Molecular Cloning, Physical Mapping, and Expression Analysis of a Novel

All articles available online at http://www.idealibrary.com on SHORT COMMUNICATION Molecular Cloning, Physical Mapping, and Expression Analysis of a Novel Gene, BCL2L12, Encoding a Proline-Rich Protein with a Highly Conserved BH2 Domain of the Bcl-2 Family Andreas Scorilas,*,† Lianna Kyriakopoulou,*,† George M. Yousef,*,† Linda K. Ashworth,‡ Aku Kwamie,* and Eleftherios P. Diamandis*,†,1 *Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, Toronto, Ontario M5G 1X5, Canada; †Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario M5G 1X5, Canada; and ‡Biology and Biotechnology Research Program, Lawrence Livermore National Laboratory, Livermore, California 94551

Received August 14, 2000; accepted November 20, 2000

loss of tissue due to ischemia, and AIDS (19). Apoptotic Members of the Bcl-2 family of apoptosis-regulating events are regulated by a number of proteins that exert proteins contain at least one of the four evolutionarily either a positive (pro-apoptotic) or a negative (anti- conserved domains, termed BH1, BH2, BH3, or BH4. apoptotic) effect. Proteins participating in these events Here, we report the identification, cloning, physical include members of the Bcl-2 family (1). Members of mapping, and expression pattern of BCL2L12, a novel the Bcl-2 family are characterized by the presence of at gene that encodes a BCL2-like proline-rich protein. least one of the BH1, BH2, BH3, or BH4 domains (14). Proline-rich sites have been shown to interact with The BH1 and BH2 domains are present in all anti- Src homology region 3 (SH3) domains of several ty- apoptotic proteins, while the BH3 domain is present in rosine kinases, mediating their oncogenic potential. the pro-apoptotic members of the family. The BH4 This new gene maps to chromosome 19q13.3 and is domain appears to be present in the N-terminal do- located between the IRF3 and the PRMT1/HRMT1L2 genes, close to the RRAS gene. BCL2L12 is composed main of the anti-apoptotic Bcl-2, Bcl-XL, and Bcl-w of seven coding exons and six intervening introns, proteins, and its function is not clear as yet. spanning a genomic area of 8.8 kb. All of the exon– In our search for genes involved in the process of apo- intron splice sites conform to the consensus sequence ptosis, we are using positional cloning techniques. A po- 2 for eukaryotic splice sites. The BCL2L12 protein is tential BH2 domain was identified in the (BAC) clone composed of 334 amino acids, with a calculated molec- BC42053, sequenced by the Department of Energy’s ular mass of 36.8 kDa and an isoelectric point of 9.45. Joint Genome Institute. Genomic sequences from this The BCL2L12 protein contains one BH2 homology do- clone were in the form of 87 contigs of different lengths. main, one proline-rich region similar to the TC21 pro- The clone containing this gene was obtained from the tein and, five consensus PXXP tetrapeptide sequences. Lawrence Livermore National Laboratory, and the BCL2L12 is expressed mainly in breast, thymus, pros- genomic DNA was isolated. The chromosome 19 EcoRI tate, fetal liver, colon, placenta, pancreas, small intes- restriction map (3) and long PCR strategies, using tine, spinal cord, kidney, and bone marrow and to a genomic DNA from this clone, were used to construct a lesser extent in many other tissues. We also identified contiguous area of the genomic area of interest. Bioinfor- one splice variant of BCL2L12 that is primarily ex- matics approaches were used to predict the presence of pressed in skeletal muscle. © 2001 Academic Press new genes, and a putative new apoptosis-related, proline- rich protein was identified. Blast search and EcoRI restriction digestion analysis of the sequence of one adja- Programmed cell death and apoptosis are highly reg- cent (telomeric) cosmid clone (R31181) allowed us to ulated physiological processes (5, 18). Disregulaton of identify the relative position of the new gene and other apoptosis results in several diseases including cancer, previously identified genes, RRAS, IRF3, and PRMT1, Sequence data from this article have been deposited with the along the same chromosomal region (19q13.3). The se- EMBL/GenBank Data Libraries under Accession No. AF289220. quence of the putative new gene was then verified by 1 To whom correspondence and reprint requests should be ad- dressed at Department of Pathology and Laboratory Medicine, 2 Abbreviations used: BCL2L12, BCL2-like 12 (proline-rich); IRF3, Mount Sinai Hospital, 600 University Avenue, Toronto, Ontario interferon regulatory factor 3; EST, expressed sequence tag; RT- M5G 1X5, Canada. Telephone: (416) 586-8443. Fax: (416) 586-8628. PCR, reverse transcription-polymerase chain reaction; BAC, bacte- E-mail: [email protected]. rial artificial chromosome; PAC, P1-derived artificial chromosome.

FIG. 1. Genomic organization and partial genomic sequence of the BCL2L12 gene. Intronic sequences are not shown except for the splice junction areas. Introns are shown with lowercase letters and exons with uppercase letters. The coding nucleotides are shown in triplets. For full sequence, see GenBank Accession No. AF289220. The start and stop codons are encircled, and the exon–intron junctions are in black square boxes. The BH2 homology domain is highlighted in black. The possible PXXP motifs are underlined, and the TC21 identical motif is doubly overlined. The putative TATA box and polyadenylation signal are in boldface type. various experimental approaches, including EST data- gene is formed of seven coding exons and six interven- base searching, RT-PCR screening of 28 tissues, and PCR ing introns, spanning an area of 8772 bp of genomic product sequencing from both directions for each tissue. sequence on chromosome 19q13.3. All of the exon– The 3Ј and 5Ј termini of the gene were veriﬁed using intron splice sites (mGT. . ..AGm) conform to the con- RACE technology. sensus sequence for eukaryotic splice sites (8). Lengths As shown in Fig. 1, the BCL2L12 (from Bcl-2-like 12) of the coding exons are 926, 115, 143, 87, 92, 273, and SHORT COMMUNICATION 219

FIG. 2. (A) Schematic representation of the BCL2L12 gene. Exons are represented by boxes, and introns are represented by connecting lines. Numbers inside the boxes or above the lines refer to basepairs, while the amino acid numbers (AA) are given in parentheses at the end of the line. Roman numerals indicate intron phases. (B) Tissue expression of the BCL2L12 gene, as determined by RT-PCR. The splice variant (lower PCR band) is highly expressed in skeletal muscle. (C) Alignment of the BH2 deduced amino acid sequence of BCL2L12 with members of the Bcl-2 multigene family. Identical amino acids are highlighted in black and similar residues in gray.

216 bp, respectively. The predicted protein-coding re- cAMP-dependent protein kinase, protein kinase C, and gion of the gene is formed of 1005 bp, encoding a casein kinase 2. In addition, several N-myristoylation deduced 334-amino-acid polypeptide with a predicted sites have been predicted (Table 1). molecular mass of 36.8 kDa and an isoelectric point of The BCL2L12 protein was found to have proline-rich 9.45. An (AATAAA) sequence is identical with a con- sites. One PPPP site as well as five PP amino acid sites sensus polyadenylation signal (13) and is followed, af- is present in this protein. Eight putative PXXP motifs ter 35 nucleotides, by the poly(A) tail not found in the were also identified (Fig. 1). Proline-rich motifs are genomic sequence. Another (ATTAAA) sequence was characterized by the presence of the consensus PXXP found 11 bases upstream of the poly(A) tail and was tetrapeptide, found in all proline-rich proteins identi- reported to occur as a natural polyadenylation variant fied to date (2). It is known that SH3 domains recognize in 12% of vertebrate mRNA sequences (15). proline-rich sequences and that all known SH3-bind- The highlighted region in Fig. 1 indicates a 16- TABLE 1 amino-acid sequence, found in the BH2 domain of several members of the Bcl-2 family of proteins (14). The Putative Posttranslational Modification Sites in the amino acid loop (WIQXXGGW) at positions 311–318 was Novel BCL2L12 Gene also found in the Bcl-2, Bax, and Bcl-XL proteins (Fig. a a 2C). No BH1, BH3, and BH4 domains were found. Bcl-2 Modification Residue BCL2L12 BCL2L12-A family genes containing only one BH domain have been O-glycosylation Thr 117 117, 151 reported (9). Although most Bcl-2 family members con- 113, 121, tain a combination of the BH1, BH2, BH3, or BH4 do- Ser 263, 273 113, 145 mains, a truncated form of the Bax protein (11) and the Protein kinase C mammalian activator of apoptosis, Hara-kiri (9), contain phosphorylation Thr 97, 204 97 Ser 151, 259, 287 — only one domain. Thus, it appears that a full complement Casein kinase II of BH domains may not be necessary to confer apoptotic phosphorylation Thr 204 — or anti-apoptotic activity. Through the use of sequence Ser 255, 325 153 analysis tools, we were able to identify various putative 82, 145, 292, 82, 130, 159, posttranslational modification sites. There are numerous N-myristoylation Gly 294, 295, 316 166, 171 potential sites for O-glycosylation. Furthermore, several a The residues are numbered according to the sequence shown in possible sites of phosphorylation have been identified for Fig. 1 and our GenBank submission (Accession No. AF289220). 220 SHORT COMMUNICATION ing proteins contain proline-rich regions with at least during events such as apoptosis or its repression, epi- one PXXP motif (2, 6, 10, 16). Proline-rich domains dermal factor signaling, cellular localization of cyto- have been identified in a number of diverse proteins plasmic proteins, and activation of the phosphatidyl- such as epidermal growth factors, phosphatidylinositol inositol 3-kinase in response to IgM cross-linking (4, 7, 3-kinase, and, more recently, the small GTPase RRAS 10, 12). The presence of the BH2 domain, present in protein and members of the RRAS superfamily such as most anti-apoptotic proteins, together with the proline- the TC21 protein (10, 17). Moreover, the amino acid rich motifs suggests that the BCL2L12 protein may loop (PPSPEP) at positions 271–276 of the BCL2L12 participate in protein–protein interactions. To our protein is identical with the PXXP motif present in the knowledge, this is the first gene identified encoding a RRAS and TC21 oncogenes (17). This motif is required protein that contains both a proline-rich domain and a for integrin activation. BH2 domain. The presence of both domains, if func- PCR screening for BCL2L12 transcripts using gene- tional, may represent the crossroads between proteins specific primers revealed the presence of two bands in involved in the regulation of apoptotic events and pro- most of the tissue cDNAs examined. The two bands teins containing SH3 domains. were gel-purified, cloned, and sequenced. The upper band represents the classical form of the gene, and the ACKNOWLEDGMENTS lower band is a splice variant. As shown in Fig. 2A, this variant (BCL2L12-A) is missing exon 3 (143 bp). 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