Proc. Natl. Acad. Sci. USAVol. 88, pp. 6595-6599, August 1991Biochemistry

Molecular identification of a major palmitoylated erythrocytemembrane protein containing the src homology 3 motif

(erythrocyte membrane skeleton/SH-3 motif/palmitoylation)

PAUL RUFF*, DAVID W. SPEICHERt, AND A. HUSAIN-CHISHTI*t*Department of Biomedical Research, St. Elizabeth's Hospital, Tufts University School of Medicine, Boston, MA 02135; and tThe Wistar Institute,Philadelphia, PA 19104

Communicated by Harvey L. Lodish, April 17, 1991

ABSTRACT The complete amino acid sequence of a 55-kDa erythrocyte membrane protein was deduced from cDNAclones isolated from a human reticulocyte library. This protein,p55, is copuriflied during the isolation of dematin, an actin-bundling protein of the erythrocyte membrane cytoskeleton.Fractions enriched in p55 also contain protein kinase activitythat completely abolishes the actin-bundling property of puri-fied dematin in vitro. The predicted amino acid sequence ofp55does not contain any consensus sequence corresponding to thecatalytic domains of protein kinases but does contain a con-served sequence found in the noncatalytic domains ofoncogene-encoded tyrosine kinases. This conserved src homology 3(SH-3) motif appears to suppress the tyrosine kinase activity ofvarious oncoproteins and has also been found in several plasmamembrane associated proteins involved in signal transduction.Northern blot analysis indicated that p55 mRNA was consti-tutively expressed during erythropoiesis and underwent 2-foldamplification after induction of K562 erythroleukemia cellstoward the erythropoietic lineage. The abundant expression ofp55 mRNA, along with protein 4.1 mRNA, was evident interminally differentiated human reticulocytes. Although p55has many features consistent with known peripheral membraneproteins, its tight association with the plasma membrane isreminiscent of an integral membrane protein. This fact may bepartly explained by the observation that p55 is the mostextensively palmitoylated protein of the erythrocyte mem-brane.

Erythrocyte dematin consists of two subunits of 48 and 52kDa that are capable ofbundling actin filaments in vitro (1, 2).During the purification of dematin by ion-exchange chroma-tography, a protein of 55 kDa, p55, was copurified (1-3). p55was subsequently separated from dematin on a Mono Qcolumn (3). When column fractions containing p55 wereadded back to purified dematin, complete inhibition of dem-atin's actin-bundling activity was observed (3). The inhibi-tory activity in p55-enriched fractions was due to the pres-ence of a protein kinase that phosphorylated dematin (3).Protein renaturation experiments suggested, however, thatthe protein kinase activity was distinct from p55 (3). Toexplore the role of p55, we have cloned and sequenced itscDNA obtained from a human reticulocyte library.§ Thepredicted primary structure of p55 contains a conservedsequence found in protooncogene-encoded non-receptor ty-rosine kinases (4-6). This sequence, called the SH-3 (srchomology 3) motif, has been found in several other proteinsthat associate with the cytoskeleton and are suspected to playimportant roles in signal transduction (6-13).

Mutations in the N-terminal SH-3-containing regions of thesrc and abl gene products are known to activate theironcogenic potential (14, 15). Indeed, the neuronal src gene

product, which has enhanced tyrosine kinase activity, has ahexapeptide insert in the SH-3 motif (16). The suppressiveactivity ofthe SH-3 sequence was further documented by theobservation that a synthetic peptide corresponding to part ofthe SH-3 motif inhibited tyrosine kinase activity of the v-srcproduct in vitro (17). The presence of the SH-3 motif incytoskeletal and signal-transducing proteins lends support tothe hypothesis that it may mediate binding to regulatoryligand(s) common to these proteins (6). Such a ligand mayblock the transforming ability of various oncogene-encodedtyrosine kinases (15). Alternatively, the SH-3 motif maymediate recognition of substrate molecules and regulate theirproximity to the plasma membrane.

MATERIALS AND METHODS

A human reticulocyte Agtll cDNA library (a gift from J.Conboy and N. Mohandas, University of California, SanFrancisco) was screened using rabbit polyclonal antibodiesagainst purified native p55 (18). Positive plaques were puri-fied and phage DNA was analyzed by EcoRI digestion (19).Agarose gel-purified, EcoRI-digested inserts were subclonedinto the plasmid pGEM-4Z (Promega) and sequenced by thedideoxynucleotide method (ref. 20; Pharmacia). Furtherclones were isolated from the library by using a central1.1-kilobase (kb) fragment as probe. The probe was labeledwith [a-32P]dCTP by using a random primer kit (Amersham).Nucleotide and deduced amino acid sequences were ana-lyzed by computer software from Dnastar (Madison, WI).

Total RNA was isolated from peripheral blood from sub-jects with idiopathic hemochromatosis by a method thatinvolves selective lysis of leukocytes (21). RNA obtained bythis method is free of RNA from contaminating leukocytes(21). RNA was fractionated in 2.2 M formaldehyde/1.4%agarose gels and blotted onto nitrocellulose (19). Northernblots were hybridized and washed under stringent conditionsprior to autoradiography for 72 hr at -70°C (19).

Solubilization and extraction studies ofhuman erythrocyteplasma membrane were carried out as described (22). Thecopy number of p55 was determined by a quantitative im-munoblot assay similar to the one developed for synapsin(23). Palmitoylation of intact erythrocytes was carried out asdescribed (24). However, fresh erythrocytes were not de-pleted of endogenous fatty acids and were immediatelyincubated with [3H]palmitate at 37°C. SDS/PAGE and im-munoprecipitations were carried out by established methods(25).

Abbreviations: SH-3, src homology 3; IOV, inside-out vesicle.tTo whom reprint requests should be addressed.§The sequence reported in this paper has been deposited in theGenBank data base (accession no. M64925).

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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6596 Biochemistry: Ruff et al. Proc. Nati. Acad. Sci. USA 88 (1991)

A SA P E P P P A P HEP A

111112 1 1 1 1 1 2 1 3 1 1 1 1 ~~~~~FIG. 1. Organization ofhumanI(II I I I ~~~~~~~~~~~erythrocyte p55 cDNA. The re-

5 ~~~~~~~~~~~~~~~~~~~~3'striction map was obtained fromtwo overlapping clones extendinga total of 2.0 kb from a humanreticulocyte Agtl cDNA library.

---'->---->-~~~~~~~~~~~~~Thecentral block (nucleotides-----(--<-------------------------------(115-1512) represents the coding

region. Both strands were se-

> > ~~~~~~~quenced, and dashed arrows indi-_______________________________ cate the sequencing strategy.

----- Ecri, EcoRI; Hin3, HindIll.

RESULTS coding for a 466-amino acid protein of 52.9 kDa with anisoelectric point of 7.0. The stop codon' is' followed by anDNAConinandSequecing Fig 1 shws cNA cones untranslated region of 488 bp with a consensu's po'lyadenyl-

encoding the complete amino acid sequence of p55. Both yainsqecATA Fg ) h igo h oastrands were sequenced as shown. The initiator codon begins cDNA (2qkb)ciscositentwit theg size ofThe miessagte froma115 base pairs (bp') from the 5' end and reveals an open human retb)iculoytsi(Fig. 3).teszeo h esaefo

reading frame of 1398 bp (Fig. 2). Evidence that this ATG Several lines of evidence show that the cDNA encodes ancodon is the initiator includes (i) the presence of a purine 3 uhni 5.()Plcoalatbde fiiyprfebp upstream (26), (it) the match of a peptide sequence agisp5 reonzdfinpotnsncedb thproduced by S. aureus V8 protease cleavage of p55 with the cDA i)Prfe 5 n h uinpoenpouepredicted sequence from amino acids 8-24, and (iii) the' lack similar one-dimensional CN~r peptide ma'ps (not shown). (ii)of any downstream methionine that could account for the Amino acid sequences of eight different peptides derivedcomplete amino acid sequence (Fig. 2). A single cDNA clone from purified p55 were identical to the predicted amino acidwas isolated that contained the entire open reading frame, sequence in Fig. 2. A hydropathy plot of the deduced amino

ccccr - '---C2GCATCACCTGCCCTGICTGC- A -cr3GrA0TGGrCGGG 1

a eq e Sq q

S is h t a 1S--y1 -CATCCATTGAATAICTISTr ', I-C G "-,--'TTAG C7~ CA 971GCCCTOCCAGGTCAAGh p 1

5--- p a

GGACAGGAGG"TGCGGr',,,l'jCT,7'-r'"-/' TA-,' "",--'- AG A' 'A"i- 2,G 'ATGG-GAATCACGCTGAAG 31g q e vr 4CTGAATGAA"."AAACACT-"''-n"-T''P, ""t 3AT AGA(AGCCTCC 4

GTGGG0Gf-ATGAGATC-T '.~A.'.2'rT-AT'),._3'~ TATAAAATT -TAGTCACC7>GAAGGCGATO 5v ibe ---AAAGAPPACCAAAGGAAA AT- A A,- -'-rr~Tk e t k - - S

ATGAGAGCGCAGTTTC~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~r~~~~~~AT 'TC'? ~~~~~~~~~~~~~~~~~~~~~~ TGAT AGTTTGCT6~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~GTGkAGTTCis. rag d d k d n -ik f a 2ACTGGGGACATTATC'-CAGATTATr-AACA'AGGA,.T'ACAGCAATTGGTCt'3"A f"A'CXGGTGGAAGGCTCCT.CCAAG -:t gd ii kidds I w w_ gr e gaSS k 2GAGTCAGCAGGATTGATlCCCmTmCCCCTGAGCTGCAe'GCAATLGG0C0AGTGC"CAAGTATGGCTCAGTCAGCTCCTAGC 821e s -a-- 1 p_ e :q E, r v a s ..i a q s a p sGAAGCC-CCGAGCTGCAGTCCCTTTOGGGAAGAACA,A A. A C AAAAAT,. CGTGTe a psSCaSpS SaTTTGAT(CAGTTGG1_ATGTTGTT-VTCCTA'C'S kA- AAGGAAGACCCTGGT 'f d q d 7v kATCGGAGCCAGTGGGGTGGTCCC AATl PI A AAATG CT'rGA ~TTT C1

CCTTTCCCATATACAAC-ACGG7_CG CCA G AGGAAGATGGGAAGGPG ACCACLTTATCTCAACGGAG h1p Sp y t P p h f laSte 3GAGATGA0GvAGGAACATCTLCTGCCAAT"AT''--i-r(,-A4X'-ITG "GC, Gf-TACcAAGGCAACATGTTTGGCACCAAA 1I2)e m tv r n s a n eC-8 q S y qg n ms Sf t ki 3ETTTGAAACAGTGCACCAGATCCATAAGrAGA"AC,,"A'AJTCTCCATCrTTGACATTGAGC fCAGA1CC1TGAAAATT 12Saet v h q i bk k 3± 3GTTCGGACAGCAGAAC7TTTC-GC-rTTTCATTG-TGrTTC'TTi -'-A,-~-TCC'7ACC'CG-'-CArTC-AGA'AGAAGCC1CTG 13 CV r t a e 1 a p 5 'q- a 1 4 ILCAGCAGCTGCAGAAGGACTCTGA-GGCCA'TCCCAGC ALTA C T'4A C , A -CTC AC TGGTCAATAAT 142

q q I q k d s ea aS4.r

GGTGTTGATGAAACCCTTLAAGAAATnTACAACAA,.'L"GCC'r".,I--'Arte'AAI,'CCCT.GCAGTTCTC, -''%-TC"I'GTGCCTGTLC 14.50g v d et 1 k k 'L q e -I -F'I : SW p 46TCCTGGGTTTACTAAGCTTGTAGAAT'-GGGGGAiACr-CC.VTGTA'T13Ci_-TCT1.-.-AGrTnGA"CC 1.'S w V y 46bsTTGCTTTAAGACAAACAGGGC"TG,'CTCCAt'ACTLAGTTTTTC-'"--I--AC TTTCACZCG AG:7TATCTAATTCAG 1652'CCAGTAAGGTTCAGTrCTTC-TTGC'rTrAGGCTCCGACGTTATCTCTATACTI '73GT2 CCACTGATCTGG 17251ATTTGAAAAGGATTTLCTArGAAATTGGGGGTA'AGJ'AACT",'AC-TACCAAAAGTAA TGCTAATCAAGGGTGATGCAC-A 18 CCGCAAAAGCAATGGACCCCATCCCTCTAAAGCCTGr(_CCCTCkCTTTG~l.CCTTC(-AACTG--TATATGCTGOCT"LATTTCATTT12GTCTTTTTATTTTGGAGAAAGCGTTTTTAACTGC AAC TTTTCTATAATGJCCAAAATGACACATC TGTGCAATAGAA 19%CTGATGTCTGCTCTAGGGAAAC(-TTrAAAAGATAA.A T JTTGTr 20 CO

FIG. 2. Complete cDNA sequence and deduced amino acid sequence of p55. Shading indicates amino acid residues confirmed by automatedgas-phase sequencing of seven peptides produced by Staphylococcus aureus V8 protease and one peptide (residues 221-236) produced bycleavage with 3-bromo-2-(2'-nitrophenylsulfenyl)skatole. The underlined region (amino acids 165-233) represents the SH-3 motif. Thepolyadenylylation consensus sequence (AATAAA) is boxed.

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Proc. Natl. Acad. Sci. USA 88 (1991) 6597

2 3

-28S

-18SFIG. 3. Northern blot analysis

of total RNA from human reticu-locytes and K562 cells. Blots were

_ probed with radiolabeled 1.1-kbfragment of p55 cDNA. Lane 1,total RNA (30 ,g) from reticulo-cytes (a single message of 2.0 kbwas detected); lane 2, total RNA(20 Ag) from uninduced K562cells; lane 3, total RNA (20 ,g)from K562 cells induced for 6 hrwith 1-f8-D-arabinofuranosylcy-tosine (ara-C). Positions of 28Sand 18S rRNA are shown at right.

acid sequence (27) revealed a predominantly hydrophiliccharacter. Analysis of the secondary structure with theChou-Fasman algorithm (28) showed a predominance ofa-sheets with little a-helical content (data not shown). Theseresults suggest that p55 is a globular protein with no trans-membrane component, a property consistent with a periph-eral membrane protein.

Northern Analysis. The mRNA of p55 is one of the mostabundant messages in human reticulocytes. A comparativeNorthern blot assay usingcDNA probes for p55, band 3, ankyrin,spectrin, proteins 4.1 and 4.2, and chicken erythrocyte dematinsuggested that p55 and protein 4.1 mRNAs are among the mostabundant messages in human reticulocytes (data not shown).Although protein 4.1 is synthesized late during erythropoiesis(29), p55 appears to be constitutively expressed throughouterythropoiesis (Fig. 3). Northern blot analysis oftotal RNA fromK562 cells, a multipotent hematopoietic precursor cell line,showed a 2.0-kb p55 message (Fig. 3, lane 2). After induction withara-C, which commits K562 cells to terminal erythroid differen-tiation (30), a 2-fold amplification of p55 message was observed(Fig. 3, lane 3). A faint 4.0-kb message was also detected andunderwent amplification with ara-C. Although the origin of the4.0-kb message is not known, it may arise from alternativesplicing.Homology with Oncogene-Encoded Tyrosine Kinases and

Other Membrane Proteins. A computer search of the Swiss-

p55

Prot data base (release no. 17) (31) revealed identities of upto 41% between a 59-amino acid region of p55 (residues165-233, Fig. 2) and the SH-3 domains in the noncatalyticregion of non-receptor tyrosine kinases (Fig. 4). This familyof tyrosine kinase genes, of which the protooncogene c-src isthe prototype, includes lyn, syn, hck, yes, blk, Ick, fgr, andabl (7, 8), as well as v-crk, a transforming oncogene whoseproduct lacks tyrosine kinase activity (32). The SH-3 motifhas also been found in proteins that associate with thecytoskeleton and play diverse roles in signal transduction (6):a yeast actin-binding protein (7), phospholipase C-y (8),myosin I (9, 10), erythroid and nonerythroid a-spectrins(33-35), calcium-channel f subunit (11), neutrophil oxidasefactor (12), and GTPase-activating protein (13).Asciation of p55 with the Erythrocyte Plasma Membrane.

Although p55 has not been studied before, some information maybe obtained from studies ofdematin (1-3). p55 does not associatewith F-actin in sedimentation assays and does not appear to bindto dematin with high affinity since the two proteins can beseparated on anion-exchange columns (2). Gel filtration failed todetect any interaction between p55 and human erythrocytespectrin (data not shown). p55 remained associated with eryth-rocyte inside-out vesicles (IOVs) after removal of spectrin andactin with low-ionic-strength buffers but was solubilized in Tri-ton/KCl solutions (Fig. 5A, lanes 4, 5, 10, and 11). Extraction ofIOVs at pH 11.0 or by 1 M KCl/KI, conditions known to releaseprotein 4.1 and ankyrin (36), caused only partial solubilizationconsistent with p55 as a peripheral membrane protein (Fig. 5B).Extraction of IOVs with 1 M KCI resulted in partial proteolysisof p55 (Fig. 5B, lane 7).A quantitative immunoblot assay determined -80,000 cop-

ies of p55 monomer per erythrocyte (23). Based on itsmobility in SDS/polyacrylamide gels, p55 may also exist asa dimer on the membrane (Fig. 5A, lane 13, arrowhead).Stoichiometrically, dimeric p55 (40,000 copies per cell) is asignificant component of the erythrocyte membrane (37).There was no evidence of cytosolic p55 and the copy numberremained constant from reticulocytes to mature erythrocytes(data not shown). Trypsin treatment of intact erythrocytesdid not alter p55 mobility in SDS/polyacrylamide gels, andindirect immunofluorescence localized p55 to the cytoplas-mic face of the erythrocyte membrane (data not shown).During separation of p55 from dematin, a protein kinase

was coeluted with purified p55 (3). The absence of anyconsensus sequences for kinase catalytic domains or a pu-tative nucleotide binding site in the amino acid sequence ofp55 suggests that p55 is not a protein kinase. Immunopre-cipitation ofp55 from metabolically 32Pi-labeled erythrocytes

AqFDYDPKkDNliPckEagLkFatGD-IIQIIN-KDDSnWWqG-R-V-EGssKE-SaGLIPSP-eL

c-src slyDYksr-D------EsdLsFmkGD-rmevI-ddteSdWW---R-V-vnlttrqe-GLIPln-fv 33%

v-crk AlFDf--K-gN----ddgdLpFkkGD-IlkIr-dKpeeqWWnaed-m-dG--K-r--GmIPvP-yv 34%

v-yes AlyDYearttd-----d--LsFkkGe-rfQIIN-ntegdWWea-Rsi-atg-K-t--GyIPSn-yv 31%

a-sp AlyDYqeK--s--P-rEvtmk--kGD-IItIIN-stnkdWW---k-V-Evndrq---GfvPaa-yv 29%

myolb AlFDYfaa-eN--P-dE--LtFneGa-vvtvIN-KsnpdWWeG-e-l-nG--q-r--GvfPas-yv 32%

mnyoll AlyDYDaqtgd-----E--LtFkeGD-tI-IvhqKDpagWWeG-e-l-nG--K-r--GwvPan-yv 35%

abpl AeyDYDaaeDN-----E--LtFvenDkIInIefv-DD-dWWlG-el--E---KdgSkGLfPSn-yv 41%

cac-Os tnvgYnPspgdevPvegvaitFepkDf-lhIke-KynndWWiG-RlVkEGc--E-v-GfIPSPvkL 34%

plc-') AlFDYkaqred-----E--LtFtksa-IIQnve-KqeggWWrgd-y-h--hkKq---lwfPSn-yv 31%

consensus A FDYDPK DN P E L F D IIQIIN KDDS WW G R V EG KE S GLIPSP L

FIG. 4. Alignment of SH-3 amino acid sequences (single-letter code). From the top: p55, p60c-src, p47gag-crk, p90V-Yes, a-10 domain ofDrosophila spectrin, Acanthamoeba myosin 1B and 1L, yeast actin-binding protein, /8 subunit of skeletal muscle calcium channel, andphospholipase C-y. The percent identity between the SH-3 motif in p55 and the SH-3 motif in each protein is indicated at right.

Biochemistry: Ruff et al.

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and kinase assays of immune complexes failed to detect anyprotein kinase activity (data not shown and ref. 25).

Palmitoylation of p55. Intact erythrocytes acylate severalmembrane proteins in vitro (24, 38, 39). More than 75% ofexogenous [3H]palmitate is incorporated into a protein of 55kDa (ref. 24 and Fig. 6, lane 3). To establish whether thepalmitoylated 55-kDa protein was p55, the following evi-dence was obtained. (i) In SDS/PAGE analysis of metabol-ically labeled erythrocyte plasma membranes, the most abun-dant [3H]palmitoylated protein migrated with mobility iden-tical to that of p55 (Fig. 6, lane 3). (it) Immunoprecipitationof solubilized membranes with polyclonal antibodies againstp55 precipitated a 3H-labeled protein with mobility identicalto that ofp55 (lane 4). (iii) All the radiolabeled, CNBr-cleavedpeptides of3H-labeled 55-kDa protein (lane 6) were containedwithin the silver-stained CNBr-generated map of purified p55(lane 5). (iv) The extraction behavior of p55 from erythrocytemembranes (Fig. 5) and the specificity and kinetics of itspalmitoylation were identical to those of [3H]palmitoylated55-kDa protein previously reported (24). These results sug-gest that p55 is the major palmitoylated protein of the humanerythrocyte membrane.

DISCUSSIONThe reported experiments were initiated to examine the role ofp55 in the inhibition of dematin's actin-bundling activity (2, 3).The deduced primary structure of p55 and immune-complexkinase assays demonstrated that p55 does not contain any activekinase activity. The inhibition of actin-bundling activity of de-matin therefore appears to be mediated by a protein other thanp55 (3). However, these studies do notpreclude an accessory rolefor p55 in the regulation of dematin's actin-bundling activity.Although the elution behavior of p55 is different from that ofdematin on ion-exchange columns, a direct association betweenthem cannot be excluded at this stage.Northern analysis revealed p55 mRNA in uninduced K562

cells, a multipotent hemopoietic cell line. Upon induction of

1 2 3 4

._01

55kDa -

5 6

_- 66kDa

45

-.v4 -36

4#1& -29

-- 24

--20.1

- 14.2

FIG. 6. Palmitoylation of erythrocyte membrane proteins. Fresherythrocytes were metabolically labeled overnight with [3H]palmi-tate at 37°C as described (24) and ghosts were analyzed by SDS/PAGE. Lane 1, pure p55; lane 2, Coomassie-stained [3H]palmitate-labeled ghosts; lane 3, fluorograph of lane 2; lane 4, fluorograph ofimmunoprecipitated p55 from labeled ghosts (polyclonal antibodiesto p55 were not as effective in immunoprecipitation experiments asin immunoblots; the lighter area under the immunoprecipitated p55band is due to coprecipitated immunoglobulins); lane 5, silver-stained CNBr-cleaved peptides ofpurified p55; lane 6, fluorograph ofCNBr-cleaved 55-kDa protein excised from lane 3.

K562 cells with ara-C, a 2-fold increase in p55 message wasobserved (Fig. 3). The constitutive expression of p55 mRNAis in contrast to protein 4.1, ankyrin, band 3, and dematin,which are expressed late during erythropoiesis prior to theirstable assembly into the membrane skeleton (29, 40).Based on hydropathy and secondary structure analysis,

p55 appears to be a peripheral membrane protein. This issupported by the observation that a significant amount ofp55

1 23 4 56 78 910 11.-- w*

12 13 B M 1 2 3 4

*,

....I..a l

4 5 6 7 8

FIG. 5. Association of p55 with the erythrocyte membrane. (A) Solubilization of erythrocyte membrane p55. Erythrocyte membrane"ghosts" were incubated in solubilization buffers on ice for 60 min. After centrifugation at 35,000 rpm (type 42.2 Ti rotor) for 60 min, supernatantwas removed and pellets were analyzed by SDS/WIM PAGE. Lane 1, ghosts; lane 2, IOVs; lane 3, Triton-extracted ghosts; lane 4, Triton plus50 mM KCl; lane 5, Triton plus 150 mM KCI; lane 6, Triton plus 500 mM KCl. Buffer contained 0.5% Triton X-100, 50 mM Tris-HCl (pH 7.5),1 mM EDTA, 1 mM EGTA and a mixture of NaF, vanadate, ouabain, and protease inhibitors. Lanes 1-6 are Coomassie-stained and lanes 7-12are corresponding immunoblots with p55 polyclonal antibodies. Lane 13 represents a longer exposure of lane 7, indicating dimeric p55(arrowhead). Note that p55 was completely solubilized in isotonic Triton solution (lane 11). (B) Extraction of p55 from IOVs. Lane M, purifiedp55; lane 1, IOVs; lane 2, IOVs plus 0.1 mM EGTA (pH 11.0 at 23°C); lane 3, IOVs plus 1 M KCl (at 37°C); lane 4, IOVs plus 1 M KI plus0.5 mM EGTA (pH 8.0 at 23°C). Samples were incubated for 60 min and centrifuged as described above. Lanes 1-4 are Coomassie-stained andlanes 5-8 are corresponding immunoblots. Note that a fraction of p55 (25%) was resistant to all extraction conditions in the absence of TritonX-100. Lane 7 shows partial proteolysis of p55 in the presence of 1 M KCI.

A

55kDa -

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Proc. Nati. Acad. Sci. USA 88 (1991) 6599

can be eluted from IOVs by high-salt extractions in theabsence of detergent (Fig. SB). However, the remaining p55is tightly associated with the membrane and its solubilitybehavior in Triton X-100 is reminiscent of an integral mem-brane protein (Fig. SA). A similar solubility behavior hasbeen observed with protein 4.2, another peripheral mem-brane protein (41). The detergent solubilization of p55 may bepartly explained by the observation that it is the mostabundantly palmitoylated protein (Fig. 6). Due to rapidturnover of bound lipid, salt-eluted p55 may contain signifi-cantly less palmitate. The properties of p55 are identical tothose of a previously described palmitoylated 55-kDa proteinof human erythrocyte membrane (24, 39). Although theidentity of that 55-kDa protein was not known, the specificityand kinetics of its palmitoylation are similar to those of p55(24). There are four cysteine residues in p55, including one inthe SH-3 motif, that may provide potential sites for palmi-toylation (ref. 42 and Fig. 2). Since a consensus sequence ofpalmitoylated cysteine is not known, identification of palm-itoylated residue(s) awaits separation and microsequencingof 3H-labeled peptides of p55.Mutation analyses and direct enzyme inhibition assays

suggest that the functional role of the SH-3 motif in non-receptor tyrosine kinases is to down-regulate their kinaseactivity (14, 15, 17). One can predict that the ligand(s) whichbinds to the SH-3 motif may modulate the tyrosine kinaseactivity of oncoproteins and by definition may have anti-oncogene activities (15). In fact, the mechanism by whichp47gagcrk, which contains only SH-2 and SH-3 sequences,transforms cells may be partly due to the depletion of such aligand via SH-3 sequences (32). Moreover, the occurrence ofthe SH-3 motif in cytoskeletal proteins suggests that thissequence may mediate interaction with ligand(s) common tosignal-transducing molecules (6).To identify ligands that interact with the SH-3 sequence, it

will be imperative to have a system where the interactions ofvarious constituents can be reconstituted and quantified. Thediscovery of an actin-binding protein with an SH-3 motif inyeast provides one such system (7). The human erythrocytemembrane provides another experimental system that hasbeen well characterized (37). Moreover, the erythrocytemembrane contains another protein with an SH-3 motif,a-spectrin (33-35). Purified a-spectrin or its SH-3 motif mayserve as a reagent to confirm the specificity of ligands for thep55 SH-3 motif. Since analogs of erythrocyte membraneproteins exist in other cells, the identification ofthese ligandsmay have consequences beyond red-cell biology.

We wish to acknowledge (i) Nathalie Garbani for help in copy-number determination and membrane extraction studies; (it) Dr.D.-H. Chen for assistance in obtaining peptide sequences; (iii) Dr. S.Kharbanda for providing RNA from K562 cells; (iv) Prof. DanielBranton (HL17411), Harvard University; (v) support from an Amer-ican Heart Association Grant-in-Aid (National) and a grant from theWhitaker Health Science Fund, Massachusetts Institute of Technol-ogy (to A.H.-C.), and from National Institutes of Health GrantsAR39158 and HL38794 (to D.W.S.).

1. Siegel, D. L. & Branton, D. (1985) J. Cell Biol. 100, 775-785.2. Husain-Chishti, A., Levin, A. & Branton, D. (1988) Nature

(London) 334, 718-721.3. Husain-Chishti, A., Faquin, W., Wu, C.-C. & Branton, D.

(1989) J. Biol. Chem. 264, 8985-8991.4. Grosveld, G. (1990) in Acute Lymphoblastic Leukemia, eds.

Gale, R. F. & Hoelzer, D. (Liss, New York), pp. 15-27.5. Hoffman, F. M., Fresco, L. D., Hoffman-Falk, H. & Shilo,

B.-Z. (1983) Cell 35, 393-401.6. Pawson, T. (1988) Oncogene 3, 491-495.

7. Drubin, D. G., Mulholland, J., Zhimin, Z. & Botstein, D. (1990)Nature (London) 343, 288-290.

8. Stahl, M. L., Ferenz, C. R., Kelleher, K. L., Kriz, R. W. &Knopf, J. L. (1988) Nature (London) 332, 269-272.

9. Jung, G., Korn, E. D. & Hammer, J. A. (1987) Proc. Natl.Acad. Sci. USA 84, 6720-6724.

10. Jung, G., Saxe, C. L., Kimmel, A. R. & Hammer, J. A. (1989)Proc. Natl. Acad. Sci. USA 86, 6186-6190.

11. Ruth, P., Rohrstein, A., Biel, M., Bosse, E., Regulla, S.,Meyer, H. E., Flockerzi, V. & Hoffman, F. (1990) Science 245,1115-1118.

12. Leto, T. L., Lomax, K. J., Volpp, B. D., Nunoi, H., Sechler,J. M. G., Nauseef, W. M., Clark, R. A., Gallin, J. I. & Ma-lech, H. L. (1990) Science 248, 727-730.

13. Vogel, U. S., Dixon, R. A. F., Scaber, M. D., Diehl, R. E.,Marshall, M. S., Scholnick, E. M., Sigal, I. S. & Gibbs, J. B.(1988) Nature (London) 335, 90-93.

14. Franz, W. M., Berger, P. & Wang, Y. J. (1989) EMBO J. 8,137-147.

15. Jackson, P. & Baltimore, D. (1989) EMBO J. 8, 449-456.16. Martinez, R., Mathey-Prevot, B., Bernards, A. & Baltimore,

D. (1987) Science 237, 411-415.17. Sato, K., Miki, S., Tacibana, H., Hayashi, F., Akiyama, T. &

Fukami, Y. (1990) Biochem. Biophys. Res. Commun. 171,1152-1159.

18. Huynh, T. V., Young, R. A. & Davis, R. W. (1985) in DNACloning: A Practical Approach, ed. Glover, D. M. (IRL, Ox-ford), Vol. 1, pp. 49-78.

19. Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989) MolecularCloning:A Laboratory Manual (Cold Spring Harbor Lab., ColdSpring Harbor, NY), 2nd Ed.

20. Sanger, F., Nicklen, S. & Coulson, A. R. (1977) Proc. Natl.Acad. Sci. USA 74, 5463-5467.

21. Goosens, M. & Kan, Y. W. (1981) Methods Enzymol. 76,805-817.

22. Husain-Chishti, A. & Branton, D. (1986) Anal. Biochem. 155,206-211.

23. Jahn, R., Schiebler, W. & Greengard, P. (1984) Proc. Natl.Acad. Sci. USA 81, 1684-1687.

24. Maretzki, D., Mariani, M. & Lutz, H. U. (1990) FEBS Lett.259, 305-310.

25. Golden, A., Nemeth, S. P. & Brugge, J. S. (1986) Proc. Natl.Acad. Sci. USA 83, 852-856.

26. Kozak, M. (1984) Nucleic Acids Res. 12, 857-871.27. Kyte, J. & Doolittle, R. F. (1982) J. Mol. Biol. 157, 105-132.28. Chou, P. Y. & Fasman, G. D. (1974) Biochemistry 13, 222-245.29. Hanspal, M. & Palek, J. (1987) J. Cell Biol. 105, 1417-1424.30. Bianchi Scarra, G. L., Romani, M., Covielo, D. A., Garre, C.,

Ravazzolo, R., Vidali, G. & Ajmar, F. (1986) Cancer Res. 46,6327-6332.

31. Pearson, W. R. & Lipman, D. J. (1988) Proc. Natl. Acad. Sci.USA 85, 2444-2448.

32. Mayer, B. J., Hamaguchi, M. & Hanafusa, H. (1988) Nature(London) 332, 272-275.

33. Lehto, V. P., Wasenius, V.-M., Salven, P. & Saraste, M. (1988)Nature (London) 334, 388.

34. Dubreuil, R. R., Byers, T. J., Sillman, A. L., Bar-Zvi, D.,Goldstein, L. S. B. & Branton, D. (1989) J. Cell Biol. 109,2197-2205.

35. Sahr, K. E., Laurila, P., Scarpa, A. L., Coupal, E., Leto,T. L., Linnebach, A. J., Speicher, D. W., Marchesi, V. T.,Curtis, P. J. & Forget, B. G. (1990) J. Biol. Chem. 265,4434-4443.

36. Tyler, J. M., Hargreaves, W. R. & Branton, D. (1979) Proc.Natl. Acad. Sci. USA 76, 5192-51%.

37. Bennett, V. (1989) Biochim. Biophys. Acta 988, 107-121.38. Staufenbiel, M. & Lazarides, E. (1986) Proc. Natl. Acad. Sci.

USA 83, 318-322.39. de Vetten, M. P. & Agre, P. (1988) J. Biol. Chem. 263,

18193-181%.40. Faquin, W. C., Husain-Chishti, A. & Branton, D. (1990) Eur.

J. Cell Biol. 53, 48-55.41. Korsgren, C. & Cohen, C. M. (1986) J. Biol. Chem. 261,

5536-5543.42. Schmidt, M. F. G. (1989) Biochim. Biophys. Acta 988, 411-

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