Ruth J. Lyons

The activation of the activating protein-1 (AP-1) family of transcription factors, including c-Fos and c-Jun family members, is one of the earliest nuclear events induced by growth factors that stimulate extracellular signal-regulated kinases (ERKs). In the case of c-Fos, the activation of ERK leads to an increased expression of c-fos mRNA. In turn, we have recently shown that ERK phosphorylates multiple residues within the carboxylterminal transactivation domain (TAD) of c-Fos, thus resulting in its increased transcriptional activity. However, how ERK-dependent phosphorylation regulates c-Fos function is still poorly understood. In this regard, it has been recently observed that the prolyl isomerase Pin1 can interact with proteins phosphorylated on serine or threonine residues that precede prolines (pS/T-P), such as the transcription factors p53 and c-Jun, thereby controlling their activity by promoting the cis-trans isomerization of these pS/T-P bonds. Here, we found that Pin1 binds c-Fos through specific pS/T-P sites within the c-Fos TAD, and that this interaction results in an enhanced transcriptional response of c-Fos to polypeptide growth factors that stimulate ERK. Our findings suggest that c-Fos represents a novel target for the isomerizing activity of Pin1 and support a role for Pin1 in the mechanism by which c-Jun and c-Fos can cooperate to regulate AP-1-dependent gene transcription upon phosphorylation by mitogen-activated kinase (MAPK) family members. The activation of the activating protein-1 (AP-1) family of transcription factors, including c-Fos and c-Jun family members, is one of the earliest nuclear events induced by growth factors that stimulate extracellular signal-regulated kinases (ERKs). In the case of c-Fos, the activation of ERK leads to an increased expression of c-fos mRNA. In turn, we have recently shown that ERK phosphorylates multiple residues within the carboxylterminal transactivation domain (TAD) of c-Fos, thus resulting in its increased transcriptional activity. However, how ERK-dependent phosphorylation regulates c-Fos function is still poorly understood. In this regard, it has been recently observed that the prolyl isomerase Pin1 can interact with proteins phosphorylated on serine or threonine residues that precede prolines (pS/T-P), such as the transcription factors p53 and c-Jun, thereby controlling their activity by promoting the cis-trans isomerization of these pS/T-P bonds. Here, we found that Pin1 binds c-Fos through specific pS/T-P sites within the c-Fos TAD, and that this interaction results in an enhanced transcriptional response of c-Fos to polypeptide growth factors that stimulate ERK. Our findings suggest that c-Fos represents a novel target for the isomerizing activity of Pin1 and support a role for Pin1 in the mechanism by which c-Jun and c-Fos can cooperate to regulate AP-1-dependent gene transcription upon phosphorylation by mitogen-activated kinase (MAPK) family members. The transcription complex AP-1 4The abbreviations used are: AP-1activating protein-1MAPKmitogen-activated kinaseTADtransactivation domainERKextracellular signal-regulated kinaseEGFepidermal growth factorGSTglutathione S-transferaseGFPgreen fluorescent proteinaaamino acidsFBSfetal bovine serumDTTdithiothreitolPMSFphenylmethylsulfonyl fluorideMOPS4-morpholinepropanesulfonic acidPBSphosphate-buffered salineMEKmitogen-activated protein kinase/extracellular signal-regulated kinase kinaseDBDDNA-binding domainmmutantwtwild-type. (activating protein-1) is a dimer composed of Jun and Fos family members whose expression and activity is tightly regulated by the mitogen-activated protein kinase (MAPK) family of serinethreonine kinases (reviewed in Refs. 1Karin M. Liu Z. Zandi E. Curr. Opin. Cell Biol. 1997; 9: 240-246Crossref PubMed Scopus (2310) Google Scholar, 2Whitmarsh A.J. Davis R.J. J. Mol. Med. 1996; 74: 589-607Crossref PubMed Scopus (1391) Google Scholar, 3Treisman R. Curr. Opin. Cell Biol. 1996; 8: 205-215Crossref PubMed Scopus (1163) Google Scholar). These kinases control the expression of AP-1, as well as its activity by the post-translational processing of preexisting or newly synthesized AP-1 proteins. For example, a critical step in the transcriptional activation of c-Jun is accomplished by the phosphorylation of a set of serines (Ser63 and Ser73) within its NH2-terminal transactivation domain (TAD) by the group of c-Jun NH2-terminal kinases (JNKs), which in turn stimulate the expression of c-Jun by regulating the c-Jun promoter as part of a positive autoregulatory loop (1Karin M. Liu Z. Zandi E. Curr. Opin. Cell Biol. 1997; 9: 240-246Crossref PubMed Scopus (2310) Google Scholar). A dual mechanism of control by MAPKs also operates over c-Fos, the prototypical member of the Fos family. Indeed, in the case of c-Fos, activation of extracellular signal-regulated kinases (ERKs) leads to the coordinated stimulation of c-fos expression by acting on transcription factors bound at the c-fos promoter (4Whitmarsh A.J. Shore P. Sharrocks A.D. Davis R.J. Science. 1995; 269: 403-407Crossref PubMed Scopus (880) Google Scholar, 5Treisman R. Curr. Opin. Genet. Dev. 1994; 4: 96-101Crossref PubMed Scopus (620) Google Scholar), and the post-translational modification of c-Fos by the direct phosphorylation of the c-Fos TAD (6Chen R.H. Abate C. Blenis J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10952-10956Crossref PubMed Scopus (258) Google Scholar, 7Murphy L.O. Smith S. Chen R.H. Fingar D.C. Blenis J. Nat. Cell Biol. 2002; 4: 556-564Crossref PubMed Scopus (761) Google Scholar), thereby enhancing c-Fos transcriptional activity (8Monje P. Marinissen M.J. Gutkind J.S. Mol. Cell. Biol. 2003; 23: 7030-7043Crossref PubMed Scopus (122) Google Scholar). However, the precise mechanism by which phosphorylation by MAPKs alters the function of these transcription factors remains not fully understood. For example, reversible phosphorylation may result in changes in the stability, nuclear localization, rate of binding to target DNA sequences, and/or the positive or negative modulation of the transactivating activity of these transcription factors (reviewed in Ref. 9Hill C.S. Treisman R. Cell. 1995; 80: 199-211Abstract Full Text PDF PubMed Scopus (1197) Google Scholar). In the latter case, it is possible that the phosphorylation of specific residues may favor the interaction with other transcription factors or with the transcriptional initiation complex, either directly or through the recruitment of co-activators. In this regard, it has been shown that c-Fos interacts with the TATA box-binding protein (10Metz R. Bannister A.J. Sutherland J.A. Hagemeier C. O'Rourke E.C. Cook A. Bravo R. Kouzarides T. Mol. Cell. Biol. 1994; 14: 6021-6029Crossref PubMed Scopus (78) Google Scholar) and the transcriptional activator cAMP-responsive element-binding protein (CREB)-binding protein (CBP/p300) (11Bannister A.J. Kouzarides T. EMBO J. 1995; 14: 4758-4762Crossref PubMed Scopus (319) Google Scholar) through its COOH-terminal domain. However, there is no evidence that MAPKs regulate these or other interactions. activating protein-1 mitogen-activated kinase transactivation domain extracellular signal-regulated kinase epidermal growth factor glutathione S-transferase green fluorescent protein amino acids fetal bovine serum dithiothreitol phenylmethylsulfonyl fluoride 4-morpholinepropanesulfonic acid phosphate-buffered saline mitogen-activated protein kinase/extracellular signal-regulated kinase kinase DNA-binding domain mutant wild-type. Recently, the peptidyl-prolyl isomerase Pin1 has emerged as a novel phosphorylation-dependent regulator of key transcription factors, including p53, NF-κB, NFAT, and c-Jun (reviewed in Refs. 12Shaw P.E. EMBO Rep. 2002; 3: 521-526Crossref PubMed Scopus (135) Google Scholar and 13Lu K.P. Liou Y.C. Zhou X.Z. Trends Cell Biol. 2002; 12: 164-172Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar). Pin1 interacts with its substrates through the recognition of specific phosphorylated serine or threonine residues adjacent to prolines (pS/T-P motifs). Its primary function is to promote conformational changes by facilitating the cis-trans isomerization of these peptide bonds (13Lu K.P. Liou Y.C. Zhou X.Z. Trends Cell Biol. 2002; 12: 164-172Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar). Thus, substrates of proline-directed protein kinases, such as the MAPK and cyclin-dependent kinase (CDK) family members, may represent likely targets for Pin1 function. This prompted us to investigate whether Pin1 regulates AP-1-mediated transcription. We found that, along with its effects on c-Jun, Pin1 contributes to the activation of AP-1 by interacting directly with c-Fos. This interaction is dependent upon the phosphorylation of c-Fos by ERK on a set of recently identified COOH-terminal phosphoacceptor sites (8Monje P. Marinissen M.J. Gutkind J.S. Mol. Cell. Biol. 2003; 23: 7030-7043Crossref PubMed Scopus (122) Google Scholar). Overall, our findings suggest that c-Fos represents a relevant target for the isomerizing activity of Pin1 and support a role for Pin1 as part of the underlying molecular mechanism by which MAPKs regulate c-Jun and c-Fos thereby promoting AP-1-dependent transcription. Materials—Recombinant human platelet-derived growth factor (rHu PDGF-BB) was purchased from Intergen Co. (New York). The anti NH2 -terminal c-Fos polyclonal (sc-52) and anti-glutathione S-transferase (GST) (sc-138) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The anti COOH-terminal c-Fos polyclonal antibody (ab2) was from Geneka Biotechnology Inc. (Quebec, Canada). Monoclonal antibody against AU-5 epitope was purchased form Covance (Berkeley, CA). Recombinant EGF and ERK2 (active) was purchased from Upstate Biotechnology (Lake Placid, NY). The full-length cDNA from the human Pin1 gene in pCMV-Sport6 was obtained from the IMAGE-EST collection (Resgen). All other reagents were of analytical grade. DNA Constructs—The full-length Pin1 cDNA was amplified by PCR and cloned as a BamHI/NotI fragment in pGEX-4T3 in frame with the coding sequence for the GST gene to produce a GST-Pin1 fusion protein in bacteria or in the pCEFL-GFP vector to express in mammalian cells as a GFP-Pin1 fusion protein. The expression vectors for c-Fos and c-Fos-m (aa 1–380) in pCEFL-AU5, and the Gal4- and polyhistidine (His6)-tagged c-Fos TAD-wt (aa 209–380), TAD-m, and TAD mutants containing single phosphorylation sites were described previously (8Monje P. Marinissen M.J. Gutkind J.S. Mol. Cell. Biol. 2003; 23: 7030-7043Crossref PubMed Scopus (122) Google Scholar). The expression vectors pCEV29-MEK EE, pCEFL-HA-ERK2, pCEFL-c-Jun, and the Gal4-driven luciferase reporter, pGal4-Luc, were also described previously (16Chiariello M. Marinissen M.J. Gutkind J.S. Mol. Cell. Biol. 2000; 20: 1747-1758Crossref PubMed Scopus (168) Google Scholar, 17Marinissen M.J. Chiariello M. Pallante M. Gutkind J.S. Mol. Cell. Biol. 1999; 19: 4289-4301Crossref PubMed Scopus (190) Google Scholar). The pAP-1 luciferase reporter plasmid was obtained from Stratagene. Cell Cultures and Transient Transfections—NIH 3T3 mouse fibroblasts were cultured in Dulbecco's modified Eagle's medium (DMEM; Invitrogen), supplemented with 10% calf serum and penicillin-streptomycin-amphotericin B (Invitrogen). HEK-293T cells were grown in DMEM containing 10% fetal bovine serum (FBS). Cells were transfected for 3 h in serum-free DMEM containing up to 2 μg of total plasmid DNA together with the Lipofectamine Plus Reagent (Invitrogen), according to the protocol suggested by the manufacturer. Preparation of Nuclear Extracts—Cells were incubated in lysis buffer (10 mm HEPES, pH 7.9, 10 mm KCl, 0.1 mm EDTA, 0.1 mm EGTA, 0.5 mm PMSF, 1 mm DTT, 0.5% Nonidet P-40) and centrifuged at low speed. The resulting nuclei (pellet) were disrupted with extraction buffer (20 mm HEPES, pH 7.9, 0.5 m NaCl, 1 mm EDTA, 1 mm EGTA, 1 mm DTT). Cell debris was separated by low speed centrifugation and nuclei aliquots (50 μg) were subjected to GST pull-down assays. Reporter Assays—Cells were transfected with different expression vectors together with 0.1 μg of each luciferase reporter and 0.01 μg of pRL-null (a plasmid encoding the luciferase gene from Renilla reniformis) that served as an internal control for transfection efficiency. The total amount of transfected DNA was normalized with pcDNAIII-β-gal, an expression vector for the enzyme β-galactosidase. Cells were lysed in passive lysis buffer (Promega) 24 h post-transfection. Cell lysates (50 μl/well) were transferred to a 96-well plate and firefly and Renilla luciferase activities were assayed using the Dual-Luciferase Reporter System (Promega). Light emission was quantified using a Microliter Plate luminometer as specified by the manufacturer (Dynex Tech, Chantilly, VA). GST Pull-down Assays—Cells were lysed in 25 mm HEPES, pH 7.5, 0.3 m NaCl, 1.5 mm MgCl2, 0.2 mm EDTA, 0.5 mm DTT, 20 mm β-glycerophosphate, 1 mm sodium vanadate, 1% Nonidet P-40, 1 mm PMSF, 20 μg/ml aprotinin, and 20 μg/ml leupeptin. Precleared cell lysates were incubated with fresh GST or GST-Pin beads (2 μg of protein) for1hat4°C.The beads were pelleted by centrifugation, washed three times with lysis buffer, denatured in SDS-loading buffer (400 mm Tris/HCl, pH 6.8, 10% SDS, 50% glycerol, 500 mm DTT, 2 μg/ml bromphenol blue), and analyzed by Western blot. To prepare the GST beads, Escherichia coli BL-21 Lys cells (Promega) were transformed with the vectors pGEX-4T3 or pGEX-4T3-Pin1 encoding for GST and GST-Pin1 proteins, respectively. Protein expression and purification were performed essentially as described previously (8Monje P. Marinissen M.J. Gutkind J.S. Mol. Cell. Biol. 2003; 23: 7030-7043Crossref PubMed Scopus (122) Google Scholar). In Vitro Phosphorylation of His6-c-Fos TAD Fusion Proteins—His6-tagged proteins were isolated using nickel-nitrilotriacetic acid magnetic agarose beads (Qiagen) as described previously (8Monje P. Marinissen M.J. Gutkind J.S. Mol. Cell. Biol. 2003; 23: 7030-7043Crossref PubMed Scopus (122) Google Scholar). The kinase reactions were performed in 12.5 mm MOPS, pH 7.5, 12.5 mm glycerophosphate, 7.5 mm MgCl2, 0.5 mm EGTA, 0.5 mm sodium fluoride, 0.5 m m sodium vanadate, containing 20 μm unlabeled ATP, 1 mm DTT, and 1 μg of substrate. The reactions were initiated by the addition of 1 μl of purified active ERK2 and allowed to proceed for 30 min at 30 °C before assaying the products in GST pull-down experiments. Western Blots and Immunoprecipitations—For co-immunoprecipitations, cellular lysates were incubated with for1hat4°C with the specific antibody against AU5. Immunocomplexes were recovered with the aid of γ-bind Sepharose beads (Amersham Biosciences). Immunoprecipitates or lysates were combined with SDS loading buffer, boiled for 5 min, and resolved by 10% SDS-PAGE. Fractionated proteins were electrophoretically transferred to polyvinylidene fluoride membranes (Immobilon-P, Millipore). The membranes were incubated with 5% nonfat-dried milk in PBS containing 0.05% Tween 20 (PBS-T) and then with dilutions of the respective primary and horseradish peroxidase-conjugated secondary antibody (ICN-Cappel) in PBS-T, 1% bovine serum albumin. Immunoreactive proteins were visualized by enhanced chemiluminescence detection (ECL+Plus System, Amersham Biosciences). Pin1 Potentiates AP-1 Activity by Increasing the Transcriptional Response of c-Fos and c-Jun to Growth Factors: a Novel Interaction between Pin1 and c-Fos—To explore the possibility that Pin1-directed prolyl isomerization of AP-1 factors controls the response of AP-1 to mitogenic signals, we examined whether Pin1 could affect the expression of an AP-1-driven luciferase reporter plasmid in response to PDGF in NIH 3T3 cells. As shown in Fig. 1A, PDGF significantly induced AP-1 transcription in these cells, and increasing concentrations of ectopically expressed Pin1 enhanced the basal AP-1 activity as well as the transcriptional response to this growth factor in a dose-dependent manner. To begin addressing the mechanism whereby Pin1 stimulates AP-1, we next examined the effect of Pin1 on the transactivating function of c-Jun and c-Fos. As shown in Fig. 1B, expression of c-Jun or c-Fos increased AP-1-dependent transcription and potentiated the AP-1 transcriptional response provoked by PDGF. This was enhanced by the co-expression of Pin1, which elevated the basal AP-1 stimulating activity of c-Jun and c-Fos, as well as increased dramatically their response to PDGF. Thus, Pin1 appears to regulate the transactivating activity of both members of the AP-1 family of transcription factors. Based on these observations, we then asked whether Pin1 may interact with c-Fos. For these experiments, we first generated a bacterial expression vector encoding a GST fusion protein of Pin1 and then used purified GST-Pin1 coupled to glutathione-Sepharose beads to pull down c-Fos from nuclear extracts of NIH 3T3 cells stimulated with PDGF. As expected, PDGF promoted the expression of c-Fos (Fig. 1C). the results from the pull-down that Pin1 binds to newly synthesized c-Fos proteins, thus a molecular to the of Pin1 to stimulate the activation of AP-1 by c-Fos. The Interaction of c-Fos and Pin1 on c-Fos the of Pin1 to to its substrates the recognition of specific phosphorylated Zhou X.Z. M. K.P. Science. 1999; PubMed Scopus Google Scholar, K.P. T. Nat. Biol. 2000; PubMed Scopus Google Scholar), we next examined whether the interaction of c-Fos with Pin1 was dependent on the of c-Fos in a we have observed that of NIH 3T3 cells to PDGF or serum leads to c-Fos phosphorylation by ERK on specific and within the COOH-terminal c-Fos TAD (8Monje P. Marinissen M.J. Gutkind J.S. Mol. Cell. Biol. 2003; 23: 7030-7043Crossref PubMed Scopus (122) Google Scholar). To explore this we c-Fos from a active in NIH 3T3 cells and performed GST-Pin1 pull-down in total lysates from cells subjected to that promote c-Fos In as shown in Fig. Pin1 was to interact with the phosphorylated of c-Fos generated by the stimulation with serum or PDGF and with c-Fos proteins from cells. the interaction was dramatically using a mutant c-Fos protein sites for ERK phosphorylation were by residues (Fig. To that c-Fos can interact with in we expressed the c-Fos and c-Fos-m together with a in HEK-293T cells. As shown in Fig. low of GFP-Pin1 were in c-Fos in these cells, a phosphorylated of c-Fos in HEK-293T cells. A to EGF provoked a in the of Pin1 with c-Fos. In no of Pin1 was in cells c-Fos-m EGF In this regard, we have previously shown that this mutant form of c-Fos phosphorylated by ERK and is in its transcriptional response to ERK-dependent (8Monje P. Marinissen M.J. Gutkind J.S. Mol. Cell. Biol. 2003; 23: 7030-7043Crossref PubMed Scopus (122) Google Scholar). The of this mutant an to whether c-Fos phosphorylation was for the effect of Pin1 on the transcriptional response of c-Fos. Fig. that this c-Fos-m a transcriptional response to Pin1, likely their and it to stimulate AP-1-dependent transcription as as the c-Fos stimulated by PDGF. these results suggest that the specific phosphorylation of a of residues within the c-Fos TAD is critical for the interaction between Pin1 and c-Fos and that this may to the enhanced transcriptional activity of c-Fos upon phosphorylation by ERK in response to growth factors. The COOH-terminal TAD of c-Fos c-Fos Interaction with for the Phosphorylation of the of c-Fos phosphorylation by ERK in the interaction between Pin1 and c-Fos, we the of phosphorylated of c-Fos, which by as electrophoretically of molecular by c-Fos together with ERK2 and EE, a active form of that ERK2 Fig. that Pin1 binds to c-Fos c-Fos is phosphorylated by ERK in the interaction of c-Fos with Pin1 was to basal the c-Fos-m protein was assayed for its to interact with (Fig. As it is still possible that c-Fos to Pin1 for by binding to Jun proteins, we to explore whether Pin1 can to the c-Fos TAD in To this we performed GST-Pin pull-down using a purified of expressed of the c-Fos COOH-terminal TAD (Fig. we first observed that the purified c-Fos TAD fusion protein was to interact with However, a interaction between these proteins was the c-Fos TAD was phosphorylated by ERK2 in to assaying for its to Pin1 (Fig. with this no interaction was observed an amount of in Fig. was used as a Pin1 in the of purified These that Pin1 can directly to c-Fos and that the COOH-terminal domain of c-Fos (aa is to this that it is first phosphorylated by ERK. To the of each ERK phosphorylation on the c-Fos TAD to the interaction between Pin1 and c-Fos, we generated mutant of the c-Fos TAD in which ERK phosphoacceptor sites were first to and then each was (8Monje P. Marinissen M.J. Gutkind J.S. Mol. Cell. Biol. 2003; 23: 7030-7043Crossref PubMed Scopus (122) Google Scholar), as in Fig. this we have observed that each of these serines or in targets for the activity of ERK2 that for the transcriptional of the c-Fos TAD to extracellular (8Monje P. Marinissen M.J. Gutkind J.S. Mol. Cell. Biol. 2003; 23: 7030-7043Crossref PubMed Scopus (122) Google Scholar). The results from Fig. that the phosphorylation of sites by ERK was not to support its binding to Pin1, that the phosphorylation of multiple sites on c-Fos to promote its interaction with Pin1 c-Fos Activity through Its on the c-Fos on these observations, we set to investigate whether the TAD of c-Fos was the direct target of Pin1 function to changes in transcription. For these experiments, we examined the effect of Pin1 on a reporter the TAD of c-Fos is expressed as a fusion protein in frame with the DNA-binding domain of the transcription factor and the activity of this is by its to stimulate transcription from a luciferase reporter plasmid Pin1 not stimulate the basal activity of the at of the concentrations not as shown in Fig. with PDGF to the transactivating function of the c-Fos the fusion protein containing in ERK to to Pin1 with an in transactivation PDGF stimulation (Fig. of which support the direct function of Pin1 on the activity of the c-Fos TAD upon its these findings suggest that the prolyl isomerization of phosphorylated peptide bonds on c-Fos by Pin1 may represent a novel step controlling c-Fos activity. In this regard, it is to that this may to a between a low and of transcriptional activity of the c-Fos TAD, as a in its may c-Fos to interact with novel molecular to changes in its to promote expression from AP-1 these also a molecular mechanism by which growth factors that stimulate ERK may control the activity of c-Fos and AP-1 dependent transcription. In this the activation of ERK by acting on cell such as an kinase may promote the expression of c-Fos and the phosphorylation of specific within the c-Fos TAD that result in the recruitment of Pin1, which may then their thus a of with other transcription factors, or of the transcriptional to investigate the of Pin1 on phosphorylated c-Fos TAD, these findings may a the still poorly by which activation of members of the MAPK family regulate the and activity of nuclear transcription factors, thereby controlling gene

Fusion genes are a major cause of cancer. Their rapid and accurate diagnosis can inform clinical action, but current molecular diagnostic assays are restricted in resolution and throughput. Here, we show that targeted RNA sequencing (RNAseq) can overcome these limitations. First, we establish that fusion gene detection with targeted RNAseq is both sensitive and quantitative by optimising laboratory and bioinformatic variables using spike-in standards and cell lines. Next, we analyse a clinical patient cohort and improve the overall fusion gene diagnostic rate from 63% with conventional approaches to 76% with targeted RNAseq while demonstrating high concordance for patient samples with previous diagnoses. Finally, we show that targeted RNAseq offers additional advantages by simultaneously measuring gene expression levels and profiling the immune-receptor repertoire. We anticipate that targeted RNAseq will improve clinical fusion gene detection, and its increasing use will provide a deeper understanding of fusion gene biology.

Growth factor regulation of the cortical actin cytoskeleton is fundamental to a wide variety of cellular processes. The cortical actin-associated protein, cortactin, regulates the formation of dynamic actin networks via the actin-related protein (Arp)2/3 complex and hence is a key mediator of such responses. In order to reveal novel roles for this versatile protein, we used a proteomics-based approach to isolate cortactin-interacting proteins. This identified several proteins, including CD2-associated protein (CD2AP), as targets for the cortactin Src homology 3 domain. Co-immunoprecipitation of CD2AP with cortactin occurred at endogenous expression levels, was transiently induced by epidermal growth factor (EGF) treatment, and required the cortactin Src homology 3 domain. The CD2AP-binding site for cortactin mapped to the second of three proline-rich regions. Because CD2AP is closely related to Cbl-interacting protein of 85 kDa (CIN85), which regulates growth factor receptor down-regulation via complex formation with Cbl and endophilin, we investigated whether the CD2AP-cortactin complex performs a similar function. EGF treatment of cells led to transient association of Cbl and the epidermal growth factor receptor (EGFR) with a constitutive CD2AP-endophilin complex. Cortactin was recruited into this complex with slightly delayed kinetics compared with Cbl and the EGFR. Immunofluorescence analysis revealed that the EGFR, CD2AP, and cortactin co-localized in regions of EGF-induced membrane ruffles. Therefore, by binding both CD2AP and the Arp2/3 complex, cortactin links receptor endocytosis to actin polymerization, which may facilitate the trafficking of internalized growth factor receptors. Growth factor regulation of the cortical actin cytoskeleton is fundamental to a wide variety of cellular processes. The cortical actin-associated protein, cortactin, regulates the formation of dynamic actin networks via the actin-related protein (Arp)2/3 complex and hence is a key mediator of such responses. In order to reveal novel roles for this versatile protein, we used a proteomics-based approach to isolate cortactin-interacting proteins. This identified several proteins, including CD2-associated protein (CD2AP), as targets for the cortactin Src homology 3 domain. Co-immunoprecipitation of CD2AP with cortactin occurred at endogenous expression levels, was transiently induced by epidermal growth factor (EGF) treatment, and required the cortactin Src homology 3 domain. The CD2AP-binding site for cortactin mapped to the second of three proline-rich regions. Because CD2AP is closely related to Cbl-interacting protein of 85 kDa (CIN85), which regulates growth factor receptor down-regulation via complex formation with Cbl and endophilin, we investigated whether the CD2AP-cortactin complex performs a similar function. EGF treatment of cells led to transient association of Cbl and the epidermal growth factor receptor (EGFR) with a constitutive CD2AP-endophilin complex. Cortactin was recruited into this complex with slightly delayed kinetics compared with Cbl and the EGFR. Immunofluorescence analysis revealed that the EGFR, CD2AP, and cortactin co-localized in regions of EGF-induced membrane ruffles. Therefore, by binding both CD2AP and the Arp2/3 complex, cortactin links receptor endocytosis to actin polymerization, which may facilitate the trafficking of internalized growth factor receptors. Subcellular compartmentalization and trafficking of signal transduction complexes and a variety of dynamic cellular responses to extracellular stimuli require regulated interactions between specific components of signaling pathways and the cytoskeleton. These interactions may be direct or mediated by particular adaptor or scaffolding proteins. Among these, cortactin was identified as a v-Src substrate associated with the cortical actin cytoskeleton approximately a decade ago, although insights into its cellular function and the underlying mechanisms have only been obtained recently (1Weed S.A. Parsons J.T. Oncogene. 2001; 20: 6418-6434Crossref PubMed Scopus (362) Google Scholar). In line with an adaptor role, cortactin is a multidomain protein, with the individual modules capable of mediating specific protein-protein interactions (1Weed S.A. Parsons J.T. Oncogene. 2001; 20: 6418-6434Crossref PubMed Scopus (362) Google Scholar). The N-terminal region mediates binding to the Arp 1The abbreviations used are: Arp, actin-related protein; ASAP, ARF GTPase-activating protein containing Src homology 3, ankyrin repeat, and pleckstrin homology domains; CD2AP, CD2-associated protein; CIN85, Cbl-interacting protein of 85 kDa; CMS, Cas ligand with multiple Src homology 3 domains; EGF, epidermal growth factor; EGFR, EGF receptor; ER, estrogen receptor; FCS, fetal calf serum; FISH, five Src homology 3 domains; GFP, green fluorescent protein; GST, glutathione S-transferase; MALDI-TOF MS, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; μLC-MS/MS, microcapillary liquid chromatography tandem mass spectrometry; PBS, phosphate-buffered saline; PVDF, polyvinylidene difluoride; RIPA, radioimmunoprecipitation assay; SH3, Src homology 3; TBS, Tris-buffered saline; WASP, Wiskott-Aldrich syndrome protein.1The abbreviations used are: Arp, actin-related protein; ASAP, ARF GTPase-activating protein containing Src homology 3, ankyrin repeat, and pleckstrin homology domains; CD2AP, CD2-associated protein; CIN85, Cbl-interacting protein of 85 kDa; CMS, Cas ligand with multiple Src homology 3 domains; EGF, epidermal growth factor; EGFR, EGF receptor; ER, estrogen receptor; FCS, fetal calf serum; FISH, five Src homology 3 domains; GFP, green fluorescent protein; GST, glutathione S-transferase; MALDI-TOF MS, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; μLC-MS/MS, microcapillary liquid chromatography tandem mass spectrometry; PBS, phosphate-buffered saline; PVDF, polyvinylidene difluoride; RIPA, radioimmunoprecipitation assay; SH3, Src homology 3; TBS, Tris-buffered saline; WASP, Wiskott-Aldrich syndrome protein.2/3 complex, a highly conserved regulator of the assembly and structure of actin networks (2Weed S.A. Karginov A. Schafer D.A. Weaver A.M. Kinley A.W. Cooper J.A. Parsons J.T. J. Cell Biol. 2000; 151: 29-40Crossref PubMed Scopus (335) Google Scholar), and contains a DDW motif characteristic of Arp2/3-interacting proteins such as WASP, Myo3p, and ActA (2Weed S.A. Karginov A. Schafer D.A. Weaver A.M. Kinley A.W. Cooper J.A. Parsons J.T. J. Cell Biol. 2000; 151: 29-40Crossref PubMed Scopus (335) Google Scholar, 3Uruno T. Liu J. Zhang P. Fan Y.-X. Egile C. Li R. Mueller S.C. Zhan X. Nat. Cell Biol. 2001; 3: 259-266Crossref PubMed Scopus (454) Google Scholar). This is followed by six and a half copies of a 37-amino acid repeat, with the fourth repeat necessary for binding to F-actin in vitro (2Weed S.A. Karginov A. Schafer D.A. Weaver A.M. Kinley A.W. Cooper J.A. Parsons J.T. J. Cell Biol. 2000; 151: 29-40Crossref PubMed Scopus (335) Google Scholar). Downstream of the repeats is a predicted helical domain and a region rich in serine, threonine, and proline residues. The latter is a target for both tyrosine and serine/threonine phosphorylation (4Huang C. Ni Y. Wang T. Gao Y. Haudenschild C.C. Zhan X. J. Biol. Chem. 1997; 272: 13911-13915Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar, 5Huang C. Liu J.L. Haudenschild C.C. Zhan X. J. Biol. Chem. 1998; 273: 25770-25776Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar, 6Campbell D.H. Sutherland R.L. Daly R.J. Cancer Res. 1999; 59: 5376-5385PubMed Google Scholar). The C terminus of cortactin is characterized by an SH3 domain. Several binding partners for this module have been identified, including CortBP1/Shank 2 (1Weed S.A. Parsons J.T. Oncogene. 2001; 20: 6418-6434Crossref PubMed Scopus (362) Google Scholar, 7Du Y. Weed S.A. Xiong W.-C. Marshall T.D. Parsons J.T. Mol. Cell. Biol. 1998; 18: 5838-5851Crossref PubMed Scopus (219) Google Scholar), ZO-1 (8Katsube T. Takahisa M. Ueda R. Hashimoto N. Kobayashi M. Togashi S. J. Biol. Chem. 1998; 273: 29672-29677Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar), and dynamin 2 (9McNiven M.A. Kim L. Krueger E.W. Orth J.D. Cao H. Wong T.W. J. Cell Biol. 2000; 151: 187-198Crossref PubMed Scopus (342) Google Scholar). Cortactin localizes with the Arp2/3 complex at sites of dynamic cortical actin assembly such as lamellipodia (2Weed S.A. Karginov A. Schafer D.A. Weaver A.M. Kinley A.W. Cooper J.A. Parsons J.T. J. Cell Biol. 2000; 151: 29-40Crossref PubMed Scopus (335) Google Scholar). Cortactin binding leads to a relatively weak stimulation of the actin nucleation activity of the Arp2/3 complex (10Weaver A.M. Karginov A.V. Kinley A.W. Weed S.A. Li Y. Parsons J.T. Cooper J.A. Curr. Biol. 2001; 11: 370-374Abstract Full Text Full Text PDF PubMed Scopus (466) Google Scholar), but it can enhance actin polymerization induced by co-activators of Arp2/3 such as WASP family proteins (3Uruno T. Liu J. Zhang P. Fan Y.-X. Egile C. Li R. Mueller S.C. Zhan X. Nat. Cell Biol. 2001; 3: 259-266Crossref PubMed Scopus (454) Google Scholar, 10Weaver A.M. Karginov A.V. Kinley A.W. Weed S.A. Li Y. Parsons J.T. Cooper J.A. Curr. Biol. 2001; 11: 370-374Abstract Full Text Full Text PDF PubMed Scopus (466) Google Scholar, 11Weaver A.M. Heuser J.E. Karginov A.V. Lee W.-I. Parsons J.T. Cooper J.A. Curr. Biol. 2002; 12: 1270-1278Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar). Furthermore, cortactin promotes the formation of branched actin networks and their stabilization (10Weaver A.M. Karginov A.V. Kinley A.W. Weed S.A. Li Y. Parsons J.T. Cooper J.A. Curr. Biol. 2001; 11: 370-374Abstract Full Text Full Text PDF PubMed Scopus (466) Google Scholar). One function of cortactin is to regulate the organization and subcellular localization of transmembrane complexes. The cortactin SH3 domain target CortBP1/Shank 2 performs a scaffolding function in the organization of receptor complexes at post-synaptic sites of excitatory synapses, whereas a further SH3 binder, ZO-1, interacts with the transmembrane proteins claudin and occludin at epithelial tight junctions (1Weed S.A. Parsons J.T. Oncogene. 2001; 20: 6418-6434Crossref PubMed Scopus (362) Google Scholar). A second functional role for cortactin is regulation of receptor-mediated endocytosis, via its interaction with dynamin 2, a mechanochemical GTPase that participates in the scission of endocytic vesicles at the plasma membrane (9McNiven M.A. Kim L. Krueger E.W. Orth J.D. Cao H. Wong T.W. J. Cell Biol. 2000; 151: 187-198Crossref PubMed Scopus (342) Google Scholar, 12Cao H. Orth J.D. Chen J. Weller S.G. Heuser J.E. McNiven M.A. Mol. Cell. Biol. 2003; 23: 2162-2170Crossref PubMed Scopus (182) Google Scholar). 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Cell. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). and to the The was One was can be in both to that the required been and of used to to proteins by chromatography PubMed Scopus Google Scholar). and proteins to A Scholar). to of with protein, the of was by to of for at proteins by with in for at and three in containing Daly R.J. A. Sutherland R.L. Oncogene. Google Scholar). The of was with the for 2 at and five in by to a and to The was to analysis of by mass by of protein with of the in to analysis by in which proteins by of was with of The in to by the and to the and MALDI-TOF mass was a with a of to the of for protein used to the and via the was by R. T. and Scholar). was a with an for with of SH3 or proteins by with for at The was by of for proteins by to a and for in in with the proteins for and in containing protein was the cells in transiently with EGFR. The cells for to stimulation with in in and in in with CD2AP in containing cells with for These and a with a and of to the SH3 of of is associated with in but R. D.H. Lee Daly R.J. R. Sutherland R.L. Oncogene. 1997; PubMed Scopus Google Scholar). In order to proteins with the cortactin SH3 region in we chromatography This line was it and a H. P. J. E.W. Cancer Res. PubMed Scopus Google Scholar). was a of a protein to investigated the of the cortactin region by the and was as a proteins by and by proteins by the SH3 domain and by the One protein of kDa the The was and proteins by and proteins identified by MALDI-TOF or Among the proteins identified to as CD2AP Li J. S. N. P. Cell. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, S. H. Sci. S. A. 1999; PubMed Scopus Google Scholar), P. T. S.A. J. 1998; PubMed Scopus Google Scholar), J. H. Cooper J.A. Mol. Cell. Biol. 1998; 18: PubMed Scopus Google Scholar), and M. H. T. 1997; PubMed Scopus Google Scholar). The mass obtained for CD2AP is in The specific binding of proteins to the cortactin SH3 domain was by of of cortactin SH3 domain with or proteins. proteins by with specific proteins identified by mass of CD2AP with Cortactin in FISH, and have been in regulation of and the three proteins of Src family S. H. Sci. S. A. 1999; PubMed Scopus Google Scholar, P. T. S.A. J. 1998; PubMed Scopus Google Scholar, J. H. Cooper J.A. Mol. Cell. Biol. 1998; 18: PubMed Scopus Google Scholar, Curr. Cell Biol. 2002; PubMed Scopus Google Scholar). These that their as cortactin SH3 targets a functional with we have been to or with cortactin at endogenous expression of the binding although was recently to with cortactin in in an SH3 H. H. H. T. Cancer Res. 2002; Google Scholar). 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Because we have the of CD2AP in cortactin that cortactin is recruited to the complex. to that the of cortactin and CD2AP was by their SH3 to the proline-rich region of dynamin 2, we investigated whether dynamin 2 be in protein complexes. dynamin 2 was in it was CD2AP This that the association between CD2AP and cortactin is to be of the for the Cortactin SH3 contains three proline-rich regions sites S. H. Sci. S. A. 1999; PubMed Scopus Google The binding for the cortactin SH3 domain been characterized as and and acid J.E. Sci. S. A. PubMed Scopus Google Scholar). of the CD2AP acid reveal an with this In order to the binding of the three proline-rich regions of CD2AP was as a A protein to the binding for the cortactin SH3 domain was as a These proteins, and as a to a with or binding of the SH3 domain to was but binding to and to was These that the cortactin SH3 domain can to proline-rich and Because both cortactin and CD2AP via SH3 it was of to the binding of the SH3 domain to with that of the cortactin SH3 The SH3 domain to and relatively to and This that in may whereas cortactin is recruited to In order to the cortactin SH3 domain binding site in proline to into in CD2AP to the cortactin SH3 domain binding and proteins transiently in cells and binding to the cortactin SH3 domain was by protein analysis of the motif binding to that of the protein, whereas of a of in a which was binding of the was compared with that of the whether association of the proteins in cells followed this binding the CD2AP proteins and for cortactin In with the was identified as the interaction with a the a role for be is that the and in their to binding of the of the protein to endogenous this site the in with the for the cortactin SH3 domain for association with CD2AP and the of the cortactin SH3 domain to to that association of the proteins in cells is mediated by direct binding of the cortactin SH3 domain to the region of the binding activity in interactions a to complex CD2AP, and the in EGF CD2AP been to lamellipodia and cytoplasmic S. 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The Grb10 adapter protein is capable of interacting with a variety of receptor tyrosine kinases, including, notably, the insulin receptor. Biochemical and cell culture experiments have indicated that Grb10 might act as an inhibitor of insulin signaling. We have used mice with a disruption of the Grb10 gene (Grb10Delta2-4 mice) to assess whether Grb10 might influence insulin signaling and glucose homeostasis in vivo. Adult Grb10Delta2-4 mice were found to have improved whole-body glucose tolerance and insulin sensitivity, as well as increased muscle mass and reduced adiposity. Tissue-specific changes in insulin receptor tyrosine phosphorylation were consistent with a model in which Grb10, like the closely related Grb14 adapter protein, prevents specific protein tyrosine phosphatases from accessing phosphorylated tyrosines within the kinase activation loop. Furthermore, insulin-induced IRS-1 tyrosine phosphorylation was enhanced in Grb10Delta2-4 mutant animals, supporting a role for Grb10 in attenuation of signal transmission from the insulin receptor to IRS-1. We have previously shown that Grb10 strongly influences growth of the fetus and placenta. Thus, Grb10 forms a link between fetal growth and glucose-regulated metabolism in postnatal life and is a candidate for involvement in the process of fetal programming of adult metabolic health.