Hem-1, a potential membrane protein, with expression restricted to blood cells

Bioclumica et Biophysica Acta, 1090 (1991) 241-244 © 1991 Elsevier Science Publishers B.V. All ~ights reserved 0167-4781/91/$03.50 ADONIS 016747819100224X BBAEXP 90265

241

Short Sequence-Paper

Hem-l, a potential membrane protein, with expression restricted to blood cells R o b e r t H r o m a s ~, Steven Collins 2,3, W e n d y R a s k i n d 3, Larry D e a v e n 4 and Ken Kaushansky 3 ! Department of Medicine IB-442, Indiana University Medical Center, Indiannlm~ IN (U.S.A.), z Departmem of Molec~dar Medicine, Fred Hutchinson Cmcer Research Center, Seattle, WA (U.S.A.), "*Delmrtm~ of Medicine, University of Washington School of Medicine, Seattle, WA ( U.S,4.) and ¢ Life Sciences Division, Los Alamos National Laboratmy, Los Alantm, NM (U.S.A.) (Received 19 July 1991)

Key words: Hematopoiesis; Membrane protein; Phosphorylation site; Chromosomal translocation

Overlapping cDNAs 3.8 kb in length containing a long open reading frame were obtained that hybridized exclusively to transcripts from hematopoietic eells. Sequence analysis found eight potential membrane domains and two possible cAMP/cGMP phesphorylation sites. This sequence exhibited no homologies with the EMBL/Genbank nucleic acid, SwissProt or GenPept amino acid data bases. The gene is located at 12qD.l, a region of eccasienul translecatious in hematopoletic neoplasia and a rare folk acid fragile site, Fra 12A. Blood cells of all lineages develop continuously from a common hematopoietic stem cell. With the advent of hematopoietic colony formation assays, the environmental signals (the colony stimulating factors) that control lineage divergement have been well described [1]. However, there are common genetic elements in all lineages. These common phenotypes might be important in the decision of hematopoietic stem cells to differentiate or proliferate. There are three genes that have been described that are ubiquitous in and yet restricted to blood cells: CD45, a surface membrane glycoprotein with tyrosine phosphorylase activity [2]; HS1, which has homology to helix-turn-helix motifs in its N-terminus and src-family tyrosine kinases in its C-terminal end [3]; and vav, which is an oncogene that also has a helix-turn-helix motif at its N.terminal and two Cys-Cys zinc fingers in the middle portion of the protein [4]. Here we report a fourth gene, designated Hem-l, whose expression is also common among blood cells.

The sequence data in this paper have been submitted to the EMBL/Genbank Data l.z'braries under the accession number M58285. Correspondence: IL Hrmnas, Department of Medicine nlB-442, Indiana University Medical Center, 975 W. Walnut Street, Indianapolis, IN 46202-5121, U.S.A.

While screening a large number of polymerase chain reaction generated products [5], we found a sequence that was expressed in only hematopoietic cells. Using this fragment, we cloned the complete Hem-1 cDNA. This cDNA has 3836 nucleotides in total ~ength and contained a long open reading frame of 10;6 amino acids (Fig. 1). The 5' most methionine is placed at the translational start site. It matches a Kozak consensus at 9/13 nucleotides [6]. There are four termination codons in frame that are 5' to this methionine. There are nine potential N-glycosylation sites according to the parameters of Marshall and Blause [7,8]. There are two cAMP/cGMP-depondant kinase phosphorylation sites according to Glass and colleagues [9]. In addition, there are eight potential membrane-associated dompins according to Eisenberg and colleagues [10]. Although there is no classic leader sequence, the first thirty amino acid residues are hydrophobic, and designated a membrane-associated domain according to Ref. 10. The sequence is not homologous to aay other in the EMBL/Genbank, PIR, GenPept or Swissprot data base. The tissue specificity of Hem-1 expression was explored using Northern blot analysis. Fig. 2 shows poly(A) + Northern blots probed with the nick-translated Hem-1 cDNA. Two mRNA species are present; there is a major transcript about 4 kb in size and a minor transcript 7 kb. There also may be another

242

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GTC TT'I" GAC ATC TGC TTA CCA G~A TAA ATT A ~ AGA GAA GCT C ~ TAT CAT C~A G ~ C~C T'rG TTC ~ ~C~ ~ A~ T~ ~ ~ ~ ~ ACT ATT TCC CAA CAT AGA TGT CGA AAC ~ ACG CAA CAT TTA GGA CCA GZA TAC CAG TCA TTT GTO CAT GTC ATG CAT TTC GG~ AT~ ATG TAT A'J~ ~ 14 D F G ! H Y H ~llJ TTC TC~ ACA CCA TTG ATG CCT GO~ AGT GCC ATT TTG ATA TCA ATC TCA ACT TTG ATT TTC ACT CGG AGT TAC CTG GAC ~ A"JrTGTA A~r iF S T P L H P q S A ~ ~. ~ ~ I S T L l F T , R :3 Y L D L I V T TAC ACC TCA GTC AFT TTA CTT CTG TCA C(~ ATT GAA CAT CGG CC~ ATA ~ ATT C;GC ATG TAC AAT TGT GCC CAT GAG ATG CTG CAT GGG Y T S V I L L L S R l E D R R l L I G H Y N C A lJ E H L H O H G D ~ S F A R L G Q H V L E Y D 14 P L K K L F G ACAAAGGCTGTGAGTGGAGCCCT~CTC~'T T T G C A T T T C C T C T T T G T C C ~ A ~ ~ ~ AGTGCC T K A V S G A L L S L fl F L F VjR R N Q G A ~ Q W R 5 A Q L CTA AGC CTC A'I"~ J~C AAC CCC CCA GCC ATG ATT ~ C CCT ~ AAT TCA GAT ACA ATG GO~ TGT GAG TAT ~ TG'r ~ GAA GTA ATG GAG L S L I S N P P A H I N P A N S D T H ~ C E Y tL S V E V H E CGC TGG ATT ATC ATT GGG TTT CT~ CTT TGT CAT GOG TGC CTC AAC TCC AAT AOC CAG ',C-C CAG /LAG CTG TGG AAG c'rG TGT ~ CAG GGC R H I I I G F L L C H G C L j P S N S Q C Q K L M K L C L Q G TCC CTC TAC ATC ACC CTT ATC CGT GAG GAT GTG CTG CAG GTU ~ AM GTC .~C GAG GAC CTG TTT AGC AGT TTG ~ GGG TAT GGC P.4G S L Y ! T L I R E D v L Q V fl K v T E D L F S S L K O Y G K AGA GTG GCA GAC ATA AAG GAG AG~ AAG GAA CAT GTA ATT GCA AAC AGT ~ CAG TTT CAT TGT CAA C3~GCOG CAA TTT C"i'G CGG AT(; GCA R V A D l K E S K E H V I A II S G O F II C O g R O F L R M A ~ o ~ o ~ o CT~ G ~ ACT ~TO TT~ OCT G~T G ~ C ~ G ~ CT^ CT~ ~ T c c r A ~ o c t CTT T~T OCT TTC ^TO GCC C ~ TO: TTC ~T~ COT v x ~ x. z T vt F ~ ~ ~ e G ~ c ~ ~= x ~ t. ~" ~ F II A L s • ~. n GAT GAG GTC ACC: TGG CTO GTT CO~ CAC ACA GAG ~ GTC ACC AAG ACA AAG ACA CCT GAG ~ TAT ~ ~ ~ ~ A~ ~ ~ ~A o [ v T . c v R . T Z V T tc T x : L= E p ~ ^ D S x A z L crr ~ 'r~ rr'; c,~ ~ ^ r r ~u~ ~c'r c'ro ~ r c ~ ^ c ~ ^TC ~ ~TC; ^T^ c ~ c,v, T~C ~ c CTT C ~ T~C TTO G ~ ~ T~ c~z L F c c z ~ x ~ s ~ v R ~ . x x v z o o ~ . ,~ O ~ ~ A R r n GCT CTT GTG CTC AGT GJ~ .TC .TT C~O ~ TTO TCT GTG TGT CC& GAG G~O GAG TCC . ~ .TC & ~ T ~ ~ TTC ~ ~ A~ ~ ~ A L V L 5 D I l L S V C ~ E E E S l X H S S F V S TCT CTG AAT CTC AAA CAA GTT GAT PAT GAA AAA TTT GAA TCC TCA GGA T~G AGG CTG GAC TOG TTC CGC CTA CAG G~q TAC CAT AGC S L N L K Q V D N G E K F E S S G L R L D W F R L Q A Y H S GTG GCT AAG GCC CCT CTG CAC CTG CAT GAG AAC CCT GAC TTA GCC /LAGGTG ATG ~ CTC ATT GTC TTC CAC TCC CGA AT(; CTG GAC TCC V A IC A P L H L il E H P D L A K V H N L X V F U S R M L D S GTA GAA AAA TTG CTG GTG GAP. ACT TCT GAT CTG TCT ACT TTC TOC TTT CAT CTT CGT ATC TTT GAG AAG ATG ~ GCC ATG ACC ~ GAG V E K L L V E T S D L S T F C F fl L R I F E K H F A 14 T L sr GAA TCT GCC AT() TTG ~;T TAT GCC ATT GCT TTC CCC C"FGATT T~r GCT CAC TTT GTC CAC TC;C ACT CAT GAG ATG TGC CCA GAG GAG TAC E S A H L R I Y A 1 A F P L I C A U F V H C T U E H C PJE E Y CCC CAC CTC AAG AAC CAT GGT CTT CAC CAC TGC AAC TCC TTC CTG GAA GAG ~ ~ ~ ~ ~ ~ ~T ~ ~ ~ ~ A~ ~ r tl L J~ N H O L It H C N S F L E E L A K O T S N C V L Z l C GCT OAG CAG C ~ ~)d~ C'fO A ~ GAG CAG CTr G ~ C:~ AAG CAC TGT C ~ ACT .~..A ATC ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ A E O R ~.) L S E O L A P K H C A T T I S K A K N K K T R [ Q AOG CAG ACT CCC ~ A.~ GGA GAG CCC GAG AGG GAC A~G CCA GGA GCT G,~ AGT C~C CGG ~ ~ CG~ AGC ATT GTC ACC AAC ATG G~K: It K O E P E R n K P G ^ E S H R K(~ R s l v T J( H n R P O T M o c~A CAC c ~ M ~ TTG ~ G ~ CTG GCA CTG ACA ATO AAT CAT GTA TA: AGT TTC TCC GTG ~ r GAA CAT ACT ATC TI~ CCT TCT GAG K L H L ~M~ L T E L A L T H N H V Y S F S V F E H T I F P $ E TAC CTC J~C AGC ~ CTG GAG GCC AGA CTC AAC AGA GCC ATT GTG TGG CTG GCT GGC TAC ~ T GCC ACG ACC CAG GAG AT~ GTA CGO ~ Y ~ s s u L z ^ ~ L . ~ ^ x v . L ^ O Y (~) A ~ ~ Q Z X V Z ~ '/~J" GAG CTG T~G GCA GGA GTC AAA GCA TAC A'/~ GGT TTC ATA CAG TCA CTG GCC CAG ~ ~ GGT GCA GAT G~r TCC AGA GTC ATC CGC s Z*L ~ ~ G v x ~ y x c) T ~ q ~; L ~ o F L ~ ~, n ~ s R v x It AAG CCC CTC CTG CAG CAG ACA CAA CCA clrG GAT TCC TGT GGG GAA CAG ACA ATC ~ ~ ~ T~ ~ ~ ~ T~ ~ ~ ~ ~ K P L L Q Q T Q P L D S C G E Q T | T T L ¥'T N W Y L E S L CrT ~ C ~ GCA ~ C ~ ' r ~ C ~ A~C ATC ~ ~ ~ ~O: ^'rG ~ GCC ~ GTC ~ CTG CCC ~ G.~ G ~ GAG C.~ . ~ TTC ~ ~ R Q A S S G T ! ! L S P A M Q A F V S L r R E G E Q ~ • S GCA GAG GAG TTC TCT GAC ATC TCT GAG ATG CGG GCC TTO OCA GAA CTC CTO G0C CCC TAT GGC AT(; AAG TTC CTG AGT GAA C~S ATG A E E F S D 1 S E' H R A L A E L L G P ¥ G H K F L S E M L N TGG CAT GTG AO~. TCI CAG ATT GTG GAG CTG AAG AAG C'~ GTG GTG GAA AAC ATG GAC ATA C , ~ C ~ A ~ AGA TCC 4 ~ ; YTT AGC AAG W H V T S O I VE LK K L V V E N H D Z L V Q X R 5 A~ F S K CCG t~C TTG ATG GCT 'rCC CTG CTG CCC CAG CTG ACA GGG GCT GAA AAT GTG CTA . ~ A~ ~ A ~ A . GGG GTT CTC AGT TTC P D L H A S L L P Q L T G A , N V L K R H m 1 I G V I L S F AGG GCC ATG GCC CAA GAG Gt'sA CTT CGG GAG GTT TTC TCC TCC CAC TGC CCA "~'.~1" ~ T A ~ GGT ( ~ ATT GAG TGC TTG AAG GAG TTT ~ R A H A Q E G L R E V F S S f l C P F : H G P I E C L K E F V ACT CCA GAC ACA GAC ATC AAG GTG ACC TTG AGT ATC TTT GAG CTG GCA ~CT GCT GCA GGT ~ ~ ~T ~ ATT GAC CCA GCC TTG GTG T ' D T D Z K V T L S|I F E L A S A A G VG C D I D P A L V GCT G(~ ATT GCT AAT CTG AAA GCT GAT ACT TeA TOT COT C~,G GAG GAA TAT AJ~ ~ ~ ~ ~ ~ ~ A'J~ TTT CTG GCT,GTT 1~CC ~ C ~ M L K A D T S S P E E E Y K|V A C L L L l F L A V S GC¢ ACT GA¢ CCT TC'r T¢¢ TTT TAT AGC ATT GAG AAG GAT GGT TA¢ AA¢ ~ AAT ATT CAT TGC TTG .~C AAA GOC Arc f L ~.~ A T O P S * S F ¥ S l E K D G ¥ U N N ! fl C L T E A X GTG OCC TCT GTC AGC CTC I Q V S A A L F T L ¥ m K U l [ T U L K [ F V V V A S V S L T~q~ CAG CTG G0C CAG GAG ACT GAC AAG CTT AAA ACC AGA AAT CGA GAA TCC ATT TCT ~ ~ A~ ~ ~ GTG GTG GAG GAG TCA TCC L Q L G Q E T D K L K T R N R E S Z S L L H It L V V E E S S TTC CTG ACC CTG GAC ATG CTG GAG TCC TOT TTC CCT TAT GTC CTG CTT CGA AAT GCC TAT CGG GAG GTG TCT CGG GCC TTC CAC CTA AAC F L T L D M L E S C F P Y V L L R N A ¥ .R E V S R A F B L II ATG (~T GCC ~ AC~ CAC TGA AGA (~C CTT TGG ~ T ~ CTA AJ~ ¢~[ TGC CAT AGT GG~ A(~ 'rGT GGT CAC TTT CGC AGG GGG TGG U H P A S T H GAA ~ GGT GGG GTC ACT AAG GAG AG~qGGG TCA GGA GCC AGA GTT CAT GAG CAG ATC TGT GGA AGA ACA Arc CAG GGC TGA GAA ATC GTA GAG CAG TGA GGC AGG CTG GGA OCA ~~,G AGG ACA GCT TAT ~ AAA AGT TAG GGC GTG GGG CCA CAT GIG TGA ATT TTA CAA TC~ A M AAG GAG TAA CGT ACA AGT ATA TTT TCT ATC TTC TGO TGA CTT GAG CTT GAG CTC TGA CAG OCA TGG GCC ~ C(~ ACC TTC AT(: ACT ATr CTT AGG ATA ATO CTG OCG GGC AGA GAT GAT CA/, TCA TCA TAT TAA ATC ATA AT(; AGC CC

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Fig. 2. Poly(A) ÷ Northern analysis of the expression of Hem-I in various tissue types. 7 ~tg of poly(A) ÷ RNA was loaded per lane in blot A, while 2 / t g of p o l k A ) + RNA was loaded per lane in blots B and C. The blots were reprohed with radiolahelled actin cDNA as a control for integrity and quantity of RNA. North¢in analysis was performed using formaldehyde agarose gels blotted onto nitrocelt~!o~e according to Ref. 17.

minor transcript at about 5.5 kb as seen in Fig. 2C. Hem-1 transcripts were present in Ju-kat lymphoblastic leukemia cells; HL-60 promyelocvtic leukemia cells; HL-60 cells stimulated retinoic acid, phorbol ester, GM-CSF, vitamin D-3, or dimetbylsulfoxide; K562 erythroleukemia cells; KG-1 myeloid teukemia cells; U937 monocytic leukemia cells; H E L erythroleukemia cells; Daudi B-lymphoma cells; C10 HTLV-l-transformed T-lymphocytes; Eskol hairy cell leukemia; JY EpstienBarr virus-transformed B-lymphocytes; and MO7E megakaryocytic leukemia cells. Neither Hem-I transcript was present in rat brain, pancreatic carcinoma, melanoma, SJ4 murine breast carcinoma cells, HepG2 hepatoma cells, placenta, smooth muscle, endothelial cells, Panc-1 pancreatic carcinoma cells, JEG placenta cells, FHS 738 embryonic lung cells, MCF-7 breast carcinoma cells, TMR-32 neuroblastoma cells, Tera-2 teratocareinoma cells, SK-Hep-I hepatoma cells, WiDr colon carcinoma cells and CV-I monkey kidney cells. Hem-I is also expressed in the marrow cells of two patients with CML and four patients with myelodysplasia (data not shown). On Southern blot analysis, Hem-1 appeared to be a single col~J gene (data not shown). Thus, we set about to ,ocalize this gene on its chromosome. First, nitrocellulose discs with flow cytometrically-sorted human chromosomes dotted onto them were hybridized to

nick-translated Hem-I cDNA [111. The autoradiographed discs revealed that Hem-I hybridized only to those dots containing human chromosome= t2 (data not shown). Next, in situ spreads of metaphase human chromosomes were analyzed for hyt'ridi~ation to the nick-translated Hem-1 cDNA. There were 132 sites of hybridization in 92 human metaphase cells. Of these 132 sites, 42 were on chromosome 12, while the rest were randomly distributed on the genome. The great majority of the chromosome 12 hybridization sites were found to be at q13 by fluorescent banding (Fig. 3). There is one reported case of M2 acute myeloid lcukcmia and one case of diffuse mL~ed lymphoma with transloca6ons in this region [12,13]. l'here is also a rare folic acid fragile site at this locale, and Yunis has postulated that this may play a role in the transiocations involving this area [13]. in addition, trisomy 12 is the most common chromosomal abnormality in chronic iympho¢5~ic leukemia [14]. However, we do not know at this time if Hem-I is involved in these chromosomal transiocations or helps generate those malignancies, in summary, Hem-I is a novel gene that appears to be ubiquitous in and yet restricted to blood cells. The putative translation of the eDNA of the most abundant transcript size found that Hem-1 codes for a potential membrane-associated, phosphorylated protein. The function of this protein is yet to be established, but

Fig. l. Hem-I cDNA sequem~c. The longest open reading frame is translated below the nucleic acid sequence. The membrane domains are underlined. The glycosylation sites are designated by circles. The cyclic nucleotide phosphorylation sites are designated by squares. The clones makirg up this sequence were obtained by probing a Agtll cDNA h'brary constructed from chronic myelogenous leukemia cell RNA ll5]. The clones were sequenced by the dideoxy method [16].

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Fig. 3. Localization of Hem-I on chromosome 12. (A) Histogram of the entire genome showing the comparative amount of hybridization signals on 12ql3 as compared to other sites. (B) An enlargement of chromosome 12 showing the regionalization of signals at 12q13.1. Hybridization of Hem-1 to mitotic chromosomes was performed according to the methods of Harper and Saanders [18l and Marth et al. [19].

analysis o f t h e r e g u l a t i o n o f H e m - 1 m a y l e n d insight into t h e c o m m o n e l e m e n t s t h a t direct h e m a t o p o i e t i c specific g e n e e x p r e s s i o n . References 1 Clark, S. and Kamen, R. (1987) Science 236, 1229-1237. 2 Thomas, ~t.L. and I_~francois, L. (1988) Immunol. Today 9, 320-326. 3 Kitamura, D., Kaneko, H., Miyagoe, Y., Ariyasu, T. and Watanabe, T. (1989) Nucleic Acids Res. 17, 9367-9379. 4 Katzav, S., Martin-Zanca, D. and Barbacid, M. (1989) EMBO J. 8, 2283-2290. 5 He, X., Treacy, M.N., Simmons, D.M., lagraham, H.A., Swanson, L.W. and Rosenfeld, M.G. (1989) Nature 340, 35-42. 6 Kozak, M (1987~ Y~cleic Acids Res. 15, 8125-8132. 7 Marshall, R.D. (1972) :~nu. R~v. Biochem. 41, 673-702. 8 Blause, E. (1983) Biochem. J. ~'O9,331-336. 9 Glass, D.B., F1 ~,laghrabi, M.R. and Pilkis, S.J. (1986) J. Biol. Chem. 261, 29~ 2993. 10 Eisenherg, D., Schwar-,:,E., Komarony, M. and Wall, R. (1984) J. Mol. Biol. 179, 125-142. 11 r .,.yen, L.I., Van Dilla, M., Bartholdi, M.F., Carrano, A.V. Cram, I S Fuscoe, J.C., Gray, J.W., Hildebrand, L.E., Moyzis, !" g and Perlman, J. (1986) Cold Spring Harbor Syrup. Quant. Biol. 5!, !59-167. 12 Yunis, J. (1984) Cancer Genet. Cytogenet. 11, 125-137. 13 Yunis, J. (1984) Cancer Genet. Cytogenet. 12, 85-88. 14 Schroder, J., Vuopio, P. and Autio, g. (1981) Cancer Genet. Cytogenet. 4, 11-21. 15 Hagen, F., Gray, C.I. and Kuijper, J.L. (1988) Biotechn~ues 6, 340-345. 16 Sanger, NicHew, W. and Coulson, A.R. (1977) Proc. Natl. Acad. Sci. USA 74, 5465-5467. 17 Maniatis, T., Fritsch, EF. and Sambrook, J. (1982) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Press, Cold Spring Harbor. 18 Harper, M.E. and Saunders, ~. (1981) Chromosoma 83, 431-439. 19 Marth, J.D., Disteche, C., Pravtcheva, D.. Ruddle, F., Krebs, E.G. and Perlmutter, R. (1986) Proc. Natl. Acad. Sci. USA 83, 7400-7404.