Cloning of AIP1, a Novel Protein That Associates with the Apoptosis-linked Gene ALG-2 in a Ca2+-dependent Reaction

Abstract

ALG-2 is a 22-kDa calcium-binding protein necessary for cell death induced by different stimuli in 3DO T-cell hybridoma. 3DO cell clones depleted of ALG-2 protein exhibit normal caspases activation, suggesting that ALG-2 function is required downstream or is independent of caspase proteases activity for apoptosis to occur. Using the yeast two-hybrid screening system, we have isolated and characterized the mouse cDNA encoding for ALG-2 interacting protein 1 (AIP1), a novel protein that interacts with ALG-2. ALG-2 and AIP1 colocalize in the cytosol and the presence of calcium is an indispensable requisite for their association. Sequence alignment shows that AIP1 is highly similar to BRO1, a yeast protein related to components of the Pkc1p-MAP kinase cascade.Overexpression of a truncated form of AIP1 protects two different cell types from death induced by trophic factors withdrawal; thus, our data indicate that AIP1 cooperates with ALG-2 in executing the calcium-dependent requirements along the cell death pathway. ALG-2 is a 22-kDa calcium-binding protein necessary for cell death induced by different stimuli in 3DO T-cell hybridoma. 3DO cell clones depleted of ALG-2 protein exhibit normal caspases activation, suggesting that ALG-2 function is required downstream or is independent of caspase proteases activity for apoptosis to occur. Using the yeast two-hybrid screening system, we have isolated and characterized the mouse cDNA encoding for ALG-2 interacting protein 1 (AIP1), a novel protein that interacts with ALG-2. ALG-2 and AIP1 colocalize in the cytosol and the presence of calcium is an indispensable requisite for their association. Sequence alignment shows that AIP1 is highly similar to BRO1, a yeast protein related to components of the Pkc1p-MAP kinase cascade. Overexpression of a truncated form of AIP1 protects two different cell types from death induced by trophic factors withdrawal; thus, our data indicate that AIP1 cooperates with ALG-2 in executing the calcium-dependent requirements along the cell death pathway. Programmed cell death (PCD) 1The abbreviations used are: PCD, programmed cell death; HA, hemagglutinin; kb, kilobase(s); GST, glutathioneS-transferase; PAGE, polyacrylamide gel electrophoresis; MAP, mitogen-activated protein. is a physiologically regulated cell type-specific deletion that takes place during various developmental stages of multicellular organisms and is essential for the establishment and the maintenance of cellular homeostasis (1Jacobson M.D. Weil M. Raff M.C. Cell. 1997; 88: 347-354Abstract Full Text Full Text PDF PubMed Scopus (2414) Google Scholar). PCD occurs by apoptosis, which refers to the morphological changes that can be observed in cells undergoing PCD. These include plasma membrane blebbing, cell shrinkage, chromatin condensation, and DNA degradation (2Wyllie A.H. Morris R.G. Smith A.L. Dunlop D. J. Pathol. 1984; 142: 67-77Crossref PubMed Scopus (1442) Google Scholar). The process eventually culminates with the fragmentation of the cells into apoptotic bodies that, in vivo, are rapidly phagocitized by the surrounding cells (3Arends M.J. Wyllie A.H. Int. Rev. Exp. Pathol. 1991; 32: 223-254Crossref PubMed Scopus (1394) Google Scholar). The suicide signal can be received and transduced into the cell through death receptors that initiate a signaling cascade, which shortly leads to cell demise (4Chinnaiyan A.M. Dixit V.M. Semin. Immunol. 1997; 9: 69-76Crossref PubMed Scopus (94) Google Scholar). A number of molecules have been identified that either regulate the death pathway or are able to modulate the death signal. Most attention has recently focused on the interleukin-1β converting enzyme-like proteases, a family of at least 10 related mammalian cysteine proteases collectively named caspases (5Cohen G.M. Biochem. J. 1997; 326: 1-16Crossref PubMed Scopus (4145) Google Scholar). These proteins are considered the executioners of mammalian apoptosis by virtue of two seminal observations: (a) ectopic overexpression of interleukin-1β converting enzyme, as well as other members of the family, results in protease activation and induces apoptosis (6Miura M. Zhu H. Rotello R. Hartwieg E.A. Yuan J. Cell. 1993; 75: 653-660Abstract Full Text PDF PubMed Scopus (1332) Google Scholar, 7Kumar S. Kinoshita M. Noda M. Copeland N.G. Jenkins N.A. Genes Dev. 1994; 8: 1613-1626Crossref PubMed Scopus (588) Google Scholar), and (b) specific inhibitors of these proteases inhibit cell death (8Enari M. Hug H. Nagata S. Nature. 1995; 375: 78-81Crossref PubMed Scopus (798) Google Scholar, 9Los M. Van de Craen M. Penning L.C. Schenk H. Westendorp M. Baeuerle P.A. Droge W. Krammer P.H. Fiers W. Schulze-Osthoff K. Nature. 1995; 375: 81-83Crossref PubMed Scopus (646) Google Scholar). Accordingly, the caspase paradigm asserts that cells undergoing apoptosis accomplish their suicide program by activating a hierarchy of caspases. However activation of caspases does not always correlate with induction of apoptosis (10Miossec C. Dutilleul V. Fassy F. Diu-Hercend A. J. Biol. Chem. 1997; 272: 13459-13462Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 11Wilhelm S. Wagner H. Hacker G. Eur. J. Immunol. 1998; 28: 891-900Crossref PubMed Scopus (103) Google Scholar), and in some circumstances, caspase activity is required for protection from apoptosis (12Bergeron L. Perez G.I. Macdonald G. Shi L. Sun Y. Jurisicova A. Varmuza S. Latham K.E. Flaws J.A. Salter J.C.M. Hara H. Moskowitz M.A. Li E. Greenberg A. Tilly J.L. Yuan J. Genes Dev. 1998; 12: 1304-1314Crossref PubMed Scopus (605) Google Scholar). In addition to proteases activation, numerous evidence demonstrates that alterations in intracellular calcium play an important role during apoptosis. For some time it has been proposed that DNA fragmentation, a general hallmark of apoptosis, is a Ca2+-dependent event that requires a Ca2+-activated endonuclease (13Cohen J.J. Duke R.C. J. Immunol. 1984; 132: 38-42PubMed Google Scholar, 14Peitsch M.C. Polzar B. Stephan H. Crompton T. MacDonald H.R. Mannherz H.G. Tschopp J. EMBO J. 1993; 12: 371-377Crossref PubMed Scopus (546) Google Scholar). Induction of apoptosis triggers an increase of [Ca2+]i as well (15Nakayama T. Ueda Y. Yamada H. Shores E.W. Singer A. June C.H. Science. 1992; 257: 96-99Crossref PubMed Scopus (65) Google Scholar, 16McConkey D.J. Hartzell P. Amador-Perez J.F. Orrenius S. Jondal M. J. Immunol. 1989; 143: 1801-1806PubMed Google Scholar), and intracellular chelators of Ca2+ inhibit cell death (17Oshimi Y. Miyazaki S. J. Immunol. 1995; 154: 599-609PubMed Google Scholar). As consequence of Fas stimulation, calcium is immediately mobilized from intracellular stores (17Oshimi Y. Miyazaki S. J. Immunol. 1995; 154: 599-609PubMed Google Scholar), and lymphoid cells rendered deficient for the calcium release channels are resistant to apoptosis induced by different stimuli (18Jayaraman T. Marks A.R. Mol. Cell. Biol. 1997; 17: 3005-3012Crossref PubMed Scopus (235) Google Scholar, 19Khan A.A. Soloski M.J. Sharp A.H. Schilling G. Sabatini D.M. Li S.H. Ross C.A. Snyder S.H. Science. 1996; 273: 503-507Crossref PubMed Scopus (245) Google Scholar). Of interest, and consistent with the literature, the antiapoptotic protein Bcl-2 decreases calcium release from the endoplasmic reticulum stores (20Lam M. Dubyak G. Chen L. Nunez G. Miesfeld R.L. Distelhorst C.W. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6569-6573Crossref PubMed Scopus (613) Google Scholar). Considering these data altogether, the existence of a Ca2+-sensitive step(s) along the cell death pathway is clearly apparent. Although a precise picture of how Ca2+ exerts these effects at the molecular level is still not available, it is likely that transduction of Ca2+-regulated signals occurs through Ca2+-binding proteins. Members of the EF hand Ca2+-binding protein family exhibit Ca2+ affinities and binding kinetics compatible with concentration and time range of Ca2+ wave (21Ikura M. Trends Biochem. Sci. 1996; 21: 14-17Abstract Full Text PDF PubMed Scopus (603) Google Scholar). The functional and structural Ca2+-binding unit is the helix-loop-helix motif commonly called EF hand domain. One of them, ALG-2, is a 22-kDa protein that was identified during a screening for genes involved in apoptosis (22Vito P. Lacana E. D'Adamio L. Science. 1996; 271: 521-525Crossref PubMed Scopus (457) Google Scholar, 23D'Adamio L. Lacana E. Vito P. Semin. Immunol. 1997; 9: 17-23Crossref PubMed Scopus (30) Google Scholar). T-cell hybridoma clones depleted of ALG-2 via transfection of the antisense cDNA are resistant to cell death induced by diverse stimuli, including T-cell receptor and Fas stimulation as well as dexamethasone, staurosporine, and ceramide treatment. Nonetheless, in these ALG-2-deficient clones caspases are normally activated upon induction of cell death, as determined by cleavage of poly(ADP-ribose)polymerase and of a fluorogenic substrate (24Lacana E. Ganjei J.K. Vito P. D'Adamio L. J. Immunol. 1997; 158: 5129-5135PubMed Google Scholar). In this paper we report the cloning of AIP1, a novel gene that interacts with ALG-2 in a calcium-regulated fashion. Overexpression of a deletion mutant of AIP1 protects HeLa and COS cells from apoptosis induced by serum starvation; thus AIP1 might mediate, at least in part, the ALG-2 requirement for apoptosis. The two-hybrid screening was conducted using the Matchmaker system fromCLONTECH according to the manufacturer's instructions. Yeast strain Y190 was transformed with the corresponding bait plasmids by lithium acetate/polyethylene glycol 4000 procedure and selected on synthetic dropout plates lacking tryptophan. Selected colonies were then analyzed for expression of the GAL4-bait fusion protein by immunoblot analysis. For library screening, yeast Y 190 expressing GAL4-ALG-2 fused protein was sequentially transformed with a mouse liver cDNA library cloned in the pGAD10 vector (CLONTECH). 2 × 106 clones were analyzed. Transformed yeast were selected on synthetic dropout/agar plates lacking leucine, tryptophan, and histidine in the presence of 50 mm 3-aminotriazol (Sigma) and grown for 5 days at 30 °C. Colonies positive for growth on selective media were blotted on filter paper (Whatman No. 5), permeabilized in nitrogen liquid, and placed on another filter soaked in Z buffer (60 mm Na2HPO4, 40 mm NaH2PO4, 10 mm KCl, 1 mm MgSO4, 37.5 mmβ-mercaptoethanol) containing 1 mm5-bromo-4-chloro-3-indolyl-β-d-galactoside. Colonies that developed color were re-streaked on selective plates to allow plasmid segregation and tested again for β-galactosidase activity. Yeast colonies were then scored as positive when a bright color developed in 2–5 h, a negative was scored when color failed to develop within 12 h. Assays were done for 5–10 independent transformant. The cDNA insert contained in clone TH 28 was used to screen phage murine liver and a 3DO T-cell hybridoma cDNA libraries. Nucleotide sequences of several cDNA clones, as well as the cDNA insert of clone TH 28, were determined on both strands with Sequenase kit (Amersham Pharmacia Biotech). The cDNA insert contained in clone TH 28 was also used to probe a mouse multiple tissue Northern blot (CLONTECH) according to the manufacturer's protocol. The cDNA insert of clone TH 28was cloned in pcDNA3 (Invitrogen) with an HA epitope the provides a Kozak consensus sequence and a methionine for translation. AIP1 full-length (3.8 kb) was cloned in pcDNA3 joining a 1.6-kbEcoRI/BsrGI fragment from a liver cDNA clone with a 2.4-kb BsrGI/NotI fragment from clone TH 28. Most of the other plasmid constructs used were made by polymerase chain reaction performed with primers containing appropriate restriction sites or epitope tags as needed. GST fusion proteins were made in pGEX vectors (Amersham Pharmacia Biotech). Plasmid constructs were confirmed by partial sequencing and immunoblot analysis. The cDNA encoding for full-length AIP1 was transcribed and translated in vitroin the presence of l-[4,5-3H]leucine with the TNT-coupled lysate system (Promega) following the manufacturer's instructions. 293T, L66, HeLa, and COS cells were cultured in Dulbecco's modified Eagle's medium, 10% fetal calf serum. 3DO cells were cultured in RPMI, 5% fetal calf serum. The polypeptide DEIKKERESLENLK was used to generate rabbit polyclonal antisera. The whole rabbit serum was affinity-purified on CNBr-activated Sepharose beads (Amersham Pharmacia Biotech) coupled with the antigenic peptide. HeLa and COS7 cells were transfected with lipofectAMINE (Life Technologies, Inc.) following the manufacturer's indications. 293T cells were transfected by the calcium phosphate method. Cell lysates were made in RIPA buffer, and protein concentration was determined by Bio-Rad protein assay; 10–20 μg of protein was subjected to polyacrylamide gel electrophoresis. Proteins were then transferred to nitrocellulose membrane and incubated with primary antibody followed by a secondary antibody horseradish peroxidase-conjugated (Promega). Blots were developed using SuperSignal (Pierce) and visualized by exposure to autoradiography film. For coimmunoprecipitation experiments, cells were lysed in lysis buffer (50 mm Tris-HCl, pH 8, 150 mm NaCl, 1 mm EDTA, 1% Nonidet P-40, and a protease inhibitor mixture). Lysates were divided and immunoprecipitated with the indicated antibodies; immunocomplexes were bound to protein A-agarose beads and resolved by SDS-PAGE as described above. L66 cells were grown in chamber slides, fixed in 4% paraformaldehyde for 15 min at room temperature, and then permeabilized in phosphate-buffered saline, 0.5% saponine. AIP1 was visualized by incubating the cells with the affinity-purified anti-AIP1 antiserum for 30 min, washing several times with phosphate-buffered saline, 0.5% saponine, and then incubating for 30 min with a fluorescein mouse L66 cells in the presence of an of as negative were done at room the were analyzed with a For cells were with a in lysis mm pH 10 mm KCl, mm 1 mm EDTA, and a of protease were by the at × 10 min, and the was at for min to the and the membrane fusion protein was times in buffer mm pH 150 mm and to of binding buffer, which of buffer with 1 mm and either or The of of these to a concentration of was with program A. PubMed Scopus Google Scholar). encoding and ALG-2 proteins were in as described above. The lysate was to the protein in binding buffer and incubated for 1 at room with In the in 5 of in and ALG-2 proteins were incubated in binding buffer containing the indicated and were immunoprecipitated with an antibody and protein beads The beads were then times in binding buffer, in buffer, and the analyzed by SDS-PAGE followed by COS and HeLa cells were transfected with 2 μg of pcDNA3 expressing the indicated cDNA with μg of (CLONTECH). transfection the cells were with 1 or 1 (Sigma) for or the fetal calf serum concentration to activity was visualized by the cells in for 10 min followed by in phosphate-buffered containing mm and and 1 for at °C. The number of cells and cells with apoptotic were in at least ALG-2 interacting we performed a two-hybrid screen with the full-length ALG-2 cDNA fused to the DNA binding A plasmid library of fusion the activation and from mouse liver was for with fusion protein in the yeast strain A of clones were isolated that activated the β-galactosidase when 2 × 106 plasmids were and sequencing of these that clones TH 28, the insert containing an for fused to were then performed with clone TH 28 in the yeast As in this library clone not β-galactosidase by or when with the vector or with a it with and was with truncated of ALG-2 at the or the full-length ALG-2 interacts with TH 28 in clone TH 28 and ALG-2 β-galactosidase activity was determined by a filter for yeast transformed with the indicated plasmid as described of the full-length ALG-2 and the deletion constructs used in the The the calcium binding the indicate the in a β-galactosidase activity was determined by a filter for yeast transformed with the indicated plasmid as described of the full-length ALG-2 and the deletion constructs used in the The the calcium binding the indicate the Northern clone TH 28 a and an The 3DO T-cell the and the of are not regulated by T-cell receptor stimulation or not Using clone TH 28 as we a mouse liver cDNA phage library and cloned the full-length that we named AIP1 for ALG-2 The sequences of several phage clones indicated that AIP1 of Accordingly, the in of full-length AIP1 cDNA a polypeptide of 1 A that several of have been in the S. Weil J. 1997; PubMed Scopus Google in the S. S.H. J. 1997; PubMed Google Scholar), and in the Mol. Cell. Biol. 1996; PubMed Scopus (65) Google Scholar). and evidence that and in signal transduction A rabbit antiserum was the polypeptide of the AIP1 which is also in the TH 28 performed on cell lysates from L66 and 3DO T-cell hybridoma that this antiserum the AIP1 protein as a with an molecular of 293T cells transfected with AIP1 cDNA exhibit a specific that with the AIP1 2 signals from murine cell lysates and transfected 293T cells were the was by with the not then tested the ALG-2 and AIP1 also occurs in mammalian As in 2 AIP1 with ALG-2 in lysates from As TH 28 with ALG-2 as that in overexpression TH 28 can inhibit the ALG-2 and In in lysates from L66 cells and the T-cell the AIP1 with the ALG-2 2 ALG-2 and AIP1 in mammalian the cellular of AIP1, L66 were permeabilized and with the anti-AIP1 that AIP1 has a consistent with a of the protein confirmed this of lysates from L66 cells that AIP1 was in the a of the protein also in the In the ALG-2 in the with the these data that ALG-2 and AIP1 colocalize in the the activity of Ca2+-binding proteins by binding to their EF hand of these proteins upon Ca2+ which the with (21Ikura M. Trends Biochem. Sci. 1996; 21: 14-17Abstract Full Text PDF PubMed Scopus (603) Google Scholar). ALG-2 is to a Ca2+-dependent (22Vito P. Lacana E. D'Adamio L. Science. 1996; 271: 521-525Crossref PubMed Scopus (457) Google Scholar), we Ca2+ modulate the with In these we used several from the of Ca2+ in cells to a concentration that is during Ca2+ from As in in AIP1 and fusion protein when at least Ca2+ is in the binding The of Ca2+ was by Ca2+ with another intracellular of not these data indicate that ALG-2 and AIP1 in a and when intracellular of Ca2+ increase the normal The of AIP1 with ALG-2 to the effects of AIP1 expression on programmed cell for AIP1 and were with a plasmid encoding the β-galactosidase gene in HeLa which were with and in cell death be and in these not when was by trophic factors a was observed on HeLa and COS cells transfected with TH 28 A was also when cells were with or In this of experiments, the of ALG-2 along with TH 28 the to cell death and from has in the of apoptosis in mammalian In lymphoid results in a increase in intracellular followed by as apoptosis or cellular activation and apoptotic cell death in a of suggesting that an increase in intracellular is to signal the cell to the apoptotic that essential for a to the of is on the activation of specific the this calcium to cellular apoptosis are ALG-2 is a Ca2+-binding protein to be involved in the of programmed cell death; the ALG-2 is still In this we have identified and characterized AIP1, a protein that interacts with ALG-2 in a Ca2+-dependent The presence of Ca2+ is essential for this to requirements necessary for which are when ALG-2 is in a The range of for the of the two proteins in is compatible with of In in cells Ca2+ is at concentration or release from intracellular stores can in an increase to the range required for the that AIP1 and ALG-2 are in cells and following stimulation that results in intracellular of a truncated form of AIP1 apoptosis by some The of these is that ALG-2 and AIP1 is required for cell death, and the polypeptide by TH 28 as a negative inhibitor of AIP1 by for binding to ALG-2. it is that the truncated protein by TH 28 might still some of the by the of apoptosis that we observed was partial and with a specific allow to the role of this in programmed cell death and in cell in AIP1 shows a of to BRO1, a S. protein of that is to AIP1 the sequence Mol. Cell. Biol. 1996; PubMed Scopus (65) Google Scholar). and functional evidence to components of the Pkc1p-MAP kinase in similar to by deletion of a gene encoding a kinase that maintenance of cell in a growth which is by calcium binding it is likely that, in the function of is regulated by an protein. The AIP1 and that by which the might programmed cell death is by activation of signal transduction to with this is the evidence that kinase is required for apoptosis induced by trophic factors J.L. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). are to Ca2+-regulated signals through AIP1, activation of of other Ca2+-dependent protein and in apoptosis has been of a Ca2+-dependent protein that in T-cell activation, rapidly induces apoptosis in the of growth factors F. F. J. Cell Biol. 1995; PubMed Scopus Google Scholar). the it is that an in gene the to apoptosis. is also by the evidence that is activated during Ca2+-dependent apoptosis, and Bcl-2 the signaling through by binding and of F. E. F. Nature. 1997; PubMed Scopus Google Scholar). AIP1 a role in this to be is the A. gene encoding for a protein which to AIP1 and in the of gene expression S. S.H. J. 1997; PubMed Google Scholar). A of AIP1 also in C. S. Weil J. 1997; PubMed Scopus Google Scholar), which for a protein. and AIP1 their a containing the consensus sequence of likely that AIP1 is involved in a of cellular of which apoptosis is of the and Ganjei for and and for of the and members of the of and and the for for and