Screen and Identification of Proteins Interacting with ADAM19

Screen and identiﬁcation of proteins interacting with ADAM19 cytoplasmic tail

Li Huang, Libing Feng, Limin Yang, Weiguo Zhou, Shouyuan Zhao & Changben Li∗ Institute of Genetics, Fudan University, Shanghai 200433, P.R. China; ∗Author for correspondence (Phone: 86-21-65642428; Fax: 86-21-65642428; E-mail: [email protected])

Received 22 January 2002; accepted 9 May 2002

Key words: ADAM, ArgBP1, yeast two-hybrid

Abstract ADAM family plays important roles in neurogenesis. The cytoplasmic tail of ADAM19 (ADAM19-CT) contains 193 residues. The presence of two putative SH3 ligand-binding sites suggests potential interactions with cytosolic proteins, which could be possibly linked to the functions of ADAM19. To address these issues, a yeast two-hybrid screen was performed in human fetal brain cDNA library to isolate proteins that interact with the cytoplasmic tail of ADAM19. Four proteins were obtained, ArgBP1, β-cop, ubiquitin and a novel protein. GST-Pulldown assay has confirmed the interaction between AdAM19 and ArgBP1. By constructing series of deletion mutants of ADAM19-CT and ArgBP1 respectively, the interaction regions have been identified. They are the SH3 binding sites in ADAM19-CT and the P4 region in ArgBP1. And the interaction is specific. ArgBP1 does not bind to ADAM22, ADAM29 or ADAM9 (mouse). ArgBP1may be the key protein, which accounts for the physiological function of ADAM19.

Introduction the development of glialcytes and axongenesis. Be- sides, ADAM19 is a novel marker for dendritic cell ADAMs (proteins containing A Disintegrin And differentiation [5]. Metalloprotease domain) are transmembrane glyco- Compared with the extracellular domains, the cy- proteins with a characteristic domain organization toplasmic tails of ADAM family are less conserved. composed of four potential conserved domains: a met- More than half of this family has long cytoplasmic alloprotease domain, a disintegrin domain, a cysteine- tails (about 200 residues) with predicted SH3 domains rich region and a cytoplasmic tail [1, 2]. They may and phosphorylation sites for PKC. These suggest play important roles in the proteolysis of the extra- that interacting with cytosolic proteins may affect cellular matrix, cell-cell and/or cell-matrix adhesion, the activity of metalloprotease and disintegrin, and/or membrane fusion and signal transduction, which are regulate their subcellular localization and process- already conﬁrmed to involve in a variety of physio- ing. ADAM19-CT has 193 residues with two putative logical and pathological processes such as histogene- SH3 binding sites. To gain insight into the biological sis, sperm-egg fusion, myogenesis, osteogenesis, and role of ADAM19-CT, we used yeast two-hybrid ap- tumor cell adhesion. proach to identify binding partners that interact with As researches go on, people increasingly real- ADAM19-CT in human fetal brain cDNA library with ize the critical status of ADAMs in neurogenesis. ADAM-CT as a bait. Kuzbanian (ADAM10) regulates the function of Notch and Delta [3]. And ADAM19 is involved in the processing of neuregulin (NRG) [4]. Diverse evidence indicates that ADAM19 contributes to glialcyte differentiation by processing NRGs, which is signiﬁcant in 318

Materials and methods Preparation, transformation of competent yeast cells and testing the transcription of β-galactosidase and Strains Leu

E. coli strains DH5-α, KC8,BL21(DE3), and Competent cells were prepared and transformed ac- AD494(DE3) were preserved in our lab. Yeast strain cording to the protocol for MATCHMAKER two- S. cerevisiae EGY48 (MATα, his3, trp1, ura3, hybrid system (Cat. #1605-1). Competent EGY48 LexAop(x6)-LEU2) was kindly provided by Dr. Tong- cells were transformed with pEG202-adam19-CT. wen Wang from Washington University, USA. Then a test was performed to investigate whether the bait protein can activate the transcription of Laz and Plasmids Leu. pEG202 was a gift of Dr. Tongwen Wang; Plas- Screen in human fetal brain cDNA library and mids containing 3 terminus cDNA of ADAM19, identification of positive clones ADAM22,ADAM29 & ADAM9 (mouse) were preserved in our lab. Yeast strain EGY48, which harbors the LexA- op-Leu reporter gene and LexA-op-LacZ plas- Primers mid ,was initially transformed with the bait plasmid and then subsequently with 50 µg of hu- pJG4-5F: 5CCA GCC TCT TGC TGA GTG GAG man fetal brain cDNA in pJG4-5. Transformants ATG 3 , were isolated on Gal/Raf ura−his−trp−leu− se- pJG4-5R: 5 GAC AAG CCG ACA ACC TTG ATT lective medium. Candidate clones were identified GGA G 3. − − by their ability to grow on Gal/Raf ura his Adam19F: 5 CAG,GAA,TTC,AGA,CAG,AAG,AAC, trp−leu− plates, but not on Glu ura−his−trp−leu− AAA,CTA 3(EcoRI) plates, and their ability to produce blue clones on Adam19R: 5 TGC,CAG,TCG,ACA,GCG,GGC,ACC, Gal/Raf ura−his−trp−X-gal plates, but not on Glu AAG,AAA,CAT3 (XhoI). ura−his−trp−X-gal plates.The specificity of interaction of candidates were tested by retransformation of positive clones into yeast harboring pLexA-ADAM19- Major reagents CT, pLexA-mpl-CT, pLexA and only the reporter gene plasmids respectively. HA monoclonal antibody and BM chemiluminines- cence blotting substrate were purchased from Roche Isolation of library plasmids from positive clones Molecular Biochemicals (CatNo. 1500 708); His-bind resin was purchased from Novagen Company; glu- The isolation of library plasmids from positive clones tathione sepharose 4B was obtained from Pharmacia was performed according to the methods of ref. [7]. Biotech; minimal SD Base/Gal/Raf was purchased The bacterial colonies containing library plasmids from Clontech Company; yeast nitrogen base was were identified by PCR with pJG4-5F and pJG4-5R purchased from Difco Company; 5-bromo-4-chloro-3- primers. The reaction mixture was annealed at 92 ◦C indolyl-β-D-galactoside(X-gal), amino acids and acid for 2 min, then amplified under the following thermal washed beads (425–600 µm) were all purchased from profile: 92 ◦C 30 sec, 65 ◦C 2 min, 75 ◦C30secper Sigma Company. cycle for 30 cycles upon completion was incubated at 75 ◦ for 5 min. Molecular cloning Expression and purification of GST-ArgBP1 Molecular cloning was performed according to the routine methods of reference [6]. Partial coding sequence of ArgBP1 was digested from pJG4-5 with EcoRI/XhoI and cloned into pGEX to yield in-frame fusion proteins. Plasmids encoding GST fusion proteins were used to transform E. coli BL21 (DE3). Fusion protein expression was induced 319 by IPTG. Soluble proteins were released by sonica- cloned into pEG202 and their interactions with tion. Debris was removed by centrifugation. GST ArgBP1 were tested in yeast two hybrid system. fusion proteins were captured from the supernatants on glutathione-sepharose beads. GST protein was expressed and purified with the same procedures. Results

Preparation of the yeast extracts Construction and identiﬁcation of target plasmid

Fresh EGY48 clones containing pLexA-ADAM19-CT The cytoplasmic tail of ADAM19 was amplified by and pLexA respectively were picked out and then PCR from the plasmids containing 3 sequence of put into 5 ml of Glu ura−his− medium. The liquid ADAM19 and then ligated to EcoRI and XhoI sites cultures were grown overnight at 30 ◦C shaking to of pEG202 (his is the marker) in the same frame OD600 ≈ 0.2. The next day, the liquid were transferred of LexA. The resulting plasmid was designated as into 10 ml of Glu ura−his− medium and cultured for pLexA-ADAM19-CT. The ability of transcriptional another 4–5 h to OD600 ≈ 0.6. Yeast extracts were activation of the fusion protein was tested before prepared according to Ref. [7]. a two-hybrid screen. As shown in Table 1 (pLexA as a control), the fusion protein cannot activate the Binding assay in vitro transcription of the reporter genes. So the pLexA- ADAM19-CT can be used as a bait plasmid for an GST and GST-ArgBP1 fusion protein were purified interactor hunt. as described above. The beads saturated with either fusion protein were incubated with cell lysates pre- Screen in human fetal brain cDNA library and pared from yeast. Proteins interacting with GST and identification of positive clones GST-ArgBP1 in the lysates will bind to the beads. − − The bound proteins were washed down by 2×-loading 126 transformants were isolated on Gal/Raf ura his − − buffer, separated by SDS-PAGE and were detected by trp leu medium. After identified on four kinds of western blotting. Western Blotting analysis was per- plates, 47 candidates were obtained. As shown in Ta- formed with anti-LexA monoclonal antibody, accord- ble 2, 9 positive transformants were screened out after ing to the protocol of chemiluminescence detection kit retransformation test. Sequencing results showed that (Cat No. 1500 780). they represented 4 different encoding sequences, as shown in Table 3. Four clones represent ArgBP1 (96– Identification of the interaction regions in 475aa, including complete C terminus). Two areβ- ADAM19-CT and ArgBP1 cop, two ubiquitin, and one unknown protein (designated as AD58). Owing to some striking features of A series of deletion mutants of ADAM19-CT and ArgBP1, we focused on ArgBP1. ArgBP1 were generated by PCR using primers de- signed to give a 5 EcoRI site and a 3 XhoI site. Expression and purification of GST-ArgBP1 The PCR products were digested with EcoRI and XhoI and then inserted into the corresponding sites The result is shown in Figure 1. GST-ArgBP1 was in pEG202 and pJG4-5, respectively, called pLexA- successfully expressed in E. coli BL21 and purified. ADAM19-CT-partial and pJG-ArgBP1-partial. Then the interactions between pLexA-ADAM19-CT-partial Identification of the interaction between ADAM19-CT and pJG-ArgBP1, pLexA-ADAM19-CT and pJG- and ArgBP1 in vitro (GST-Pulldown) ArgBP1-partial were inspected. Interactions were scored by nutritional selection and β-galactosidase To further confirm the association observed by yeast production as outline above. two-hybrid, an in vitro assay for interaction with the binding partners expressed in E. coli were performed. The interaction between ArgBP1 and ADAM22, Figure 2 shows that GST-ArgBP1 is able to bind to ADAM29 & ADAM9 (mouse) LexA-ADAM19-CT fusion protein in vitro.

The cytoplasmic tails of ADAM22, ADAM29 and ADAM9 (mouse), other members of ADAMs, were 320

Table 1. Test of transcriptional activity of the target protein

− − − − Medium Glu Ura His Glu Ura His − − Glu Ura His − Phenotype Leu X-gal

plexA-adam19-CT +−× plexA +−×

+ indicates clones growth, − no detectable colne growth; in X-gal containing ∗ medium, indicates clones turning blue, × clones not turning blue.

Table 2. Further identiﬁcation of positive clones

Medium G/R-3 X-gal Glu-3 X-gal Phenotype G/R-4 Glu-4

plexA-adam19-CT+the library plasmids +−∗ × plexA+ the library plasmids −−× × + the library plasmids −−× × plexA-mpl-CT+ the library plasmids −−× ×

+ indicates clones growth, − no detectable colne growth; in X-gal containing medium, ∗ indicates clones turning blue, × clones not turning blue.

Figure 1. Expression and purification of GST-ArgBP1 fusion protein. GST fusion protein expression was induced by IPTG in E. coli BL21 (DE3). Soluble proteins were released by sonication. Proteins were separated bySDS-PAGE. Molecular weight markers are shown Figure 2. Analysis of the interaction between ADAM19-CT and to the left. 1. ArbBP1 protein purified by glutathione-sepharose ArgBP1 in vitro (GST-Pulldown) in yeast lysates background. beads; 2. induced for 4h; 3. induced for 2h; 4. induced for 0h; 5. GST-ArgBP1 fusion proteins coupled to gluthathione sepharose GST protein beads were incubated with translated LexA and LexA-ADAM19 -CT fusion proteins in yeast lysates respectively. LexA-ADAM19- CT fusion proteins as control are shown to the right. 1. Identification of the interaction between ArgBP1 and GST-ArgBP1 + LexA proteins (in yeast lysates); 2. GST-ArgBP1 other members of ADAM family + LexA-ADAM19 -CT fusion proteins (in yeast lysates); 3. LexA-ADAM19- CT fusion proteins (in yeast lysates) In yeast two-hybrid assays, ArgBP1 cannot interact with the cytoplasmic tail of ADAM22, ADAM29 Identification of the interaction regions in or ADAM9 (mouse), which suggest a certain ex- ADAM19-CT and ArgBP1 tent specificity in the interaction of ArgBP1 and ADAM19-CT. According to the procedures mentioned above, the minimal regions required for interaction are deter- mined. As shown in Figure 3 (a)(b), the SH3 binding 321

Table 3. Four positive clones isolated by yeast two-hybrid

No. Length Homologous Identity Gene Biological functions (bp) gene Accession No.

A246 1600 X95633 100% ArgBP1(Arg may regulate the activity of Arg, binding protein 1) an oncoprotein. A122 1200 XM_006211 99% Beta-subunit of involed in the transport between Coatomer ER and Golgi apparatus protein(COPβ) A58 1000 AK000375 99% HEP18272clone unknown A23 750 AB009010 100% Ubiquitin related with proteolysis and cell cycle control

sites in ADAM19-CT and the P4 domain in ArgBP1 ArgBP1 (Arg binding protein 1) is a novel pro- are the key regions for their interaction. tein, which binds to Arg strongly and specifically. It was first identified in yeast two-hybrid system and then the interaction was confirmed by GST-Pulldown Discussion and co-immunoprecipitation in mammalian cells [12]. Arg is a ubiquitously expressed protein that repre- Two SH3 domain-containing proteins, endophilin I sents the mammalian member of the Abelson family of and a novel SH3 domain and phox homology (PX) nonreceptor protein tyrosine kinases (PTKs) [13, 14]. domain-containing protein termed SH3PX1, can in- There is a SH3 domain and a conserved SNARE teract with the cytoplasmic tails of ADAM9 and motif [Soluable N-ethylmaleimide-sensitive factor ADAM15 [8]. ADAM12 binds to the SH3 domain of (NSF) attachment protein (SNAP) receptor] located Src and then activates Src tyrosine kinase in C2C12 in the C-and N-terminus of ArgBP1 respectively. cell [9]. Moreover, the binding of ADAM12 cyto- SNAREs are involved in vesicle fusion [15–17]. Three plasmic tail and α-Actinin-2 is required for myoblast PEST sequences and four proline-rich sequences (P1, fusion [10]. The protease activity of ADAM9, which P2, P3 and P4) are distributed between C-terminus processes HB-EGF-like substrates, can be regulated and N-terminus, as shown in Figure 4. PESTs, which by the binding of PKCdelta and the cytoplasmic do- function as signals for rapid intracellular proteolysis, main of itself [11]. All these reports suggest that the have been identified in several signaling proteins, such cytoplasmic domains of ADAM family may affect as c-Myc, c-Fos and CDC25 [18]. Two pairs of SH3 the functions of their ectodomains. ADAM19 has a domain/proline-rich motif mediate the interaction of long cytoplasmic tail of 193aa with two putative SH3 ArgBP1 and Arg. Why we concentrate on ArgBP1 is binding sites. It may play important roles in signal also partially due to this striking SH3 domain and the transduction and in autoregulation. At present, there SNARE motif. Out of our expectation, ADAM19-CT are few reports on this issue. To elucidate the function does not bind to the SH3 domain of ArgBP1, but the of ADAM19 cytoplasmic domain, we have sought to P4 region. It is reported that ADAM family can bind identify proteins that interact with it. Using a yeast to the SH3 domain of Src homologous proteins, in- two-hybrid screen, we obtained four proteins(ArgBP1, stead of the Abl family [19]. Our observation provided β-cop, ubiquitin andAD58). Among the 9 positive evidence for it. clones, 4 represent ArgBP1. It is the human ho- In view of the fact that ArgBP1 interacts strongly mologue of a CNS-specific Xenopus gene Xlan4, a with both Arg and ADAM19, is it possible these developmentally regulated gene that is expressed in three proteins form a complex? Firstly, ArgBP1 and the central nervous system. We notice that ADAM19 Arg are both located in the cytoplasm, supporting a also plays an important role in neurogenesis. These physiological interaction. Secondly, there is also sim- impel us to pay attention to ArgBP1. ilarity in their expression patterns. Arg and ArgBP1 are both ubiquitously expressed, Arg with the highest 322

level in brain and ArgBP1with the highest levels in brain and heart. ADAM19 is detected at the highest level in heart and at an intermediate level in brain. The SNARE hypothesis provides an explanation for specificity in vesicular transport. Enlightened by it, we are considering such a hypothesis. During vesicular transport, mediated by ArgBP1, ADAM19 closes up with Arg and then processes it, thus regulates the activity and concentration balance of Arg in cells. The effects of the secretory pathway inhibitors suggest that ADAM19 participates in the intracellular processing of NRGs rather than the cleavage on the cell sur- face [4]. Other researches also demonstrated that some ADAMs are processed and activated in the trans-Golgi apparatus [20, 21]. The Abl SH3 domain has been implicated in autoinhibition [22], but the mechanism of Arg regulation is unknown. Further studies should determine whether Arg is a substrate of ADAM19. Although ADAM19 is a metalloprotease, it does not cleave the MMP (Matrix Metalloprotease) substrates. Its proteolytic activity cannot be blocked by TIMP-1, 2 (tissue inhibitors of metalloprotease). These facts indicate that ADAM19 may have a specific substrate profile, which may account for its special physiological functions [23]. β-cop is the βsubunit of COPI(coat protein I), involved in the transport from ER to Golgi and the re- verse.Ubiquitin has diverse biological functions. Dur- ing protein degradation, multiple ubiquitins bind to the unstable amino acid residues in the N-terminus. Then the protein is hydrolyzed by proteosome. It is reported that members of the HERC family may function as E3 ubiquitin ligases. One of this family, HERC3 is located in the cytosol and in vesicular-like structures containing β-cop, ARF and Rab5 proteins. HERC3 can interact with ubiquitin and is a substrate of ubiq- uitination being degraded by the proteasome [24]. In view of our observations that ADAM19 can also in- Figure 3. Identification of the interacting regions in ArgBP1 and teract with both β-cop and uniquitin, it is tempting ADAM19-CT in yeast two-hybrid assays. The SH3 binding sites in ADAM19-CT and the P4 region in ArgBP1 are responsible for to speculate a fine regulation of ADAM19 and sug- the interaction. (a)Summary of ArgBP1 deletion constructs used gest a role in vesicular traffic and ubiquitin-dependent and their ability to interact with ADAM19CT. (b)Summary of processes. ADAM19-CT deletion mutants used and their ability to interact with Among the four proteins, three are important in ArgBP1. vesicle transport and protein degradation. Consider- ing the role of ADAM19 in shedding of cytokines, we inferred that, the interactions between ADAM19 and these proteins are physiologically significant. ADAM19 may pilot the secretory proteins nearby itself and then hydrolyze them specifically. Figure 4. Structure of ArgBP1. The area indicated by arrows (from 96 to 475aa) represents the partial sequence we obtained in yeast two-hybrid screen. 323

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