also act as allosteric activators. The diversity of Gab interaction partners also implies that there are distinct types of Gab signalosomes present in the cell, which differ in their subcellular localisation and function. Thus, more refined biochemical approaches will be required to characterize the composition and stoichiometry of the different Gab signalosomes. It is also becoming evident that Gab proteins mediate the crosstalk between various signalling pathways and thereby provide the basis for the synergistic action of various receptors [in vivo situation as the cells in our bodies are simultaneously exposed to a plethora of biologically-active ligands. However, in order to fully understand the signalling roles of Gab proteins, it is clear that various disciplines will need to cooperate and utilize a systems-based approach that integrates structural and biophysical studies on regulation of protein-protein interactions, mathematical and computational modelling of the Gab signalling network and functional analyses that exploit the genetics of appropriate model organisms. Such an endeavour is likely to provide exciting new insights into the mechanisms and functions of Gab signalosomes.Since their discovery, Gab docking proteins have emerged as critical players in many physiological processes as well as pathologies such as cancer and inflammatory diseases. It is becoming more and more evident that their versatile roles in signal transduction extend beyond the original and relatively static definition of a docking protein. We are starting to appreciate that docking proteins play a central role in the management of entire signalling subsystems and that they are, at the same time, subject to complex spatiotemporal control by the same network, e.g. eceptors ,204,239,3: Inositoltrisphosphate; IRS: Insulin receptor substrate; JAK: Janus kinase/Just another kinase; JMML: Juvenile myelomonocytic leukemia; kDa: Kilodalton; KSR: Kinase suppressor of ras; LAT: Linker of activated T cells; LPA: Lysophosphatidic acid; LS: LEOPARD syndrome ; MAPK: Mitogen activated protein kinase; MBD: Met binding domain; MEFs: Mouse embryonic fibroblasts; MEK: Mitogen activated protein/extracellular signal regulated kinase kinase; MONA: Monocytic adaptor; NCFC: Neuro-cardio-facious-cutaneous syndromes; NF: Neurofibromatosis; NF-κB: Nuclear Factor kappa B; NGF: Nerve growth factor; NK: Natural killer; NRG: Neuregulin; NS: Noonan syndrome; PAG: Phosphoprotein associated with glycosphingolipid-enriched microdomains; PAK: p21-activated kinase; PDGF: Platelet-derived growth factor; PH: Pleckstrin homology; PI3K: Phosphatidyl-inositol-3 kinase; PI3KCA: gene encoding the catalytic subunit of phosphatidyl-inositol-3 kinase; PIP: Phosphatidyl-inositol-phosphate; PKB: Protein kinase B; PLC: Phospholipase; PTB: Phospho-tyrosine binding; PTEN: Phosphatase and Tensin homolog; PTK: Protein tyrosine kinase; PTP: Protein tyrosine phosphatase; PTPN: Protein tyrosine phosphatase: non-receptor; Raf: Rapidly growing fibrosarcoma; RANK: Receptor Activator of NF-κB; Ras: Rat sarcoma; RNA: Ribonucleic acid; RNAi: RNA interference; ROK: Rho kinase; RTK: Receptor tyrosine kinase; SCF: Stem cell factor; Sea: S13 erythroblastosis oncogene homolog; siRNA: Small interfering RNA; SH2: Src homology 2; SH3: Src homology 3; SHIP: SH2-containing inositol 5-phosphatase; SHP: SH2 domain-containing protein-tyrosine phosphatase; SLP: SH2 domain containing leukocyte protein; SOC: Suppressor of clear; Src: Sarcoma viral oncogene homolog; STAT: Signal transducer and activator of transcription; Syk: Spleen tyrosine kinase; ZAP-70: Zeta-chain associated protein of 70 kDa.Aa: Amino acid; AD: Alzheimer's disease; AML: Acute myeloid leukemia; BCR: B cell antigen receptor; Bcr: Breakpoint cluster region; bFGF: Basic fibroblast growth factor; BMMCs: Bone marrow derived mast cells; BTAM: Bi-phosphoryl tyrosine activation motif; BTK: Bruton's tyrosine kinase; CBP: Csk-binding protein; CD: Cluster of differentiation; CFC: Cardio-facious-cutaneous syndromes; CML: Chronic myeloid leukemia; CNS: Central nervous system; Crk: Sarcoma virus CT10 oncogene homolog; CS: Costello syndrome; CSF: Colony stimulating factor; CSW: Corkscrew; DAG: Diacylglycerol; DNA: Deoxyribonucleic acid; DOS: Daughter of sevenless; EGF: Epidermal growth factor; ERK: Extracellular signal regulated kinase; EST: Expressed sequence tag; EPO: Erythropoietin; FGF: Fibroblast growth factor; FRS: Fibroblast growth factor receptor substrate; Gab: Grb2-associated binder; GADS: Grb2-related adaptor downstream of Shc; GAP: GTPase activating protein; Grb2: Growth factor receptor-bound protein 2; Gsk: Glycogen synthase kinase; GST: Glutathione S-transferase; GTP: Guanine nucleotide trisphosphate; HER: Human epidermal growth factor receptor; HGF: Hepatocyte growth factor; IL: Interleukin; IPThe authors declare that they have no competing interests.FUW, RJD and TB surveyed and selected the literature for this review. FUW prepared the figures and tables with input from her co-authors. All three authors were involved in writing the manuscript, which was compiled by TB All three authors read and approved the final manuscript.


The dopamine transporter (DAT) plays a critical role in terminating the action of dopamine by rapid reuptake into the presynaptic neuron. Previous studies have revealed that the DAT carboxyl terminus (DAT-CT) can directly interact with other cellular proteins and regulate DAT function and trafficking.Here, we have identified that carboxypeptidase E (CPE), a prohormone processing exopeptidase and sorting receptor for the regulated secretory pathway, interacts with the DAT-CT and affects DAT function. Mammalian cell lines coexpressing CPE and DAT exhibited increased DAT-mediated dopamine uptake activity compared to cells expressing DAT alone. Moreover, coexpression of an interfering DAT-CT minigene inhibited the effects of CPE on DAT. Functional changes caused by CPE could be attributed to enhanced DAT expression and subsequent increase in DAT cell surface localization, due to decreased DAT degradation. In addition, CPE association could reduce the phosphorylation state of DAT on serine residues, potentially leading to reduced internalization, thus stabilizing plasmalemmal DAT localization.Taken together, our results reveal a novel role for CPE in the regulation of DAT trafficking and DAT-mediated DA uptake, which may provide a novel target in the treatment of dopamine-governed diseases such as drug addiction and obesity. The dopamine transporter (DAT) is a presynaptic plasma protein found on dopaminergic nerve terminals that terminates dopamine signaling by rapidly sequestering dopamine released into the synaptic cleft ,2. DAT bMorphological studies reveal that functional DATs are localized on the plasma membrane of axons, pre-synaptic axon terminals, and dendrites of dopaminergic neurons, with the highest expression in the axonal processes in striatum and dendrites in ventral tegmental area -10. SpecThe increasing reports of proteins that interact with DAT ,19-26 anPCR was used to generate CPE-activating domain (AD) fusion constructs in either pACT2 or pGADT7 and binding domain(BD) fusion constructs with the DAT intracellular amino-terminal (NT: Met1-Lys66), carboxyl-terminal , and carboxyl-terminal fragments  in pAS2 or pGBKT7. Appropriate pairs of AD and BD constructs were cotransformed into the yeast strain Y187 grown at 30°C for 3 days on Leu-/Trp- medium lacking leucine and tryptophan, followed by assay of lacZ reporter activation, present downstream of a GAL4 binding sequence in Y187, as described by the manufacturer (Clontech). All control transformations were negative for β-galactosidase activity except for BD/p53 with AD/T antigen, used as a positive control. All constructs were sequenced to determine appropriate splice fusion and absence of spurious PCR-generated mutations.Coimmunoprecipitation, affinity pull-down, and Western blot analyses were performed as previously described -29. Rat ® T7 Quick Coupled Transcription/Translation System  and [35S] methionine . Glutathione beads carrying 20 μg GST-DAT-NT, GST-DAT-CT or GST alone were incubated at room temperature for 1 hour with [35S] methionine-labeled CPE probe, respectively. The beads were then washed six times with PBS containing 0.1–0.5% (V/V) Triton X-100 and eluted with 10 mM glutathione elution buffer. Eluates were separated by SDS-PAGE and visualized by autoradiography using BioMax (Kodak) film.A probe was made with CPE subcloned into mammalian expression vector pCDNA3 using TNT5 cells seeded per well), medium was removed and wells were rinsed with 0.5 ml of uptake buffer . Cells were then preincubated in duplicate with the indicated concentrations of dopaminergic agents (10-13 to 10-4M) 5 min before the addition of 0.25 ml of 20 nM [3H]dopamine  and incubated for 10 min at room temperature in a total volume of 0.5 ml. Nonspecific [3H] dopamine (37–53 Ci/mmol) uptake was defined in the presence of 10 μM GBR-12909. Wells were rinsed twice with 0.5 ml of uptake buffer; cells were solubilized in 0.5 ml of 1%SDS and collected to measure incorporated radioactivity using a Beckman liquid scintillation counter (LS 6000SC). For [3H] WIN binding, the differences from uptake are using [3H] WIN  and incubating for 2 to 3 h at 4°C before adding 1% SDS.We measured dopamine uptake on intact cells as described previously . Two to 6 cells), distributed equally to two six-well plates (35 mm/well), and grown for 2–4 d. The cotransfected cells were fixed in 2% paraformaldehyde for 10 min in the absence (nonpermeabilized conditions) or the presence (permeabilized conditions) of 1% TritonX-100. Cells were incubated with anti-DAT (Chemicon) for the purpose of labeling the receptors on the cell surface under nonpermeabilized conditions or the entire receptor pool under permeabilized conditions. After incubation with the corresponding horseradish peroxidase (HRP)-conjugated secondary antibodies (Sigma), the HRP substrate o-phenylenediamine  was added to produce a color reaction that was stopped with 3 N HCl. The cell surface and total expression of DAT was presented as the colorimetric readings under nonpermeabilized and permeabilized conditions and then normalized to the control groups  respectively.Cell-ELISA assays (colorimetric assays) were done essentially as previously described ,29. HEK-HEK 293 T cells were transiently transfected with DAT or together with CPE, grown to near-confluency over 12 hr, pre-incubated in extracellular solution , for 1 h at 37°C, and labeled in fresh medium containing 0.2 mCi/ml promix [35S] methionine/cysteine (Amersham Biosciences). After incubation for a further 4 h at 37°C, the pulse was terminated by washing cells twice and incubating with MEM (supplemented with 10% FBS) for the indicated times. Immunoprecipitation of DAT was carried out.After tranfection (as above) for 2–4 d, HEK-293 cells were used for double-label immunoconfocal microscopy. CPE mouse Abs (Pharmacia) and DAT Rabbit Abs (Santa Cruz) were used as primary antibodies in overnight incubations at 4°C and detected using secondary antibodies (Sigma) conjugated to FITC or Cy3 fluorophores. Control cells in which no primary Ab was included did not reveal any immunoreactive staining.DAT CT cDNA-encoding fragment was amplified by PCR from full-length cDNA clones . DAT CT Acute hippocampal slices (350 μm) were prepared from Sprague-Dawley rats using a McIlwain tissue chopper . Under deep anesthesia, brains were rapidly removed and striatum was dissected out and leave for 5 mins in ice-cold artificial cerebrospinal fluid (ACSF) containing (mM) NaCl, 126; KCl, 2.5; MgCl2, 1; CaCl2, 1; KH2PO4, 1.25; NaHCO3, 26; and glucose, 20 that was bubbled continuously with carbogen (95%O2/5%CO2) to adjust the pH to 7.4. Freshly cut slices were placed in an incubating chamber with carbogenated ACSF and recovered from stress at 37°C for 1 hr. Slices were then treated with/without PMA for 30 mins and harvested for Co-immunoprecipitation and Western blotting analysis.To search for DAT-interacting proteins, we employed the entire DAT-CT as bait to screen a human substantia nigra library using the yeast two-hybrid system Fig . InteracIn vitro binding assays were performed to provide evidence that DAT and CPE can directly interact with each other. As shown in Figure in vitro translated [35S]-CPE probe hybridized with GST-DAT-CT but not GST-DAT-NT or GST alone, indicating the specificity of the direct protein-protein interaction between CPE and DAT-CT. Taken together, these data suggest that CPE is involved in a direct protein-protein interaction with DAT-CT.To further confirm the interaction between DAT and CPE, we performed GST affinity pull down assay using purified GST fusion peptides containing sequences from DAT-CT (GST-DAT-CT). The results shown in Fig. Next, we examined in solubilized rat striatum, whether DAT could coimmunopreicipiate with CPE thus providing additional evidence of a physical association between DAT and CPE. As illustrated in Fig. 3H] DA by ~50% , whereas no significant change in the estimated Km of the DAT could be observed (DAT: Km = 6.6 ± 0.7 μM in cells expressing DAT vs 6.7 ± 1.6 μM in cells expressing DAT and CPE). To further exclude the possibility that Vmax differences might result from the affinity changes, we performed [3H] WIN 35,428 competitive binding assay. As depicted in Fig. IC50 = 986 ± 24 nM in cells expressing DAT vs 1001 ± 87 nM in cells expressing DAT and CPE). The effect of CPE on DAT was not restricted to HEK-293 cells but was also seen in COS-7 and Ltk- cells (data not show).DAT plays a determinant role in dopaminergic neurotransmission by rapidly removing released dopamine from the synaptic cleft. To explore the functional importance of the DAT-CPE interaction, we examined the DAT-mediated DA uptake activity in HEK-293 cells coexpressing CPE. As illustrated in Fig 3H] DA uptake. However, coexpression of the MG encoding the peptide DAT-CT583–620 significantly inhibited the increasing effects of DAT-mediated DA translocation by CPE. Taken together, these data indicated that the binding of DAT-CT tail583–620 sequences to CPE was both sufficient and necessary for the expression of CPE- mediated functional regulation of the DA translocation process.Next, to confirm that the selective increase in maximal DAT-mediated DA uptake resulted from DAT-CPE interaction, we tested whether a minigene (MG) encoding sequences of the DAT-CT could block these effects, since coexpressioin of CT tail peptide would act as a competitive inhibitor to the CPE-DAT interaction. As described in Fig. Since no change in DA uptake affinity could be observed upon coexpression of CPE with DAT, the increase in Vmax may reflect a change in the population of cell surface DAT. Therefore, we next examined if DAT cell surface expression levels change upon coexpression of CPE by using a cell-based ELISA assay to quantify changes at the cell surface and total DAT levels -29. As sTo directly examine the cellular effects of CPE on DAT, we analyzed subcellular localization of DAT and CPE in transfected HEK-293 cells using confocal immunofluorescence microscopy. The results showed that DAT and CPE displayed disperse distribution patterns when expressed separately in HEK-293 cells. DAT could be found both at the cell surface and within cell, whereas CPE located throughout the cytoplasm Fig. . HoweverCell surface DAT may derive from newly synthesized DAT or from recycled DAT that have been re-inserted into the membrane after being internalized. Thus, changes in both cell surface and total DAT levels may reflect changes in DAT protein turnover rates in the presence of CPE. To test this possibility, we carried out pulse-chase studies to examine DAT protein half-life in the presence of CPE. As showed in Fig. Cell surface expression and degradation of DAT  is modulTo provide further evidence that the expression and functional affects on DAT were mediated by phosphorylation changes induced by the CPE-DAT interaction, we explored the relationship of DAT phosphorylation with DA translocation velocity and surface expression under phosphorylating conditions mentioned above. As depicted in Fig Recent studies have identified new properties of the DAT, including the regulation of cell excitability through leak current conductance and channel mechanism ,39, as wThe present finding highlighted the importance of CPE in trafficking and metabolic processing of DAT. DAT undergoes cyclical traffic to and from the plasma membrane and changes in transporter surface presentation can result from modulating endocytosis, recycling or de novo biosynthesis. Coexpression of CPE with DAT changed the diffuse DAT expression throughout the cell to stronger plasma membrane localization and suggests the interaction occur intracellularlly. CPE exists in both a soluble form and a membrane form ,42. SoluAlthough not in paired form, Arg/Lys residues are also present in the C-terminus of hDAT, rendering the possibility that CPE would be involved in the processing of DAT. Interestingly, CPE band did show a slightly lower on pulse-chase assay coexpressed with DAT, which deserved further investigation. Immunogold labeling detects DAT in tubulo-vesicular structures and small electron-lucent vesicles in dopaminergic neurons from the dorsal striatum and nucleus accumbens -10. AlsoDAT possesses consensus sequences for phosphorylation by protein kinases ,13,51,52The exact mechanism for CPE's effects on DAT phosphorylation and its relationship with cell surface DAT expression require further investigation. Foster and colleagues recently report that the amino terminus of DAT, which contains a cluster of serine residues, is the primary site of phosphorylation by PKC . It is pTaken together, data from our present study and previous reports indicate that protein-protein interactions appear to be important for DAT function. The association of DAT with CPE may have critical impact on DAT phosphorylation state and be involved in cyclic trafficking. While our findings shed new light on the trafficking mechanisms of DAT, several questions still remain to be addressed. It remains to be determined in which cellular compartment this interaction occurs and the details of mechanisms involved that may regulate the CPE-DAT interaction. Furthermore, both CPE and the DAT may have roles in obesity and it will be interesting to investigate if this interaction contributes to the pathophysiology of this disease. Further investigation into characterizing this interaction and understanding the regulatory factors involved, will lead to a better understanding of DAT trafficking and may have important physiological and pathophysiological implications.The authors declare that they have no competing interests.HZ carried out all experiments, with the exception of the in vitro binding, the uptake assay, WIN binding and the assays of the phosphorylation level of DAT. SL conducted the immunoassays of the phosphorylation level of DAT and contribute to the writing of the manuscript. MW participated in the in vitro binding and helped to draft the manuscript. BV participated in the cell culture and performed the statistical analysis. ZBP provided the cDNA of the dopamine transporter and conducted the dopamine uptake and WIN binding assays. FL conceived of the study, and participated in its design and wrote the manuscript. All authors read and approved the final manuscript.


Using western blot, we found that QA treatment increased the phosphorylation of tau at serine 199/202, threonine 231 and serine 396/404 in a dose dependent manner. Increased accumulation of phosphorylated tau was also confirmed by immunocytochemistry. This increase in tau phosphorylation was paralleled by a substantial decrease in the total protein phosphatase activity. A substantial decrease in PP2A expression and modest decrease in PP1 expression were observed in neuronal cultures treated with QA. These data clearly demonstrate that QA can induce tau phosphorylation at residues present in the PHF in the AD brain. To induce tau phosphorylation, QA appears to act through NMDA receptor activation similar to other agonists, glutamate and NMDA. The QA effect was abrogated by the NMDA receptor antagonist memantine. Using PCR arrays, we found that QA significantly induces 10 genes in human neurons all known to be associated with AD pathology. Of these 10 genes, 6 belong to pathways involved in tau phosphorylation and 4 of them in neuroprotection. Altogether these results indicate a likely role of QA in the AD pathology through promotion of tau phosphorylation. Understanding the mechanism of the neurotoxic effects of QA is essential in developing novel therapeutic strategies for AD.Some of the tryptophan catabolites produced through the kynurenine pathway (KP), and more particularly the excitotoxin quinolinic acid (QA), are likely to play a role in the pathogenesis of Alzheimer's disease (AD). We have previously shown that the KP is over activated in AD brain and that QA accumulates in amyloid plaques and within dystrophic neurons. We hypothesized that QA in pathophysiological concentrations affects tau phosphorylation. Using immunohistochemistry, we found that QA is co-localized with hyperphosphorylated tau (HPT) within cortical neurons in AD brain. We then investigated  NFTs are composed of paired helical filaments (PHF)In vitro, chronic exposure of rat corticostriatal cultures to nanomolar QA concentrations leads to ultrastructural changes in neuronsWe have previously shown that the KP is activated in AD brain, leading to an increase in QA productionin vitro treatment of human foetal neurons with pathophysiological concentrations of QA significantly increased tau phosphorylation at multiple phosphorylation sites. Our data also show a decrease in the expression and activity of serine/threonine protein phosphatase(s), as the mechanism for tau hyperphosphorylation. We also found that QA induces at least ten neuronal genes, with some of them known to be strongly associated with Alzheimer pathology. This study provides a new insight into the mechanism of QA neurotoxicity and its important role in the pathogenesis of AD. This work has opened a new avenue for research on AD, in particular the application of a novel therapeutic target.We found that QA accumulates in neurons and co-localizes with tau in cortical sections from AD patients. In addition, All neuronal cell culture media and additives were from Invitrogen  unless otherwise stated. DAPI and mouse monoclonal antibodies (mAb) for β-actin and all chemicals used for Western blots, immunoprecipitation, Phosphatase (PP) activity and immunoblots (unless otherwise mentioned) were obtained from Sigma-Aldrich Chemical Co. . IFN-γ was purchased from R&D Systems , pAb tau from DAKO, mAbs anti-tau 5 from Abcam, tau AT8 and tau AT180 from Pierce Endogen. mAb PHF-1  and mAb AT270 (Pierce Endogen) were generously provided by Prof Jurgen Goetz . Protein phosphatase antibodies PP1c and PP2Ac were kindly provided by Alistair Sim . Secondary anti-mouse IgG, anti-rabbit Alexa -conjugated antibodies (594 and 647) and streptavidin-Alexa (594 and 647) were purchased from Molecular Probes . Biotinylated anti-mouse and anti-rabbit antibodies were obtained from Vector Laboratories, Burlingame CA USA). All commercial antibodies were used within the concentration ranges recommended by the manufacturer.Human foetal tissue was obtained following informed written consent. This has been approved by the Human Ethics Committees from the University of New South Wales . Blocks of human brain tissue were obtained from the National Neural Tissue Resource Centre (NNTRC) and the Australian Brain Bank Network. This study had the approval of the Human Ethics committee of the University of Sydney (10-2005/4/8616).µg/ml streptomycin sulphate and 5 mM glucose. Cultures were kept at 37°C at 5% CO2 in a humidified atmosphere. The medium was changed once or twice per week. Neurons were maintained for up to 10 weeks.Human foetal brains were obtained from 16 to 19 week old foetuses (HREC 03187). Primary cultures of human neurons were prepared as previously described6 in Permanox chamber-slides (NUNC) for 2 weeks. Then, cells were fixed with acetone/methanol (vol/vol) for 20 min at –20°C. Cells were then rinsed 3 times with PBS and a gentle membranous permeabilization was performed by incubation with 0.025% Triton X 100 in PBS for 10 min at room temperature. After washing, cells were incubated with 5% normal goat serum (NGS) in PBS for 45 min at room temperature, rinsed twice with PBS and incubated for 1 h at 37°C with selected primary antibody Tau mAbs  diluted in 5% NGS. Cells were then washed with 5% NGS solution and incubated for 1 h at 37°C with the appropriate labelled secondary antibodies (goat anti-mouse IgG or goat anti-rabbit coupled with Alexa 594). Nuclear staining was performed using DAPI at 1 µg/ml for 5 min at room temperature. After several washings with PBS at 37°C, the cover slips were quickly mounted on glass slides with Fluoromount-G, and examined using an Olympus BX60 fluorescence microscope associated with a digital SensiCam. The following three controls were performed for each labelling experiment: 1) isotypic antibody controls for mAbs and serum control for pAbs, 2) incubation with only the secondary labelled antibodies, 3) estimation of auto-fluorescence of unlabelled cells.The method has been previously described2 Leica microtome, blocks were sectioned at 45 µm into 0.1 M Tris buffer (pH 7.4) containing 0.01% azide. Serial sections were labelled for QA  and tau . Free floating sections were washed three times for 15 minutes each in 50% ethanol then placed in blocking buffer  for 20 minutes, then incubated in 1° antibody for 24 hrs at 4°C. Sections were washed three times for 15 minutes each in 0.1 M Tris and incubated in species-specific biotinylated antibody for 1 hr, washed and placed in streptavidin-Alexa 647 or 594  diluted 1∶100 in 0.1 M Tris with 0.01% TritonX for 1 hr. For double-immunostaining, the sections were washed for one hour, incubated in blocking buffer, then transferred to the second primary antibody solution. For viewing, sections were mounted on gelatinized glass slides, cover-slipped in gelatine/glycerol and examined with a Zeiss Axioplan microscope. Optical sections were taken through the sections at 2 µm intervals and reconstructed using Zeiss Axiovision software and ImageJ .Increased QA expression was confirmed in AD tissue. Brain tissue was acquired through the NSW and Victorian Brain Bank Networks supported by the National Health and Medical Research Council. The study has the approval of the Human Ethics Committees at the University of Sydney. Two age matched controls and 8 AD cases with a range of neuropathological severity were examined. Blocks of tissue were taken from 3–5 mm formalin-fixed coronal slices of the medial temporal lobe, anterior cingulate and superior frontal cortex. On a CO6 cells per well in supplemented Neurobasal medium supplemented (as described above). Neurons were incubated with various concentrations of QA (ranging from 50 to 1200 nM) for 72 hours. Control cultures were kept in the medium alone. Okadaic acid (100 nM) was used as positive control for tau phosphorylation. In a separate experiment, neurons were cultured with 500 nM each of NMDA receptor agonists  and antagonists  for 72 hrs. Cells were lysed in 500 µl of RIFA buffer, . The cell lysate was centrifuged at 16000 g for 10 minutes. Protein concentration in the supernatant was determined by the modified Lowry method and stored at −20°C until used.For Western blots, cells were cultured in Matrigel coated 12-well plates at a density of 1×10http://rsb.info.nih.gov/ij/, 1997–2008).20 µg lysate protein was resolved on 10% SDS-PAGE  in sample buffer. Protein was transferred onto PVDF membranes; the membranes were blocked with 5% milk in TBS for one hour and incubated with primary antibody overnight at 4°C. The primary antibodies used are described in 2, 1 µM calmodulin and 3 mM MnCl2. The reaction mixture was incubated at 30°C for 30 min and the absorbance measured at 405 nm. PP2A activity was indirectly measured by using 10 nM OA in the phosphatase assay buffer and calculating the OA-inhibited activity.For phosphatase activity, cells were cultured as described above and lysed in the phosphatase activity buffer , 2 µg/ml aprotinin, 2 µg/ml pepstatin and 5 µg/ml leupeptin. Total phosphatase activity was determined using pNPP (10 mM) as a substrate in 50 mM Tris-HCl, pH 7.4, 0.1 mg/ml BSA, 3 mM CaCl2 and 10 mM pNPP. The reaction mixture was incubated at 30°C for 30 min with occasional mixing. At the end of incubation, the beads were centrifuged and the clear supernatant was collected into the wells of a 96-well ELISA plate and the absorbance measured at 405 nm. To measure the amount of immunoprecipated PP2A, 20 µl of beads were boiled in 4× sample buffer and subjected to SDS-PAGE and Western blots as described earlier.PP2A was immunoprecipitated according the protocol provided by Upstate, New York. Briefly, 4 µg of anti-PP2A (Clone 1D6)  was incubated with 100 µl Protein A Sepharose beads (Amersham/Pharmacia) for 4 hours at 4°C on a rotor. The beads were washed with 750 µl TBS 3 times and 2 times with the phosphates lysis buffer. The beads with absorbed anti-PP2A were incubated with 400 µg lysate protein in a total volume of 500 µl overnight at 4°C. Unbound proteins were separated, and the beads were washed (by a brief pulse in table-top centrifuge) with 750 µl of TBS 3 times. For phosphatase activity, 20 µl beads were pelleted and resuspended in 200 µl of phosphatase assay buffer containing 50 mM Tris-HCl, pH 7.4, 3 mM MnCl2 Profiler™ PCR Array kit were obtained from SuperArray Bioscience Corporation . The Human Signal Transduction PathwayFinder™ RT2Profiler™ PCR Array profiles the expression of 84 key genes representative of 18 different signal transduction pathways. Using real-time PCR, we analysed the expression of a focused panel of genes related to any of the 18 signal transduction pathways involved in the response of human primary neurons to QA 150 nM for 24 h.RTIn order to validate the relevance of QA in the mechanism of tau phosphorylation in the human disease, AD and control tissue were labelled for QA and tau . We confirmed that QA is found within cortical neurons in AD and, to a substantially lesser degree, in cortical neurons of elderly controls Figure 2QA 500 and 1200 nM significantly increased not only the level of total tau . Our data also suggest that the apparent observed increase in total phosphatase activity in the neuronal lysate treated with relatively lower concentrations of QA (up to 200 nM) may be due to an increase in PP2B expression and/or activity.Since QA is an NMDA receptor agonist, we investigated the effect of other NMDA receptor agonists (glutamate and NMDA) on tau phosphorylation and tested whether the NMDA receptor antagonists can abrogate QA-induced tau phosphorylation. The results are shown in We investigated the expression of genes related to several signalling pathways in neuronal cultures treated with QA for 24 hours. Of the 84 genes tested, QA induced the expression of 10 genes  by more We previously found that QA accumulates in AD brain and interestingly was seen in dystrophic hippocampal neurons Tau is a phosphoprotein that can be phosphorylated at multiple serine and threonine residues. Hyperphosphorylated tau is assembled into paired helical filaments (PHF) leading to the formation of NFTsin vitro human primary neurons can take up exogenous QA  detected with antibody AT270, which recognizes pT181. This suggests that QA-induced phosphorylation at this site may increase the aggregation of tau. It has been suggested that phosphorylation of tau at S199, S202, T205, T212, S235, S262 and S404 is required for the sequestration of normal tau, whereas further phosphorylation at positions T231 and S396 is needed for self-assembly. Phosphorylation at T181 and T217 are also suggested to be involved in self-assemblyTaken together these observations led us to investigate whether elevated concentrations of QA may contribute to AD neuropathology by leading to tau hyperphosphorylation. We found that The alternative splicing of the tau gene produces six isoforms of tau in adult brain. The six isoforms of tau differ from each other by the presence or absence of one or two inserts in the N-terminal part and by the presence of either three or four repeats in the C-terminal region. Whereas all six isoforms are seen in adult brain, only the shortest isoform of tau is expressed in foetal brain The major serine/threonine phosphatases in the brain are PP1, PP2A and PP2BWe measured the expression level of PP1, PP2A, PP2B and PP5. QA treatment decreased the expression of PP1, PP2A and PP5 in a dose dependent manner. These data suggest that the observed reduction in phosphatase activity and subsequent tau phosphorylation is due to a decrease in the expression of the major tau phosphatases. The expression of PP2B, on the other hand, was markedly increased. The observed increase in total phosphatase activity with lower concentrations of QA (100–200 nM) could be due to an increase in PP2B activity. Liu et al. 52 and this observation is consistent with our findings. In this study memantine also reversed the OA-induced decrease in PP-2A activity, increase in calcium, calmodulin-dependent protein kinase II (CaMKII) and cyclin AMP-dependent PKA activities, the decrease and aggregation of MAP-2 and the phosphorylation and aggregation of neurofilament heavy/medium (NF-H/M) subunits and inhibited the neurodegeneration associated with these changes. Memantine is a low to moderate affinity non-competitive NMDA receptor channel blocker that leads to functional improvement and reduces care dependence in moderate to severe AD patientsSince QA is an NMDA receptor agonist, we investigated whether other agonists had a similar effect on tau phosphorylation. Both glutamate and NMDA increased tau phosphorylation at sites similar to QA. An increase in tau phosphorylation by glutamate has been reported previouslyFinally, we used PCR array to determine which of the genes associated with tau phosphorylation may be switched on by QA. We found that QA at the sub-physiological concentrations found in amyloid plaquesIn conclusion, we showed in this study that QA over-production by activated microglia/macrophages and consequent accumulation in AD brain is likely to be involved in two mechanisms 1) promotion of tau pathology by inducing tau hyperphosphorylation and 2) direct excitotoxicity . We propFigure S1QA uptake by human neurons in vitro. These data demonstrate that QUIN is taken up by human primary neurons in vitro.(0.29 MB PDF)Click here for additional data file.Figure S2Determination of QPRTase activity in human neuron. We developed a new method to quantify QPRTase activity and we found that neuronal QPRTase activity starts to be saturated at concentrations above 300 nM.(0.15 MB PDF)Click here for additional data file.Figure S3Intracellular co-localization of neurofibrillary tangle and QA in a human neuron. Serial optical sections were taken through the neuron at 2 µm intervals. QA  and tau .(0.60 MB PDF)Click here for additional data file.