VoL 4. 4T5-482, June 1993 Cell Growth & Differentiation 475

Members of the src Family of Nonreceptor TyrosineKinases Share a Common Mechanism forMembrane Binding’

Lauren Silverman, Marius Sudol, and Marilyn D. Resh2

Department of Cell Biology and Genetics, Memorial Sloan-Kettering

Cancer Center EL. S., M. D. R.], and Laboratory of Molecular Oncology,The Rockefeller University [M. S.), New York, New York 10021

Abstract

The src family of nonreceptor protein tyrosine kinasesshare extensive sequence homology, except for 80NH2-terminal amino acids, thought to comprise a“unique” domain. This region is presumed to mediateinteractions specific to each kinase. Recently, weidentified three NH2-terminal lysine residues, crucialfor pp60�� membrane association. Surprisingly, theselysines are conserved among several src familymembers. Since their mechanism of membraneassociation is unknown, it was of interest to determinewhether other tyrosine kinases also utilize their NH2-terminal domain. Here, we demonstrate that pp6Ovp62�YeS, and p59�#{176}polypeptides compete with eachother for membrane binding, whereas p56Itk, whichlacks the NH2-terminal �ysine motif, has no effect.Moreover, myristylated peptides corresponding to theNH2 termini of src, yes, lyn, and fyn inhibit membraneassociation of pp6Ot.’.rt, p62C�Y�S, and p59�. Our resultssuggest that src family members share a commonmechanism for membrane binding, and they provide amolecular explanation for the ability of other src familymembers to complement pp6Oc.’.rc function.

Introduction

Protein tyrosine kinases mediate transduction of signalsfrom the extracellular milieu to the interior of the cell,

ultimately participating in processes such as control ofcell growth and cell differentiation (reviewed in Ref. 1).They can be subdivided into two groups, the transmem-brane receptor tyrosine kinases and the nonreceptortyrosine kinases, the latter being largely composed ofproteins highly homologous to pp60�” (2). Members ofthe src family share sequence homology in the SH3, SH2,and tyrosine kinase domains (3). Adjacent to the myris-tylated NH2-terminal region, the amino acid compositionof these kinases diverges, defining a “unique region” ofapproximately 80 amino acids for each protein. Fourclosely related tyrosine kinases, pp60�#{176} (4 5) p62’5�(6), p56” (7), and p590/fl (8) are expressed in a large

Received 2/19/93; revised 3/20/93; accepted 3/24/93., This research was supported by grants from the American Cancer

Society and the Pew Foundation. M. D. R. is a Rita Allen Foundation

Scholar. L. S. is a Bristol-Myers Squibb Pharmaceutical Research InstituteFellow of the Life Sciences Research Foundation.2 To whom requests for reprints should be addressed, at Department of

Cell Biology and Genetics, Memorial Sloan-Kettering Cancer Center, 1275

York Avenue, New York, NY 10021.

number of cell types and are localized to the cytoplasmicface of cellular membranes. Expression of pp60�#{176} isvirtually ubiquitous (9, 10). The c-yes gene is also ex-pressed in most cells, including fibroblasts and epithelialand neuronal cells (1 1). Like pp60’#{176}”, p56�� is widelyexpressed, particularly in hematopoietic cells includingmacrophages, monocytes, and B-cells (12). Similarly,p59iYfl is observed in fibroblasts and endothelial cells, aswell as those of the hematopoietic system (13). Further-more, all four of these kinases are expressed at significantlevels in platelets (14). In contrast, the other four srcfamily members, p56�’ (15), p55t� (16), p59f�(k (17, 18),and p551)ik (19), are expressed primarily in cells of he-matopoietic origin.

In an effort to understand the role of the widelyexpressed pp60� protein, Soriano et a!. (20) recentlycreated mice containing a homozygous null mutant ofthe c-src gene. It had been presumed that pp60�r wasessential for growth and development because of itswidespread expression. The report that these animalswere viable led to the suggestion of functional redun-dancy among the nonreceptor tyrosine kinases, particu-larly p62’5t’#{176}and p59Y�, which are also widely expressedand most similar to pp6O’ #{176}“. However, enzymatic activityalone is insufficient to explain this complementation; srcfamily members must also be capable of interacting withat least a subset of those proteins with which pp60�#{176}normally associates. Thus, the suggestion of “cross-talk”implies that src family members share a similar molecularmechanism for subcellular membrane localization. It isimportant to note that proteins of the src family oftyrosine kinases are membrane associated despite thelack of a transmembrane domain. Although the mecha-nism of pp60�Sr( membrane interaction has been inves-tigated, little is known about how the related protoon-cogenes are targeted to membranes.

Membrane association of pp6O#{176}#{176}”,the oncogeniccounterpart of pp60(s�, has been shown to be mediated,in part, by its modification with myristic acid. Althoughmyristylation of pp6Ov5r is necessary for association withthe plasma membrane (21, 22), it is not sufficient, asthere are myristylated variants of pp6O#{176}#{176}”that fail toassociate with the plasma membrane (23, 24).

We have recently demonstrated that three amino-terminal lysmnes at positions 5, 7, and 9, in conjunctionwith the myristate moiety, form a binding motif essentialfor the interaction of pp6O#{176}#{176}”with the plasma mem-brane: Myr-Gly-X--X-Lys-X-Lys-X-Lys (25). TheNH2-terminal 12 amino acids of the cellular homologuepp6O’ -sr, are identical to that of pp60�s� Here weshow that a number of the nonreceptor tyrosine kinases,pp60��, p62’5�#{176},p56�, and p59iVfl, exhibit a strikinglysimilar amino-terminal motif within their so-called“unique domain,” suggesting a hitherto unsuspected re-lationship between the membrane targeting and the bi-ological properties of these molecules. Using in vitro

r�0FT;��‘.“b -

�� lvi

80

60

40

20

0

ft.)

ft.)

v-src c-src

Fig. 2. Membrane association of in vitro synthesized pp60�”, �p62’ #{176}‘�‘. p59”.’, and p56i�k. src family members, labeled with [35S]methi-onine by translation of the encoding mRNA in reticulocyle lysates, were

incubated with (+) or without (-I plasma membrane-enriched fractions(P100) from vole cells for 30 mm at 20#{176}C.Following ultracentrifugation

at 100,000 x g for 15 mm at 4#{176}C,the pellet (membrane-bound; Mb) andsupernatant (unbound) fractions were analyzed by gel electrophoresis,autoradiography, and scintillation counting. Each column represents the

average of 6 experiments; standard deviations of the data were ±5#{176}!’.orless within each experiment.

yes fyn ick

476 Membrane Binding of sri Family Members

2 3 4 5 6 7 8 9 10 11

src myrGLY SER SER LYSSERFLYS� PRO LffIi&SP PRO

lyn myr-GLY CYS ILE LYS SER LYS GLYLYSIASP SER

yes myr-GLY CYS ILE LYS SER LYS GLY ASP LYSGLY

fyn � myr’.GLYCYS VAL GLN �

hck myr-GLY CYS MET LYS SER LYS PHE LEU GLN VAL

bik � myr..GLY� LEU LEU SER SER :LYS ARG GLN VAL SER

ick � m�GLY�CYSVAL CYS SER SER ASN PRO GLU ASP

Fig. 1. The myristale + Iysine mu-

hf is shared by several src family

members. The amino-terminal res-idues of the src family membersare indicated, with conservation ofthe Myr-GIy-X-X-Lys-X-Lys-X-

Lys motif, as well as other aminoacids, highlighted.

binding assays, we demonstrate that several src family

members utilize their NH2-terminal domain for mem-brane binding, indicating a general mechanism for mem-

brane interaction.

Results

Identification of a Conserved “Myristate plus Lysine”

Motif within the src Family. Interaction of pp60’�” with

cellular membranes is initiated by its myristylated amino-terminal domain (22, 26). We have recently establishedthat the critical determinants within this domain includethe myristate moiety and 3 lysmne residues at positions 5,7, and 9 (25). Interestingly, several other members of thesrc family of tyrosine kinases share this motif (Fig. 1).Specifically, src, lyn, and yes contain 3 NH2-terminallysines in similar positions, whereas fyn and hck contain2. It was therefore of interest to determine whether src-related kinases containing the “myristate + lysmne “ motifshared a common mechanism for membrane binding.

Membrane Association of in Vitro TranslatedppGOv.’.rt, pp6OC.StC p62C.YeS, p59fYn, and p56�. In orderto investigate the membrane association of p62�5�,pp60#{176}�, and p59�, we expanded upon an in vitrobinding assay previously designed to study the interac-tion of pp60”� with its membrane-bound receptor (26,27). In this assay, in vitro translated pp6Ovsr( has beendemonstrated to bind to plasma membranes in a satura-ble and specific manner. A similar methodology was usedto determine the mechanism by which src family mem-bers couple to the plasma membrane and to uncover theamino-terminal motif mediating this interaction.

pp6O”#{176}’t,pp60�#{176}”, p62t�#{176}, p59iYfl, and p56�’ weresynthesized by in vitro transcription of the appropriatecomplementary DNA clone (see “Materials and Meth-ods”), followed by translation of the correspondingmRNA in a reticulocyte lysate. Binding was compared inthe presence and absence of membranes (Fig. 2), thelatter being a measure of background aggregation. Uponaddition of a vole fibroblast fraction enriched for plasmamembranes (P100), approximately 55% of the pp60vs�and pp60�, and 25% of p59iY�i and p62t5� becamemembrane associated, based upon cofractionation withmembranes following ultracentrifugation. In contrast,membrane binding by p561�”, which lacks the NH2-ter-minal lysine motif, was negligible. Identical data wereobtained for binding to P100 membranes from NIH 313fibroblasts. These results were reproducible among var-ious membrane preparations, with pp60�� and pp60�#{176}�always being membrane bound to comparable levels,

A B

..----______--� \ --.

0 ��

:-. src.: Ick

.-------� - 0

1)

I..

.0

>Cso

0.0.

/11 cold polypeptide competitor es,�l cold p62��� competitor

Cell Growth & Differentiation 477

I ig. I. �-\, inhibition of I���”#{176}illeml)rane binding by pp6O’”.

P100 membranes were incubatedwith [ ‘#{176}S}methionine-labeled, in

S itr(i translated 1���’#{176}#{176}Protein in

the resence of increasing concen-tralions of unlabeled, in vitro Irans-

lated P060’” or pS6i�k polypep-tide. The amount of radiolabeled

membrane-bound p59#{176}’ was

c�uantitated as described in Fig. 2.The amount of pS9’#{176}#{176}bound in the

absence of PP6O’” competitorwas normalized to 100%. B, inhi-bition of pp6O’” membrane bind-

ing I)y p62’�#{176}�’. P100 membraneswere incubated with [#{176}#{176}S]methio-nine-labeled, in vitro translated

PP6O’” in the presence of in-creasing concentrations of unla-t)eled P62’’�’ Quantilation was asdescribed in .4.

and p62’ � and p59iSfl being bound about 50% lessefficiently than the src proteins. The difference in mem-brane binding cannot be attributed to differences inmyristylation. All proteins were as efficiently myristylatedas pp60#{176}� , as determined by incorporation of [3H]my-ristate with normalization to labeling by [35S]methionine(data not shown).

src Family Members Utilize a Similar Mechanism forMembrane Binding. If other src family members specifi-cally interact with the src receptor, it should be possibleto displace the polypeptides from the plasma membraneby addition of pp6O#{176}srt to the membrane binding assay.This supposition was verified by the data in Fig. 3A, inwhich in vitro translated [35S]methionine-labeled p59ivfl

was added to membranes in the presence of increasingconcentrations of unlabeled, in vitro translated pp6O#{176}#{176}”.As a negative control, addition of unlabeled p56� to thebinding assay had no significant effect on pp59� mem-brane binding.

A similar experimental strategy was used to test com-petition between pp60’�” and p62’ 5�.s As illustrated inFig. 38, unlabeled p62�s�s protein functioned as a com-petitor of pp6O’.#{176}’tbinding. Likewise, unlabeled pp6Ov5rinhibited binding of [35S]methionine-labeled p62’ � (Ta-

ble 1). Several additional pieces of data suggest that thecompetition among src family members is due to specific

Table 1 Polypeptide competition of pp60�” membrane binding

[#{176}#{176}S}Methionine-Iabeled p62’” and pp6O’5” were synthesized by insitro translation and incubated with equimolar concentrations of the

indicated polypeplide in the presence of P100 vole membranes. The

amount of membrane-bound material appearing in the pellet after ultra-centrifugalion was quantitaled as described in the text; binding in the

presence of unprogrammed lysate (no competitor) was normalized to1 00%.

Polypeptide competitor% memb

p62(Vr�

rane bound

pp6o�.’.�(

None 100 100NY31S-src 108 102

p56� 90 98pp6O” 50 40

p60�” 94 92

utilization of the NH2-terminal domain. A mutant ofpp60v�, lacking lysines 5 and 9, NKN-src (25), was a lessefficient competitor of p62’5� binding than was wild-type pp6O#{176}.#{176}”,requiring 3-4-fold higher molar amountsto achieve the same degree of inhibition (data notshown). Moreover, neither p56�’ nor NY315-src, a var-iant of pp6Ovsr( in which amino acids 2-14 have beendeleted, competed for p625’.’#{176}membrane binding (Table1). Finally, p60”t#{176}”,a chimeric myristylated protein con-taming the first 14 amino acids of calcineurin B subunitfused to residues 15-526 of v-src (25), does not inhibitassociation of pp60’� or p625’.s with membranes (Table1). The ability of pp6O�”, p59i5fl, and p62’5’.� to cross-compete implies that these src-related proteins are lo-calized to the plasma membrane via interaction with acommon receptor, consistent with the utilization of acommon targeting domain.

The NH2-terminal pp605� Binding Motif Is Utilizedby Other src Family Members. Previously, it has beenshown that binding of pp60vs� to plasma membranescan be competed by a myristylated peptide identical insequence to the NH2 terminus of pp60”�, MGYsrc (26).If pp6O(sit, p62�5�, and p595#{176}associate with the srcreceptor, through the NH2-terminal pp60vsr� binding mo-tif, it should be possible to inhibit their membrane bind-ing with MGYsrc. Indeed, upon the addition of increasingconcentrations of MGYsrc peptide, binding of p62’#{176}�#{176}

and p59Y�� was efficiently competed, as evidenced bytheir displacement from the plasma membrane fraction(see below).

We next tested the abilities of myristyl peptides cor-responding to the amino termini of other src familymembers to inhibit membrane binding. Three 12-merpeptides, containing the NH2-terminal sequences of lyn,yes, and fyn, were synthesized in NH2-terminally myris-tylated form and added to the in vitro membrane bindingassay. The amount of in vitro translated protein in thepellet and supernatant fractions was quantitated, as be-fore, in the presence and absence of membranes. The“minus membranes” control is always included to ensurethat material appearing in the pellet fraction is dependenton the presence of membrane components. In theseexperiments, as well as our previous work (25, 26),

5)

5)

0.

5)

5)

Os

0.

�u � � myr-yes peptide

� . -tubs

::� ��

0 � � - -� � -�-- -___0 50 100 150

[peptide] pM

40Tg�-i � �� ‘-, +myr-fynpeptide

20 � � H-� - --- .---#{149} � 5)

�- .--. �

� I� �j

[peptide] ,�i

.;: � �--�--� � �� B + myr-src peptide� . -mbs

30 �-� V +mbs Fig. 4. Effect of myrislylated pep-

� tides on p59”. and pp6o” aggre-� �.-.-.-- galion. [35SjMethionine-Iabeled, in

20 �- � . vitro translated p59155 (A-C) or�V-___� pp6O�’.’� (0) was incubated with

increasing concentrations of myr-

istylated src family peptides, in the

; 0 t- _____�_____.-.. absence (#{149})or presence (V) of

� � membranes. Following ultracen-� _____---..- �- � � trifugation at 100,000 x g, the pel-� let and supernatanl fractions were

0 � ‘ � analyzed by gel electrophoresis,0 50 1 00 1 50 auloradiography, and scintillation

F e tide1 M counting. The amount of radiola-LP P i /� beled protein which fractionated

in the pellet was quantitated andexpressed as a fraction of the total.

r � T � A, fractionation of p59iW� in the

70 �- D +myr-lyn peptide presence of myr-yes peptide� (GCIKSKEDKGPY). B, fractionation

6.0 - ___-v - of p59” in the presence of myr-

� v- � src peptide (GSSKSKPKDPSY). C,50 � � fractionation of p591” in the pres-

� ________. ence of myr-fyn peptide (GC-

40 L- �------- VQCKDKEATY). 0, fractionation

� .- -� � of pp60�’.” in the presence of_�0 - � -. myr-lyn peptide (GCIKSKGK-

� . DSLY). Note the peptide-depend-

_0� L -� ent increase in protein aggregation(- membranes) induced by myr-

� 0 Q -� fyn and myr-lyn peptides.

.� � ‘ )0 � 0( � 1 20 200

[peptide] 1iM

478 Membrane Binding of src Family Members

MGYsrc peptide had no significant effect on the amountof pp60vs�, p59iYfl or p625(s that appeared in the pelletfraction in the absence of membranes. The myristylatedyes peptide behaved in a similar fashion to MGYsrc (Fig.4, A and B). Inhibition of membrane binding was ob-served with both peptides.

In contrast, myristyl-lyn and fyn peptides induced ag-gregation of in vitro translated protein, as evidenced bythe appearance of p591#{176}�(Fig. 4C) and pp60v� (Fig. 4D)in the pellet fraction in the absence of membranes. Theformation of protein aggregates obscured any potentialeffect of the peptides on membrane binding, as theaggregates cofractionated with the membrane pellets.We therefore designed a density-based centrifugationstep to separate peptide-induced protein aggregatesfrom membrane-bound material. As illustrated in Fig. 5,centrifugation of the binding assays through a 40% su-crose cushion separated aggregated protein, which pel-lets to the bottom of the tube (“gradient pellets”; Fig. 5,Lanes 7-12), from membrane-associated material, whichremains floating on top of the sucrose. The net amountof membrane-bound material was calculated by subtract-ing the amount of radiolabeled protein in the “gradientpellet” from that in the total “membrane pellet.” Thevalidity of the gradient subtraction method was verifiedusing an alternate assay (see “Materials and Methods”).With this additional gradient step, it is clear that syntheticpeptides corresponding to the myristylated amino ter-mini of src, yes, fyn, and lyn inhibited association ofp59ivn, p625(s, and pp60�” proteins with membranes (Fig.

6, A-C, and data not shown). Myristylation was necessary,as neither the nonmyristylated version of the NH2-ter-minal src peptide, GYsrc (Fig. 6, A and C), nor thenonmyristylated c-yes peptide (data not shown) com-peted in the assay. These data are quite comparable tothose previously obtained by this laboratory (25, 26),where half-maximal inhibition of pp60v�s� membranebindingwas obtained at approximately 100-150 zM myr-istyl-src peptide.

To test the importance of lysine residues in membranebinding by p62cY�s and p59�, a myristylated peptidecorresponding to the NH2 terminus of pp60v�( was syn-thesized, in which all three lysines were replacedby asparagine, NNN-src. This peptide fails to inhibitpp605� and pp60� membrane binding (25) and hasbeen shown to be an excellent predictor of membraneassociation in vitro and in vivo. Use of the myristylatedNNN-src peptide provides a versatile molecular tool thatallows one to bypass the limitations of site-directed mu-tagenesis at Iys-7, a residue required for myristylation ofpp605�. Experiments with the NNN-src peptide, com-bined with mutagenesis, established that lysine residueswere required for membrane targeting of pp60’�5’� (25).Consistent with our previous results, NNN-src failed toabrogate binding by p59f5�, p62’�, and pp60�t� in thein vitro binding assay (Fig. 68, and data not shown). Asan additional negative control, we attempted to assessthe effect of a myristylated Ick peptide on membranebinding. Unfortunately, the Ick peptide induced an ex-cessive amount of aggregation, with 75% of the total

Membranes

Myr-fyn Peptide

Membrane

Pellets

- - - +++

- + ++ - + ++

�4

GradientPellets

- - - +++

- + ++ - + ++

� �

7 8 9 1011 12

w � w

123456

A �59f�fl

�0

� .

0 #{149}

�)

�0

� . 11.‘.r V

�.. .u�vr�r

V (,i

V

V

0

. 5)

I-.0

..

. 5)

. ..

B �62Yes

�

V.

. V

...

.--.. V

. �iVi v-sV r�i.r vu

: \��N

[peptide] uM

.�

5)

.�

5)

_c � p�6OC�

VV

.

.. ... .- .

. myr-yes

. .... . V myr-tyn

- GYsrc

(peptidej uM[peptidel uM

Cell Growth A Differentiation 479

I ig. :;#{149} Separation i)t mvristvl r�p-tide-indu ed aggregates bs su-( rose gra(li(’nt c entritugation. Ag-gr(’gatii)n (if in s iro tr.inslated pro-

teiii ssas observed upon addition

of sonie p#{128}’ptides even in the ab-se’n e (if membranes, as is illus-trated with in sitro translated

01)60’” and mvr-fvn. To removeaggregat(’d material, l),sr.Illel hind-

ing assays were overlaid on a 40%

sucrose gradient, whic h prevents

plasma membrane sedimentation,and centrifuged at 200,000 x g for

1 h. Less than 10#{176}/oi)t the nwm-l)rane-l)ouil(l ni,itt’ri,il pelk’tedthrough t(�’ sucri)s(’ ( ushion (com-

pare L,inc’� 4 and 10). The amountof m,iterial that )(‘ll(’fed through40#{176}/osuc rose was defined as ot’p-

tide-indin (‘d aggregates I gradient

peIIe(� ) ,iri(l ssas sul)tracted fromthe amount in the standard

1 00,001) x g membrane Pellet

(n�ernbr,ine pellets I Ii) derive net

values for membrane-hound ma-

terial see Materi.ils and Meth-ods’ . All numerical s ,ilues svere

normaliied to 100#{176}�in the al)sc’ncs’ of )(‘ptide. Lanes I . 4, 7, and1(1 contain no peptick’: Lanes .1.8, ,in(l I I ( ontain f�n peptide at100 �g/rnl (ir 63 �isi: Lanes 1, 6, 9,

and 1.1 ( iintain f�n l)(’Pfide at 200lAg/mI or 1 31) MM.

pp6O’ �‘ appearing in the minus membranes pelletat 100

�LM Ick peptide, and 92% at 200 .tM Ick peptide. It was

therefore not possible to obtain reliable quantitation of

pp6O’ “ membrane binding in the presence of this pep-

tide. Taken together, these data indicate that src, yes,

fyn, and lyn exhibit cross-talk at the level of membrane

binding. The striking similarity in binding parameters

strongly suggests that pp6O’ �‘ , p62’ “S, p59ivfl and p56”

use the NH2-terminal myristate + lysine motif as a com-

mon mechanism for mediating membrane interaction.

Discussion

In this manuscript, we have investigated the mode ofinteraction of several src family members with cell mem-

branes. Using an in vitro membrane binding assay, it wasshown that p59iifl and p62�s polypeptides bind to fibro-

fig. (� Inhibition of p59”, p62’”. and pp6O’” membrane binding by src-related myristyl peptides. A, pS9’�; B, p62�; C, pp6O’”. P100 membranefractions sse’re’ incubated with [“S]methionine-laheled, in vitro translated p59�’, p62�r�, or pp6O’ “ in the presence of the following src family member

peptid#{128}’s: m\r-sr, niyr-lyn, mvr-yes, NNN-src a myrislylated src peptide in which lysines 5, 7, and 9 have been replaced with asparagine; myr-GSSNSNPNDPSY) or GY-sr (a PePtide identical in sequence to myr-src, but in a nonmyrislylaled form). The quanlitation of membrane-bound materialwas as described in legend to Fig. 5. [Nearly identical results were obtained for inhibition by myr-src and myr-yes peplides using the standard membranel)inding ass.iv.] The an)(iunt of radiolaheled prole’in bound to membranes in the absence of peptide was normalized to 100%.

480 Membrane Binding of src Family Members

blast membranes in a manner similar to that of pp6Ovsr(.Each of the polypeptides competes with one another formembrane binding, whereas p56��’ is without effect.Moreover, peptides corresponding to the NH2-terminaldomains of src, lyn, yes, and fyn also inhibit membraneassociation.

Several lines of evidence support the contention that,like pp605”, interaction of specific src family membersoccurs via the myristylated NH2-terminal domain con-taming the myristate + lysine motif. First, membranebinding of v-src, c-src, c-yes, and fyn polypeptides canbe reproduced in vitro, whereas lck, which lacks theNH2-terminal lysine residues, remains largely soluble (Fig.2). Myristylated src, yes, and fyn proteins compete witheach other for membrane binding; related proteins whichlack the NH2-terminal lysine motif (NKN-src, p56�, andcal-src) have limited or no effect. Second, myristylatedpeptides containing the NH2-terminal sequences of src,yes, fyn, and lyn inhibit membrane binding (Fig. 6). Third,replacement of the lysines with asparagine residues abol-ishes the inhibitory effect of the src peptide not only onpp605� membrane binding (25), but also on p625e5 (Fig.68), pp60sr, and p5915fl (data not shown). Finally, non-myristylated peptides derived from the src or yes se-quences do not inhibit membrane association of theaforementioned src kinases (Fig. 6, A and C). It is alsoimportant to note that several other residues are highlyconserved within the NH2 termini of these kinases, in-cluding cys-3, ser-6, and asp/glu-9/10. Although the ly-sine residues seem to be major determinants, one cannotexclude an additional role for these other NH2-terminalamino acids.

Taken together, these results demonstrate that

pp60�’#{176}, p62(.v��s, p59tvn, and p56� utilize a commonbinding motif in what was presumed to be a uniquedomain and suggest that localization to the plasma mem-brane occurs via interaction with a common “receptor”(or receptor family). In fibroblasts, a 32kDa3 membraneprotein has been identified which specifically binds themyristylated NH2-terminal sequence of src (28). Incuba-

tion of i2sllabeled myristylated src, yes, fyn, and lynpeptides with fibroblast membranes, followed by chem-ical cross-linking, resulted in the radiolabeling of the32kDa protein (data not shown). Incorporation of radio-label was inhibited by nonradioactive MGYsrc peptide,but not by nonmyristylated GYsrc peptide. Althoughthese data indicate that the src-related peptides displaysimilar properties in cross-linking to the 32kDa protein,definitive evidence that the 32kDa protein functions asa src protein receptor, in addition to a src-peptide recep-tor, is not yet available. Nevertheless, the ability of thevarious peptides to be recognized by the 32kDa proteinimplies that they have similar chemical properties.

Another feature of myristylated peptides that becameapparent during these studies is their differential tend-ency to cause protein aggregation. We therefore devel-oped a sucrose gradient protocol, which, when usedwith the standard membrane binding assay, allows effec-tive separation of aggregated protein from membrane-bound material. This methodology should also prove

3 The abbreviations used are: kDa, kilodalton(s); DM50, dimethylsulfoxide.

useful to other investigators using myristylated peptidesfor in vitro assays.

The data illustrated in Fig. 6 indicate that inhibition ofmembrane binding of pp6Osr(, p62(.Yes and p59tY1� withNH2-terminal peptides is incomplete; i.e., 20-40% of themolecules remain membrane bound even in the pres-ence of high peptide concentrations. The most likelyexplanation for this observation is the presence of sec-ondary membrane binding domains within the src familypolypeptides. It is well documented that downstreamdomains of pp60v� also contribute to membrane local-ization (25, 29). It is therefore likely that, like src, addi-tional domains within yes, fyn, and lyn (SH2, SH3) me-diate distinct secondary contacts with membrane-boundsubstrates. Alternatively, it is possible that two popula-tions of pp60� exist: one that is sensitive to peptideinhibition and one that is resistant. Furthermore, fibro-blasts may harbor the receptor(s) that is optimized forpp60� binding, whereas in other cell types, binding byfamily members may be equivalent, or one of the othertyrosine kinases may be preferentially utilized.

There is precedence for localization of multiple srcfamily members to a single protein. p56’��, p59�, andp621�’ all have been found to be associated with gly-coprotein IV (CD36) in human platelets (14); pp60(s�,p5&��, and p62Y can associate with the high affinitylgE receptor after engagement (30); and pp60�’t, p56�,and p62�’�’ interact with the platelet-derived growthfactor receptor after stimulation (31). In the latter case,recruitment to the receptor is through the SH2 domainrather than through the amino terminus. However, priorto activation of these specialized receptors, the src-related tyrosine kinases are most likely membranebound. Thus, the src family members may be tetheredto a general membrane receptor via their amino-terminaldomain. The NH2 terminus may contribute to specializedfunction(s) or alternatively may serve a traffickingfunction.

The amino-terminal domain of p56�’ is also utilizedfor membrane localization by interaction with the cyto-plasmic domain of two of the surface glycoprotein recep-tors of mature peripheral T-cells. Interaction with CD4and CD8 is mediated by cysteine residues (32) not pres-ent in pp6Oc.Src. Curiously, lysine residues are absent fromthe amino terminus of p56�. Although interaction basedupon cysteine residues may be a unique feature of p56kt�,it is consistent with the model that the amino terminusof the nonreceptor tyrosine kinases is important for sub-cellular localization.

We propose a general mechanism for membrane bind-ing in which amino-terminal residues in conjunction withmyristate are utilized by several members of the srcfamily for localization to the plasma membrane. Use of aubiquitous targeting apparatus has many implications forthe biological properties of these molecules and impliesa degree of cross-talk between signaling pathways thatmight otherwise appear distinct.

Materials and Methods

Cell Culture and Membrane Fractionation. Normal fieldvole cells were maintained in tissue culture as previouslydescribed (33). Plasma membrane-enriched fractions(P100) were prepared by hypotonic lysis, homogeniza-tion, and ultracentrifugation as described (33) and resus-

Cell Growth & Differentiation 481

pended to a protein concentration of 0.3 mg/mI in NTEbuffer [100 mM NaCI-lO m�,i Tris (pH 7.4)-i m�i EDTA].

Plasmid Construction. The plasmids pGEM-src (27)and pGEM-NKN-src (25) encode wild-type pp60� anda variant of pp60’�’ in which lysines 5 and 9 have beenreplaced with asparagine. pGEM-yes was created bytreatment of pGEM-src with NcoI and EcoRI to removethe coding region of v-src, but to retain the 5’ untrans-lated leader. c-yes was excised from the plasmid p6a (6)by digestion with NcoI and partial digestion with EcoRland ligated into the site of the v-src deletion in pGEM-src. pGEM-fyn was created in a similar fashion, by diges-tion of the plasmid pSP6SNcolfyn [a kind gift of P. Espino(Genzyme) and S. Courtneidge (EMBL)] followed by Ii-gation into the site of the v-src deletion. This was doneto remove the 5’ untranslated leaders of c-yes and fyn,which are inhibitory to translation. pGEM-lck was a gen-erous gift from N. Rosen (Sloan-Kettering Institute).

In Vitro Synthesis and Membrane Binding. mRNA syn-thesized by in vitro transcription was translated in rabbitreticulocyte lysates as described (26). A 20-zl aliquot ofthe translated lysate, equilibrated to 0.075% Triton X-100, was incubated with 30 �l of NTE buffer or P100membranes for 30 mm at 20#{176}C.Following ultracentrifu-gation at 100,000 X g, the pellet and supernatant wereanalyzed by gel electrophoresis (34) andautoradiography.

Peptide-induced aggregates were removed by pellet-ing a duplicate sample of the in vitro translated materialthrough a 40% sucrose cushion (40% w/w sucrose inNTE) at 200,000 x g for 1 h. The gradient pellets weresolubilized with sample buffer and were analyzed alongwith the standard membrane pellets by gel electropho-resis and autoradiography. �The following numbers rep-resent the amount of in vitro translated pp60”� [(cpm ingradient pellet/cpm total) x 100] that was collected inthe gradient pellet in the presence of 100 jzM and 200 zM

peptide, respectively: no peptide, 1%; myr-src: 2%, 4%;myr-yes: 5%, 8%; myr-fyn: 17%, 30%; myr-lyn: 16%,37%; myr-Ick: 57%, 73%.l The amount of material in thegradient pellet was subtracted from the amount in the100,000 x g membrane pellet to derive net values formembrane-bound material. To ensure the validity of this“subtraction” method, an alternate assay was performed.The reaction mixture was overlaid on a sucrose gradientand centrifuged as above, and the material at the top ofthe gradient (supernatant) was re-spun (after dilution withNTE buffer) at 100,000 x g to re-isolate the membranes.The results obtained were nearly identical to those of thesubtraction method.

Peptides. Dodecapeptides containing the NH2-termi-nal amino acids of src (GSSKSKPKDPSY), NNN-src(GSSNSNPNDPSY), lyn (GCIKSKGKDSLY), c-yes (GCIK-SKEDKGPY), fyn (GCVQCKDKEATY), and Ick (GCV-CSSNPEDDY) were synthesized, with myristic acid co-valently linked via an amide bond to the amino-terminalglycine residue (Multiple Peptide Systems, San Diego,CA), and were purified to greater than 80% by reverse-phase high performance liquid chromatography. Theamino acid sequences corresponding to wild-type srcand c-yes were also prepared in a nonmyristylated form.All peptides contain the first 1 1 amino acids ofthe maturepolypeptide with a COOH-terminal tyrosine residue ap-pended. The peptides were dissolved at a concentrationof 3.3 mM in DMSO; control experiments contained an

equivalent volume of DMSO alone. No effect of DMSOon the membrane binding assay was noted.

Acknowledgments

We thank M. Ramkishun for expert technical assistance, and P. Besmer,H. Hanafusa, and D. Wilson for helpful discussion and critical reading ofthe manuscript.

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