MOLECULAR AND CELLULAR BIOLOGY, Jan. 1990, p. 361-3700270-7306/90/010361-10$02.00/0Copyright © 1990, American Society for Microbiology

Early Activation of Endogenous pp60Orc Kinase Activity duringNeuronal Differentiation of Cultured Human Neuroblastoma Cells

CATARINA BJELFMAN,1 GABRIELLE MEYERSON,' CHRISTINE A. CARTWRIGHT,2t KARIN MELLSTROM,lULF HAMMERLING,' AND SVEN PAHLMAN1*

Department of Pathology, University Hospital, University of Uppsala, Uppsala, Sweden,' and Molecular Biology andVirology Laboratory, Salk Institute for Biological Studies, San Diego, California 921382

Received 1 May 1989/Accepted 3 October 1989

The proto-oncogene product pp60cs`c is a tyrosine-specific kinase with a still unresolved cellular function.High levels of pp60C-cs in neurons and the existence of a neuronal pp6OCSrC variant, pp60c-srCN, suggestparticipation in the progress or maintenance of the differentiated phenotype of neurons. We have previouslyreported that phorbol esters, e.g., 12-O-tetradecanoylphorbol-13-acetate (TPA), stimulate human SH-SY5Yneuroblastoma cells to neuronal differentiation, as monitored by morphological, biochemical, and functionaldifferentiation markers. In this report, we describe activation of the pp6(jrC (pp60csrc and pp60csrcN) kinaseactivity observed at 6 h after induction of SH-SY5Y cells with TPA. This phenomenon coincides in time withneurite outgrowth, formation of growth cone-like structures, and an increase of GAP43 mRNA expression,which are the earliest indications of neuronal differentiation in these cells. The highest specific src kinaseactivity (a three- to fourfold increase 4 days after induction) was noted in cells treated with 16 nM TPA; thisconcentration is optimal for development of the TPA-induced neuronal phenotype. During differentiation,there was no alteration in the 1:1 ratio of pp60`csrc to pp60csrN found in untreated SH-SY5Y cells. V8 proteaseand trypsin phosphopeptide mapping of pp6(Y"' from in vivo 32P-labeled cells showed that the overallphosphorylation of pp6(Wt' was higher in differentiated than in untreated cells, mainly because of an intenseserine 12 phosphorylation. Tyrosine 416 phosphorylation was not detectable in either cell type, and no changeduring differentiation in tyrosine 527 phosphorylation was observed.

Neuroblastoma is a neural crest tumor of early childhood(25). In rare cases, this tumor spontaneously differentiates invivo and forms nonproliferating cells with morphologicalfeatures resembling those of mature neurons (24). Theseclinical observations form the basis for the use of culturedneuroblastoma cells as in vitro models for neuronal differ-entiation.The adrenergic human neuroblastoma SH-SY5Y cells (8)

differentiate further along a neuronal pathway in the pres-ence of nanomolar concentrations of biologically activephorbol esters, of which 12-O-tetradecanoylphorbol-13-ace-tate (TPA) is one of the most potent (49). Signs of differen-tiation include neurite outgrowth and increases of neu-rosecretory granules, noradrenaline concentration, andacetylcholine esterase activity. There is also a partial switchin the enolase profile toward an increased expression ofneuron-specific enolase (NSE; y-enolase) (1, 49, 50). TPA-treated cells are functionally differentiated, as manifested byan increased membrane potential and an ability to be depo-larized by high external concentrations of potassium (2).Furthermore, when triggered with acetylcholine, the differ-entiated cells release stored noradrenaline (55). An impor-tant feature of the differentiated cells is a pronouncedreduction of the proliferation rate compared with that ofnontreated SH-SY5Y cells (49). The mechanisms underlyingthis event appear to involve differentiation-stage-dependentdown regulation or uncoupling ofgrowth factor receptors, asdemonstrated for the insulin and somatomedin receptors (45;

* Corresponding author.t Present address: Division of Gastroenterology, Department of

Medicine, Stanford University School of Medicine, Stanford, CA94205.

M. E. K. Mattson, U. Hammerling, E. Mohall, K. Hall, andS. Pahlman, Growth Factors, in press).

Postmitotic neurons of rat brain and chicken retina showhigh expression of src protein and kinase activity (11, 14, 22,57), and the specific src kinase activity increases during invitro differentiation of mouse embryo carcinoma cells (42)and rat striatal neurons (14). The src protein in rat brainneurons is modified in the N terminus (pp60-srcN) and has aspecific tyrosine kinase activity higher than that of thenonneuronal form, pp60c-src (10, 11). However, pp6Oc-srcNisolated from established human neuroblastoma cell linesappeared not to have a specific kinase activity higher thanthat of pp6Oc-src prepared from fibroblasts or glioblastomacells (48, 66). Recently, c-srcN cDNA clones from chickenand rat brains have been isolated (40, 44). Sequence datarevealed that the c-srcN transcript, via alternative splicing,has 18 extra nucleotides transcribed from the intron betweenexons 3 and 4. Whereas high src expression is found in manydifferent types of cells, e.g., neurons, neuroendocrine tumorcells, bovine chromaffin cells, and blood platelets (11, 28, 46,51), c-srcN expression has, with the exception of its detec-tion in AtT20 pituitary tumor cells (30), so far been foundonly in cells differentiated along a neuronal lineage, e.g.,neurons and neuroblastoma cells (11, 46). In peripheralneural tissue, though, pp6Oc-srcN appears to be considerablyless abundant than in different regions of the brain (38).SH-SY5Y neuroblastoma cells express high src protein

levels, with approximately 50% in the neuronal form (46). Inthis report, we show that the specific src kinase activity inSH-SY5Y cells increases threefold during neuronal differen-tiation induced by TPA. This increase does not involve ashift in the ratio of expressed c-src to c-srcN products.Activation of the pp6&Src (pp6Jcsrc pp6JcsrcN or both)

361

Vol. 10, No. 1

362 BJELFMAN ET AL.

kinase activity is apparent 6 h after induction and coincidesin time with the onset of neurite and growth cone formation,as evidenced morphologically and by the pronounced in-crease in expression of the growth cone-associated proteinGAP43. Thus, activation of the pp6Osrc kinase is a compar-atively early phenotypic change in differentiating SH-SYSYcells.

MATERIALS AND METHODS

Cell cultures. The SH-SY5Y cell line is an adrenergic cloneof the human neuroblastoma cell line SK-N-SH (7, 8) andwas kindly provided by June Biedler (Sloan-Kettering Insti-tute for Cancer Research, New York, N.Y.). The cultureconditions for these cells and the conditions for inductionand culture of TPA-differentiated cells have been describedin detail previously (50). A TPA concentration of 16 nM wasfound to result in optimal neuronal differentiation of SH-SY5Y cells (50).

Biochemical assays. NSE was measured with a radioimmu-noassay (Pharmacia AB, Uppsala, Sweden) and correlatedto total cell protein, determined by a modified Lowry pro-cedure (50).

src kinase assays. Cells were washed in phosphate-bufferedsaline and solubilized on ice for 15 min in the presence orabsence of 100 ,uM boiled sodium orthovanadate in 10 mMTris hydrochloride (pH 7.2) containing 0.16 M NaCl, 1%(wt/vol) Triton X-100, 1% sodium deoxycholate, and 0.1%sodium dodecyl sulfate (SDS) (RIPA buffer) and supple-mented with 1 mM EDTA and 1 mM ethylene glycol-bis(P-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA).After centrifugation (30,000 x g, 30 min), the protein con-centrations of the supernatants were normalized. The srcprotein(s) was immunoprecipitated from 0.1 to 0.2 mg oftotal protein (1 mg if subsequent Western [immunoblot]analysis was conducted) with either the monoclonal anti-body (MAb) 327 or MAb 273 (41) (gifts from Joan Brugge,State University of New York, Stony Brook), using 2 ,ul ofascites fluid per mg of total protein. The two antibodies gavecomparable results. The immune complexes were precip-itated with formaldehyde-fixed Staphylococcus aureus (giftfrom Lars Rudin, Pharmacia AB), preincubated with anti-mouse immunoglobulin antiserum (Dakopatts, Copenhagen,Denmark), and washed three times in RIPA buffer. Theimmune complex src kinase assay was performed on ice in10 mM Tris hydrochloride (pH 7.2) containing 5 mM MgCl2,0.5 puM ATP, and 5 puCi of [y-32P]ATP (Dupont, NENResearch Products, Boston, Mass.). As an exogenous sub-strate for the src kinase, acid-activated rabbit muscle eno-lase (Sigma Chemical Co., St. Louis, Mo.) was used asdescribed previously (18). Under these assay conditions, thereaction was linear for 20 min. The addition of 20 puM coldATP to the reaction mixture did not affect the pp60c-srckinase activity measured in colonic tissues (C. A. A., A.Meisler, and W. Eckhart, Proc. Natl. Acad. Sci. USA, inpress). After 20 min of incubation, the reaction was stoppedby SDS-polyacrylamide gel electrophoresis (PAGE) samplebuffer. The boiled samples were analyzed by SDS-PAGEunder reducing conditions (37), using a 10% gel. The phos-phorylated proteins were visualized by autoradiography andidentified by their molecular weights.

[35S]methionine labeling of cells. Cells were labeled for 4 hin low-methionine (0.1 times the normal [7.5-mg/liter] methi-onine concentration) RPMI 1640 medium containing 0.1 mCiof [35S]methionine per ml. The cultures were prepared forimmunoprecipitation as described above. Labeled pp6Osrc

was analyzed by SDS-PAGE, using a 7.5% polyacrylamidegel as described previously (10).

Immunoblotting. Immunoprecipitates (see above) from 0.5mg of total protein were subjected to SDS-PAGE (without2-mercaptoethanol in the sample buffer), and the proteinswere then electrophoretically blotted onto nitrocellulosefilters (61). They were blocked overnight at 4°C in 1% nonfatdry milk (Semper) dissolved in 10 mM Tris hydrochloride(pH 7.5) containing 0.17 M NaCl (buffer A). The filters wereincubated at room temperature for 2 h with MAb 327 (1:500dilution) in buffer A containing 0.05% (wt/vol) Nonidet P-40,0.25% gelatin, and 0.5% dry milk (buffer B). After fivewashings in buffer A supplemented with 0.05% Nonidet P-40and 0.5% gelatin, the filters were incubated at room temper-ature for 2 h with 0.25 ,uCi of iodinated anti-mouse immu-noglobulin antibodies (The Radiochemical Centre, Amer-sham, United Kingdom) per ml in buffer B. Finally, thefilters were washed five times in buffer A with 0.05%Nonidet P-40, and after autoradiography, pp6Osrc was iden-tified by its molecular weight.

Quantification of specific pp6)Src kinase activity. For deter-mination of the specific pp60src kinase activity, cell lysateswere precipitated as described above and divided into twoparts, one for kinase assay (0.15 mg of total protein) and onefor immunoblotting (0.5 mg of total protein). After the kinasereaction and electrophoresis, the phosphorylated src andenolase protein bands (identified by autoradiography) wereexcised, and incorporated radioactivity (Cerenkov counts)was measured. Similarly, pp60src levels, analyzed by immu-noblotting with '25I-labeled protein A as the detection sys-tem, were quantified by counting the excised nitrocellulosefilter pieces corresponding to pp60src. The relative specificautophosphorylation and enolase activities were calculatedas the ratio of relative kinase activity to relative src proteinlevel. The specific kinase activity in control cells, lysed inthe presence of orthovanadate, was set at 1.0.

Peptide mapping. pp60src labeled in vivo with 32Pi for 4 hwas prepared for V8 protease mapping and analyzed asdescribed previously (46) except that the cells were lysed inthe presence of 100 puM orthovanadate. Alkali treatment ofthe gels was carried out as described elsewhere (20). Briefly,the gel was fixed overnight, soaked in 1 M KOH for 2 h at60°C, and washed in destaining solution.For two-dimensional tryptic peptide mapping, cells were

labeled for 14 h at 37°C in phosphate-free Dulbecco modifiedEagle medium supplemented with 10% dialyzed, phosphate-free fetal calf serum and 2.5 mCi of 32Pi per ml as describedpreviously (14). pp6Osrc was immunoprecipitated as de-scribed previously (14) except that the RIPA buffer con-tained 100 puM orthovanadate. The proteins were separatedon a 7% polyacrylamide gel, extracted, oxidized, and di-gested with trypsin as described elsewhere (6). Trypticpeptides were separated in two dimensions on 100-pumcellulose thin-layer plates by electrophoresis at pH 1.9,followed by chromatography (14).

Northern (RNA) hybridization. Poly(A)+ RNA prepara-tion, RNA blotting, and Northern hybridization were per-formed by standard procedures (33). cDNA probes forGAP43 and human glyceraldehyde-3-phosphate dehydroge-nase (pHcGAP3) were kindly provided by Pate Skene,Stanford University, Stanford, Calif., and Ray Wu, CornellUniversity, Ithaca, N.Y., respectively. The latter probeserved as a quantitative mRNA standard.

MOL. CELL. BIOL.

ACTIVATION OF pp60S'rc KINASE ACTIVITY 363

2d 4d 8d

<:- 4:-0. 0. 0a.

o C.) I 0 ~

pp6O _ _ e-s

enolase -

FIG. 1. src kinase activity in control (Ctrl) and 16 nM TPA-treated SH-SYSY cells grown for 2, 4, and 8 days. The cells werelysed in RIPA EGTA-EDTA buffer containing 100 FLM sodiumorthovanadate. The protein content of the lysates was normalizedbefore immunoprecipitation with MAb 327 and subsequent srckinase assay, in which rabbit muscle enolase was used as anexogenous substrate. The immunoprecipitates were separated bySDS-PAGE, followed by autoradiographic visualization of the au-tophosphorylated pp6Osrc and the phosphorylated enolase. Theproteins were identified by their molecular weights.

RESULTS

Increased src kinase activity in differentiated SH-SY5Ycells. The pp6Osrc kinase activity was measured in cellsgrown for 2, 4, or 8 days with or without 16 nM TPA (Fig. 1).Cell lysis was accomplished in the presence of orthovana-date, and the samples were normalized for volume andprotein content. The specificity of the immunoprecipitationwas checked by omitting the pp60rc_specific antibodies. Thekinase activity levels at each time point were higher indifferentiating than in control cells. The kinase activity in theuntreated cells also increased with time in culture (Fig. 1).This observation is analyzed in more detail below. In cellstreated with TPA for shorter time periods, a distinct increasein src kinase activity was noted as early as 6 h afterstimulation (Fig. 2). In a previous study, we found that 16nM TPA is the optimal concentration for induction ofdifferentiation of SH-SY5Y cells (50). The src kinase activitywas therefore measured in cells treated for 4 days with 0.16nM, 16 nM, or 1.6 ,uM TPA, and we observed that aconcentration of 16 nM resulted in the highest pp6Osrc kinaseactivity level (Fig. 3A). Furthermore, there was virtually noactivation in cells treated with 0.16 nM or 1.6 ,uM TPA.

c-src and neuronal c-src protein levels in differentiatedSH-SY5Y cells. SH-SY5Y cells express both pp60`csrc andpp6Oc-srcN in approximately a 1:1 molar ratio (46). In view ofthe observations by Brugge et al. (11) that the neuronal formof the src protein has specific kinase activity higher than thatof pp6Ocsrc, we investigated whether the level of pp60csrcNalone was increased in differentiated SH-SYSY cells. Forthis purpose, pp60src was immunoprecipitated from un-treated and TPA-treated [35S]methionine-labeled cells andanalyzed by SDS-PAGE. It was found that the ratio betweenthe amount of labeled c-src and c-srcN proteins was appar-ently unaffected by TPA treatment when analyzed 4 and 8

C4Dco N r co('4 4t

-a <<+ <<++ r

Mab 327 + - + + + + +

pp6O - _ _U M0

FIG. 2. src kinase autophosphorylation in SH-SYSY cellstreated with TPA for 2 to 48 h. All cells were grown for 48 h, and 16nM TPA was added to the cells for the last 2, 6, 12, 24, or 48 h ofculture. After lysis and immunoprecipitation (see legend to Fig. 1),src kinase activity was compared with that of untreated cells grownfor 48 h (Ctrl). The specificity of the immunoprecipitation wasverified by omitting the anti-pp60src antibodies.

days after induction of differentiation (Fig. 3B). Further-more, these cells contained less labeled src proteins than didthe untreated cells. From the Western blot analysis dis-cussed below (see Fig. 4B and Table 1), it seemed that thedivergence between the amount of 35S-labeled pp6OsrC innondifferentiated and differentiated SH-SY5Y cells (Fig. 3B)merely reflected a difference in [35S]methionine incorpora-tion or src protein metabolism between these cell typesrather than a true difference in src protein level.

Increased specific kinase activity of pp6(src in differentiatedSH-SY5Y cells. A number of serine and tyrosine residues inpp6Osrc are known to be phosphorylated in vivo. Some ofthese sites appear to be involved in the regulation of srckinase activity, as exemplified by Tyr-527 in chickenpp6Ocsrc (12, 19, 21, 23, 36, 53, 54), equivalent to Tyr-530 inthe human c-src molecule (60), and N-terminal serine ortyrosine residues in platelet-derived growth factor-stimu-lated fibroblasts (31). Since orthovanadate inhibits phos-phatases (primarily tyrosine phosphatases [23, 58]), wetested whether the phorbol ester-induced activation ofpp6Osrc in SH-SY5Y cells was affected by the presence oforthovanadate in the lysis buffer. pp6Osrc immunoprecipi-tates from control and differentiated cells, obtained in theabsence or presence of orthovanadate during lysis, weresubjected to both kinase assay and Western blot analysis(Fig. 4). No significant difference in pp6Osrc level betweenthe two cell types and the two lysis protocols was observed(Fig. 4B). There was a pronounced difference in the kinaseactivity levels between control and TPA-treated cells whenthe lysis buffer was supplemented with orthovanadate (Fig.4A). However, in the absence of orthovanadate during lysis,the difference in src kinase activity levels between controland differentiated cells was small (Fig. 4A). To quantitate theapparent changes in specific src kinase activity, the auto-phosphorylation and enolase kinase activities were deter-

VOL. 10, 1990

364 BJELFMAN ET AL.

TABLE 1. Specific kinase activity of pp6OSrc in SH-SYSY cells2 2 :E

'c .,-Lo D Lt:

c< <: <Q. 0. 0.

A

pp6O --- _f*1

enolase - t IIu.

4d 8d

_ cL S- C.v

B

b Autophosphorylation Enolase kinase

Activityc Sp actd Activityc Sp actd

Control 6,386 (1.1) 285 (2.1) 2.0 780 (1.8) 1.7Control + V 6,060 (1.0) 135 (1.0) 1.0 430 (1.0) 1.0TPA 5,643 (0.93) 356 (2.6) 2.8 1,403 (3.3) 3.5TPA + V 5,611 (0.93) 438 (3.2) 3.5 1,439 (3.3) 3.6

a Cells were grown for 4 days with or without 16 nM TPA and lysed in theabsence or presence of orthovanadate (V).

b Expressed as counts per minute per 0.5 mg of total protein and determinedby counting Western blot bands, using '25I-protein A as the detection system.Numbers in parentheses are values relative to that for the control-plus-orthovanadate treatment, which was arbitrarily set at 1.0.

c Expressed as counts per minute per 0.15 mg of total protein. Numbers inparentheses are values relative to that for the control-plus-orthovanadatetreatment, which was arbitrarily set at 1.0.

d Ratio of relative kinase activity to relative src protein level. The value forthe control-plus-vanadate treatment was arbitrarily set at 1.0. Relative specific

pp60 _ kinase activities in different experiments varied up to 25% from the valuesgiven.

FIG. 3. src kinase activity in SH-SY5Y cells treated with TPA invarious concentrations (A) and c-src and c-srcN expression incontrol (Ctrl) and TPA-treated cells (B). (A) Cells were exposed for4 days to 0.16 nM, 16 nM, or 1.6 ,uM TPA, and src kinase activitylevels were then compared with the level in untreated cells as

described in the legend to Fig. 1. The optimal TPA concentration forfunctional differentiation is 16 nM (50). (B) Cells grown for 4 or 8days with or without TPA were labeled for 4 h with [35S]methionine(0.15 mCi/1.5 ml and 10-cm-diameter dish). After solubilization andnormalization for protein content, the labeled src proteins were

immunoprecipitated, separated by SDS-PAGE, and visualized byfluorography. The doublet band consists of the c-srcN (upper band)and c-src (lower band) proteins.

Mab 327

A

I- CL

pp6 (.'s _m

.> I>1.

<

B <

i. l., -.. r.1-

is .i} &WilSf._ _, _-.

''s..

-e

ernoJase - __

FIG. 4. Comparison of the src kinase activity (A) and proteinlevel (B) in untreated SH-SY5Y cells (Ctrl) and cells treated withTPA for 4 days. Cells were lysed in the presence or absence of 100,uM sodium orthovanadate (V). (A) Kinase assay (see legend to Fig.1); control immunoprecipitation without MAb 327 was performed as

indicated. (B) Immunoblot analysis of src protein levels, using thesamples shown in panel A.

mined in a separate experiment and correlated to pp6Osrclevels. There was a 1.4- to 2-fold increase in specific kinaseactivity in TPA-treated cells compared with control cellslysed in the absence of orthovanadate (Table 1). In thepresence of orthovanadate, however, there was a 3.5-foldincrease in both specific autophosphorylation and enolasekinase activities. This analysis confirmed that the level ofpp6Osrc was not significantly altered in the differentiatedSH-SY5Y cells, and the enhancement in pp6Osrc kinaseactivity in these cells could possibly be explained by achange in the phosphorylation of pp60csrc or pp60c-srcN invivo.

In vivo phosphorylation pattern of pp6wc from control anddifferentiated SH-SY5Y cells. Phosphorylation of the C ter-minus of pp6Osrc (Tyr-527 in chicken) is a major mechanismfor the suppression of src kinase activity (14, 36, 53, 54). Weused V8 protease mapping to investigate whether there wasa significantly lower degree of phosphorylation of the pp6OsrcC terminus from differentiated compared with untreatedSH-SY5Y cells (Fig. 5A). However, the V2 fragment (i.e.,the C terminus) of pp6Osrc was more strongly phosphorylatedin differentiated than in control cells, as was the N terminusof the molecule (Vl, V3, and V4 fragments), although the netphosphorylation of pp6Osrc was higher in the differentiatedcells (Fig. 5C). Samples treated and electrophoresed inparallel but subsequently alkali treated to remove serinephosphate were exclusively phosphorylated on the V2 frag-ment, and no significant change occurred in the phosphory-lation ratio between control and differentiated cells (Fig.SB). In some primary gels and V8 protease maps (includingthe one shown in Fig. 5A), the pp60c srcN and the derived Vi,V3, and V4 fragments were difficult to detect without pro-longed exposure times. This was particularly the case whencontrol cells were analyzed, indicating that the neuronalform was underphosphorylated compared with pp6Oc-src(Fig. 5A and unpublished results).To further investigate the phosphorylation pattern of

pp6fOsrc we performed tryptic peptide mapping on the in vivo32P-labeled total pp6Osrc from TPA-differentiated and un-treated SH-SY5Y cells. The overall phosphopeptide patternof pp6Osrc from both TPA-treated and untreated SH-SY5Ycells (Fig. SD and E) closely resembled that obtained withdifferentiated, primary cultured rat neurons (14). Of themajor phosphopeptides found, peptides 1 and 2 containedphosphorylated Tyr-527 (19), peptides 5 and 6 contained

MOL. CELL. BIOL.

ACTIVATION OF pp6Osrc KINASE ACTIVITY 365

A - M<-

4. i

.H

B =1-- <H C H

-PP60

-VI

* * -V2 *-

pp60= a

-V2

-V3t - V4

D7

3

E

.73

6 *5

&f-05

6*

Ftyr 527

02.-

0 70 3

tyr416

I,' 4

serl7

O 5

FIG. 5. Analysis of the in vivo phosphorylation pattern of pp605sc from TPA-treated and untreated SH-SY5Y cells. (A and B) V8 proteasemapping of pp6Osrc. Control (Ctrl) and TPA-treated SH-SY5Y cells were grown for 4 days, radiolabeled with 1 mCi of 32p; for 4 h, lysed, andimmunoprecipitated in the presence of orthovanadate after normalization for protein concentration. Two parallel samples from control andTPA-exposed cells were separated by SDS-PAGE; pp6Wrc was excised and reelectrophoresed in the presence of 50 ng of V8 protease. Thegel was then divided in two halves; one was dried immediately and subjected to autoradiographic analysis (A), and the other was fixed andtreated with 1 M KOH (B). The V2 fragment (26 kDa) represents the C terminus of the src molecule; Vl (34 kDa), V3 (18 kDa), and V4 (16kDa) are N-terminal peptides. The alkali-treated gel was exposed three times longer than the untreated counterpart. (C to F) UntreatedSH-SY5Y cells (Ctrl) and cells treated with TPA for 2 days were labeled with 32p; (2.5 mCi/ml) for the last 14 h before lysis. (C) Total pp6AYrcwas immunoprecipitated and separated on an SDS-7% polyacrylamide gel. The whole-pp6&C bands were taken for tryptic digestion. (D andE) The phosphopeptides obtained were separated two-dimensionally, first by electrophoresis at pH 1.9 (from left to right, with the anode onthe left) and then by chromatography (from bottom to top). Ctrl (D) and TPA (E) tryptic maps were loaded with 300 and 320 Cerenkov countsper min, respectively. Exposure time for the tryptic maps was 6 days at -70'C with an intensifying screen. (F) Schematic map of knowntryptic pp60frc phosphopeptides.

Ser-17 (17, 35, 52) and Ser-12 (14, 31, 32), respectively, andpeptides 3 and 7 contained a phosphoserine originating fromthe N terminus (14). We could not detect any novel pp6srcphosphorylation site in the tryptic maps of TPA-differen-tiated compared with untreated cells (Fig. 5D and E). Themost pronounced difference between the two maps was therelative increase in Ser-12 phosphorylation in the TPA-differentiated cells. Furthermore, peptide 7 was slightlymore phosphorylated in the TPA-differentiated cells than inthe untreated control, which was more evident when thephosphorylation ratio between peptides 7 and 3 was com-pared in the two maps. Tyr-527 was phosphorylated to

similar extents in both differentiated and control cells,whereas the peptide containing Tyr-416 (12; Fig. SF) was notdetectable in either the differentiated or the untreated cells.These results together with the V8 protease mapping datasuggest that dephosphorylation of Tyr-527 is not involved inthe enhancement of pp6Osrc kinase activity during differen-tiation of SH-SY5Y cells.

Activation of pp6&Src appears early during the differentia-tion process and coincides in time with the onset of morpho-logical differentiation. The earliest indication that the TPA-treated SH-SY5Y cells had entered a program of furtherneuronal differentiation was an alteration in the cell mor-

VOL. 10, 1990

It1

* 2

6'60

serl2,,

366 BJELFMAN ET AL.

A B

D

0 1 2 4 6 48 h 1 6 48 hGAP 431.88kb4

-'1.4kb

phc GAP 3

-1.5 kb

FIG. 6. Early phenotypic changes associated with neuronal differentiation in TPA-treated SH-SY5Y cells. (A) Phase-contrast microscopyof untreated SH-SY5Y cells after 2 days in culture; (B) parallel cells treated for 4 h with TPA. Growth cone-like structures are indicated byarrows. Bars, 50 p.m. (C) GAP43 mRNA expression in control cells grown for 48 h (0) and parallel cells treated with TPA for the last 1 to 48h. A 5-±g sample of poly(A)+ RNA was used for Northern blot analysis. Human glyceraldehyde-3-phosphate dehydrogenase (pHcGAP3)mRNA served as a quantitative mRNA standard. (D) Three mRNA preparations analyzed as for panel C were reelectrophoresed todemonstrate more clearly the expression of a new, slowly migrating GAP43 transcript (1.8 kilobases [kb]), which appeared transiently duringthe early phase of differentiation.

phology. Within 3 to 4 h after addition of TPA, the first cellsbegan to round up, extend neurites, and display growthcone-like structures (Fig. 6A and B). During the same timeperiod, an increase in the expression of a growth cone- andaxon-associated protein, GAP43 (29), was noted, as deter-mined at the mRNA level by Northern'hybridization (Fig.6C). In addition to an increase in the level of the 1.5-kilobaseGAP43 transcript over that found in control cells, a moreslowly migrating (1.8-kilobase) GAP43 transcript appeared 4h after TPA induction (Fig. 6C and D). This new mRNAspecies was transiently expressed and could not be detectedin cells treated with TPA for 4 to 12 days, although theGAP43 expression in these cells remained considerablyhigher than in time-matched controls (Fig. 6C and 7B). Thus,the development of growth cone-like structures and theaccompanying GAP43 expression appeared during the sametime interval as did activation of the src kinase. Thesechanges are early events, since the major phenotypic

changes associated with the differentiated phenotype appearlater (50) and continue to develop for more than 2 weeksafter the cells have been induced, as exemplified by theincreased expression of NSE accompanying the differentia-tion (Fig. 7; 50).

Culture conditions and c-src kinase activity in untreatedSH-SY5Y cells. In relation to total protein, the src kinaseactivity increased with time in culture also in untreatedSH-SY5Y cells (Fig. 1). There was a nearly twofold differ-ence in kinase activity between, for example, untreated cellscultured for 4 and 8 days (Fig. 8A). Furthermore, immuno-

blot analysis of samples'run in parallel with those assayed forkinase activity in Fig. 8A revealed that the increase in src

activity with time was primarily due to an elevated proteinlevel and was essentially not the result of activation of theenzyme (Fig. 8A and B). The subcultivation treatment(EDTA and trypsin) per se did not affect the levels of pp6 src(data not shown). We conclude that for proper comparison

C

MOL. CELL. BIOL.

VOL. 10, 1990

AC 3 *-* Control A

o A--A TPA ,, -

F.- -0,aL 2 I. oE A//ecy 1 _- .

Xi X..AC --v

0-

-- --12T4 81d2Time, days

B

GAP 43

TPA CMi

4 8 12 4 8 12 d

1.4 kb -

phGAP 3

1.5kb -

FIG. 7. NSE and GAP43 expression in SH-SYSY cells during thelate phase of TPA-induced differentiation. (A) NSE concentration incontrol and TPA-treated SH-SY5Y cells, determined by an NSEradioimmunoassay and correlated to total protein concentration; (B)GAP43 mRNA expression in cells grown with or without TPA for 4,8, or 12 days as described for Fig. 6C.

of src kinase activities and protein levels in different cul-tures, time-matched controls must be included in the exper-iment. Since TPA-induced differentiation of SH-SY5Y cellsleads to inhibited proliferation, it was important to knowwhether the increase in src expression in untreated cells wasdue to the higher cell densities and thus some growthinhibition in these cultures. To investigate this question,cells were seeded at different densities and grown for 4 days;pp60 src was then immunoprecipitated from cell lysates,normalized for protein, and analyzed for src kinase activity.The level of src kinase activity was virtually unalteredirrespective of the cell density (Fig. 8C). Another possibleexplanation for the increase in src expression with time incontrol cells would be that these cells spontaneously differ-entiate. This explanation appeared to be unlikely, sinceexpression of neither of the two neuronal differentiationmarkers GAP43 and NSE was induced during long-termculture of untreated SH-SY5Y cells (Fig. 7).

DISCUSSION

In this study, we observed distinct activation of cellularpp60src kinase activity in maturing human neuroblastomaSH-SY5Y cells. Compared with most changes associatedwith the induced neuronal phenotype, the activation ofpp60 src is an early event following exposure to TPA. Theelevated kinase level was displayed both in autophosphory-

ACTIVATION OF pp605' KINASE ACTIVITY 367

4d 8d

A

pp6O -

4d 8d

B1 2 3 4 5

C

enolase

FIG. 8. src kinase activities (A and C) and protein levels (B) inSH-SY5Y cells grown for various times (A and B) and at differentdensities (C). (A) src kinase activity in cells seeded 106 per dish andgrown for 4 or 8 days. The cell lysates were normalized for totalprotein content before immunoprecipitation. One half of the immu-noprecipitate was used for kinase assay, and the other half (B) was

immunoblotted. (C) src kinase activity in cells grown for 4 days.Cells were seeded per dish as follows: 105 (lane 1); 3 x 105 (lane 2);106 (lane 3); 3 x 106 (lane 4); and 107 (lane 5).

lation assays and in tests using rabbit muscle enolase as asubstrate. Significantly, the optimal phorbol ester concentra-tion in these experiments was identical to that required forthe most pronounced differentiation, as manifested by NSEexpression, neurite formation, noradrenaline synthesis, andgrowth inhibition (49, 50). An increased specific pp6ffsrckinase activity during in vitro differentiation has also beenreported in rat embryo striatum neurons (14) and in munneembryo carcinoma cells, which was paralleled by a limitedelevation of the src protein levels (42). In accordance withour findings in SH-SY5Y cells, the ratio between neuronaland fibroblast-type pp6Osrc was essentially unaltered duringdifferentiation of the striatal neurons (14), whereas a shift tothe 61-kilodalton (kDa) pp60c-srcN accompanies maturationin embryo carcinoma cells (42).Developing neurons accumulate large amounts of a hydro-

philic protein designated GAP43 in their growth cones (34,56). We found that there was a substantial increase of theGAP43 mRNA level starting 4 h after TPA treatment, whichcoincided with the time of pp6 src activation, axon exten-sion, and growth cone formation. It has recently beenobserved that both forms of pp6Osrc are enriched in growingaxons, especially in growth cones, whereas synapses mainlycontain the neuronal form of pp60src (43). From theseindependent findings, it may be conjectured that pp6fyrcparticipates in the regulation of axonal- and growth coneformation. GAP43 protein expression is regulated at thetranscriptional level (5), and a corresponding increase in thetranslation product in differentiated SH-SY5Y cells wouldtherefore be expected. Interestingly, an additional, moreslowly migrating mRNA species appeared concomitantlywith the increase in GAP43 transcription. We do not knowwhether this larger mRNA molecule is translated into apolypeptide product.Large amounts of the cellular pp6Osr' have been observed

in neural tissues of various species: chicken retinal neurons(57, 62), chicken brain (22), rat brain (11), and human braintissue (39) and neuroblastoma cells (9, 46, 48, 66). We founda strict correlation between pp60src activity and neuronal or

- dm4w offoo

368 BJELFMAN ET AL.

neuroendocrine development (46). It is concluded that theinvariably high pp6Osrc kinase activity found in these celltypes favors the idea of a functional involvement of cellularsrc protein(s) in the development of neuronal or neuroendo-crine cells. Apparently discordant with this concept is thegradual elevation of src kinase activity (via raised proteinlevels) that occurred with time in the SH-SY5Y cells aftersubcultivation, entirely without manifestations of differenti-ation. These results indicate that although necessary, highsrc expression is not sufficient for neuronal maturation. Inaddition, the subcellular localization of pp6Osrc might beimportant for the regulation of enzymatic activity and cellu-lar function(s) of the src products. The enrichment of pp6Osrcin growth cone and synaptosomal preparations from ratembryonal and adult brains, respectively, favors this view(43).The biosynthesis of a pp60src variant (pp60CsrcN), whose

expression is detected primarily in neurons and neuroblasts,was recently found to depend on an alternative processing ofthe c-src transcript (10, 40, 44). The slowly migrating bandthat we have previously identified in highly differentiatedneuroblastoma cell lines with use of immunoprecipitationswith pp60src_specific antibodies (46) is most likely the humanequivalent of that found in murine and chicken brains. Cellsexpressing pp6OC-srcN have been reported to display higherspecific kinase activity than those in which only pp6Ocsrc isfound (11, 14, 42). Our findings indicate that both variants ofthe cellular pp60src are present in the SH-SY5Y cell linethroughout the in vitro maturation program, and the kinaseactivation was not accompanied by alteration of the equimo-lar pp6Osrc protein ratio (Fig. 3). Therefore, we cannotattribute the increased enzymatic activity to any particularpp6f4rc form, but it is possible that pp60C-srcN exclusivelycontributes to the altered activity.

In contrast to the elevated specific kinase activity ofpp60 c-srcN observed in mature central nervous system neu-rons that preferentially express this form (11, 13, 14, 26, 43,65), O'Shaughnessy et al. (48) and Yang and Walter (66)found no significant difference in specific activity betweenthe two pp60csrc forms when isolated from cultured humanneuroblastoma cells. One explanation for these apparentlycontradictory results might be that the specific pp6csrcN(and possibly also pp60csrc kinase) activity is lower inproliferating neuroblasts, e.g., neuroblastoma cells, than innondividing mature neurons. The data presented in thisreport would fit such a model, since activation of the srckinase activity in differentiating SH-SY5Y cells is accompa-nied by an inhibited growth rate.Orthovanadate is a potent phosphotyrosine-phosphatase

inhibitor (58), and Courtneidge (23) has shown that theabsence of orthovanadate during cell lysis results in in-creased pp60-csrc activity. The requirement of this compoundfor a clear discrimination between pp6Osrc kinase activity incontrol and TPA-treated SH-SY5Y cells indicates that reg-ulation at the phosphotyrosine level has a greater signifi-cance in control than in TPA-exposed cells. Tyr-527 is thepredominantly phosphorylated tyrosine in the c-src protein,and low pp6Oc-src enzymatic activity correlates with a highdegree of phosphorylation at this position (19, 21, 36).Analysis of V8 protease-digested 32P-radiolabeled pp6Osrc indifferentiated SH-SY5Y cells showed rather a net increase inphosphorylation in the amino terminus (Fig. 5). Trypticdigestion of 32P-labeled pp60 src from these cells does notindicate a participation of Tyr-527 in the phorbol ester-mediated activation of the src protein, as is true also for theTyr-416 position, which is in agreement with findings in

other cell systems containing an activated pp60-csrc (15, 31,40). However, we cannot rule out the possibility that afraction of pp6Osrc in control cells, not treated with ortho-vanadate, lacks Tyr-527 phosphorylation and thus contrib-utes to the higher specific activity than in control cells lysedin the presence of vanadate. Interestingly, a minor unphos-phorylated fraction of pp6c-src in AtT20 pituitary cells hasbeen demonstrated (30).The major change in phosphorylation of pp6Osrc upon TPA

induction was on Ser-12, which indicates that protein kinaseC in these cells still is active during the 48-h period oftreatment with 16 nM TPA. Phorbol ester-induced elevationof the pp6Osrc Ser-12 phosphorylation in nonneuronal cells iswell documented but generally without a parallel change inenzymatic activity (27, 31, 32, 59). In fibroblasts exposed toa variety of agonists known to stimulate phosphatidylinositolturnover, platelet-derived growth factor was the only com-pound that could activate the pp6Oc-src kinase (31). Thiseffect was not primarily a result of Ser-12 phosphorylation,since all tested agonists induced this particular modification,but appeared rather to be dependent on phosphorylation ofother N-terminal serine and tyrosine residues. Moreover, inthe human neuroblastoma cell line LA-N-5, expressingpp6fc-srcN as the predominant src product (46), the specificpp60src activity increased about 1.5-fold upon exposure toTPA, whereas the activity in SK-N-MC cells (no pp6Oc-srcN[46]) remained constant when the cells were treated in thesame way (data not shown). Thus, in these cells TPAinduced neither a pronounced activation of pp6Osrc normorphological or biochemical neuronal differentiation (un-published observations). pp60-srcN in differentiated rat neu-rons has been found to contain serine-phosphorylated trypticpeptides in addition to the peptide that harbors Ser-12 (14).In differentiated SH-SY5Y cells, a slightly increased phos-phorylation was observed in peptide 7 upon exposure toTPA (Fig. SE). This peptide has been demonstrated tocontain an amino-terminal serine phosphorylation in the ratneuronal src protein (14). In conclusion, the activation of thepp6src kinase that we observed in SH-SYSY cells might atleast partly be a result of an increased phosphorylation in theSer-12 position together with a corresponding change inadditional amino-terminal serine(s) or threonine(s).

Several reports describe disturbances in the normal inter-cellular membrane communication after aberrant src expres-sion. For example, high pp60vsrc levels reduce intercellularattachment and gap-junctional communication of culturedrat kidney cells (3), NIH 3T3 cells (16), chicken embryo lenscells (47), and Madin-Darby canine kidney cells (64). Asimilar abnormality of junctional cell communication hasbeen observed in NIH 3T3 cells in association with increasedexpression of cellular src (4, 64). Interestingly, pp60-src andpp60c-srcN are highly enriched in growth cones, in the motilestructures of growing axons, and in synapses in the form ofpp60 c-srcN (43, 63). Thus, further investigations of the in-volvement of pp6Osrc in growing or communicative mem-brane structures might elucidate the important function(s) ofthis proto-oncogene.

ACKNOWVLEDGMENTS

We thank Irja Johansson for expert technical assistance and TonyHunter for suggestions and constructive criticism.

This work was supported by the Swedish Cancer Society, theSwedish Natural Science Research Council, the Royal SwedishAcademy of Sciences (to G.M.), the Children Cancer Foundation ofSweden, HKH Kronprinsessan Lovisas forening for barnsjukvard,

MOL. CELL. BIOL.

ACTIVATION OF pp6Os'r KINASE ACTIVITY 369

Hans von Kantzows, Magnus Bergvalls, and Ollie och Elof Erics-sons stiftelser.

LITERATURE CITED1. Adem, A., M. E. K. Mattsson, A. Nordberg, and S. Pihlman.

1987. Muscarinic receptors in human SH-SY5Y neuroblastomacell line: regulation by phorbol ester and retinoic acid-induceddifferentiation. Dev. Brain Res. 33:235-242.

2. Akerman, K. E. O., I. G. Scott, and L. C. Andersson. 1984.Functional differentiation of a human ganglion cell-derivedneuroblastoma cell line SH-SY5Y induced by a phorbol ester(TPA). Neurochem. Int. 6:77-80.

3. Atkinson, M. M., A. S. Menko, R. G. Johnson, J. R. Sheppard,and J. D. Sheridan. 1981. Rapid and reversible reduction ofjunctional permeability in cells infected with temperature-sen-sitive mutant of avian sarcoma virus. J. Cell Biol. 91:573-578.

4. Azarnia, R., S. Reddy, T. E. Kmiecik, D. Shaioway, and W. R.Loewenstein. 1988. The cellular src gene product regulatesjunctional cell-to-cell communication. Science 239:398-401.

5. Basi, G. S., R. D. Jacobson, I. Virag, J. Schilling, and J. H. P.Skene. 1987. Primary structure and transcriptional regulation ofGAP-43, a protein associated with nerve growth. Cell 49:785-791.

6. Beemon, K., and T. Hunter. 1978. Characterization of Roussarcoma virus src gene products synthesized in vitro. J. Virol.28:551-566.

7. Biedler, J. L., L. Helson, and B. A. Spengler. 1973. Morphologyand growth, tumorigenicity and cytogenetics of human neuro-blastoma cells in continuous culture. Cancer Res. 33:2643-2652.

8. Biedler, J. L., S. Roffler-Tarlov, M. Schachner, and L. S.Freedman. 1978. Multiple neurotransmitter synthesis by humanneuroblastoma cell lines and clones. Cancer Res. 38:3751-3757.

9. Bolen, J. B., N. Rosen, and M. A. Israel. 1985. Increasedpp60csrc tyrosyl kinase activity in human neuroblastomas isassociated with amino-terminal tyrosine phosphorylation of thesrc gene product. Proc. Natl. Acad. Sci. USA 82:7275-7279.

10. Brugge, J., P. Cotton, A. Lustig, W. Yonemoto, L. Lipsich, P.Coussens, J. N. Barrett, D. Nonner, and R. W. Keane. 1987.Characterization of the altered form of the c-src gene product inneuronal cells. Genes Dev. 1:287-2%.

11. Brugge, J. S., P. C. Cotton, A. E. Queral, J. N. Barrett, D.Nonner, and R. W. Keane. 1985. Neurons express high levels ofa structurally modified, activated form of pp60csrc. Nature(London) 316:554-557.

12. Cartwright, C. A., W. Eckhart, B. Simon, and P. L. Kaplan.1987. Cell transformation by pp60fcsrc mutated in the carboxy-terminal regulatory domain. Cell 49:83-91.

13. Cartwright, C. A., R. Simantov, W. M. Cowan, T. Hunter, andW. Eckhart. 1988. pp60c-src expression in the developing ratbrain. Proc. Natl. Acad. Sci. USA 85:3348-3352.

14. Cartwright, C. A., R. Simantov, P. L. Kaplan, T. Hunter, andW. Eckhart. 1987. Alterations in pp60csr' accompany differen-tiation of neurons from rat embryo striatum. Mol. Cell. Biol.7:1830-1840.

15. Chackalaparampil, I., and D. Shalloway. 1988. Altered phos-phorylation and activation of pp60f-src during fibroblast mitosis.Cell 52:801-810.

16. Chang, C.-C., J. E. Trosko, H.-J. Kung, D. Bombick, and F.Matsumura. 1985. Potential role of the src gene product ininhibition of gap-junctional communication in NIH/3T3 cells.Proc. Natl. Acad. Sci. USA 82:5360-5364.

17. Collett, M. S., E. Erikson, A. F. Purchio, J. S. Brugge, and R. L.Erikson. 1979. A normal cell protein similar in structure andfunction to the avian sarcoma virus transforming gene product.Proc. Natl. Acad. Sci. USA 76:3159-3163.

18. Cooper, J. A., F. S. Esch, S. S. Taylor, and T. Hunter. 1984Phosphorylation sites in enolase and lactate dehydrogenaseutilized by tyrosine protein kinases in vivo and in vitro. J. Biol.Chem. 259:7835-7841.

19. Cooper, J. A., K. L. Gould, C. A. Cartwright, and T. Hunter.1986. Tyr-527 is phosphorylated in pp60c-src: implications forregulation. Science 231:1431-1434.

20. Cooper, J. A., and T. Hunter. 1981. Changes in protein phos-

phorylation in Rous sarcoma virus-transformed chicken embryocells. Mol. Cell. Biol. 1:165-178.

21. Cooper, J. A., and C. S. King. 1986. Dephosphorylation orantibody binding to the carboxy terminus stimulates pp6f-src.Mol. Cell. Biol. 6:4467-4477.

22. Cotton, P. S., and J. S. Brugge. 1983. Neural tissues expresshigh levels of the cellular src gene product pp60csrc. Mol. Cell.Biol. 3:1157-1162.

23. Courtneidge, S. A. 1985. Activation of the pp6fj-src kinase bymiddle T antigen binding or by dephosphorylation. EMBO J.4:1471-1477.

24. Cushing, H., and B. B. Wolbach. 1927. The transformation of amalignant paravertebral sympaticoblastoma into a benign gan-glioneuroma. Am. J. Pathol. 3:203-215.

25. Evans, A. E. 1980. Natural history of neuroblastoma, p. 3-12.Raven Press, New York.

26. Fults, D. W., A. C. Towle, M. Lauder, and P. Maness. 1985.pp60c-src in the developing cerebellum. Mol. Cell. Biol. 5:27-32.

27. Gee, C. E., J. Griffin, L. Sastre, L. J. Miller, T. A. Springer, H.Piwnica-Worms, and T. M. Roberts. 1986. Differentiation ofmyeloid cells is accompanied by increased levels of pp60c-srcprotein and kinase activity. Proc. Natl. Acad. Sci. USA 83:5131-5135.

28. Golden, A., S. P. Nemeth, and J. S. Brugge. 1986. Bloodplatelets express high levels of the pp60c"src-specific tyrosinekinase activity. Proc. Natl. Acad. Sci. USA 83:852-856.

29. Goslin, K., D. J. Schreyer, J. H. P. Skene, and G. Banker. 1988.Development of neuronal polarity: GAP-43 distinguishes axonalfrom dendritic growth cones. Nature (London) 336:672-674.

30. Gould, K. L., L. M. Bilezikjian, and T. Hunter. 1989. AtT20cells express modified forms of pp60c-src. Mol. Endocrinol.3:79-88.

31. Gould, K. L., and T. Hunter. 1988. Platelet-derived growthfactor induces multisite phosphorylation of pp60csrc and in-creases its protein-tyrosine kinase activity. Mol. Cell. Biol.8:3345-3356.

32. Gould, K. L., J. R. Woodgett, J. A. Cooper, J. E. Buss, D.Shalloway, and T. Hunter. 1985. Protein kinase C phosphory-lates pp60src at a novel site. Cell 42:849-857.

33. Hammerling, U., C. Bjelfman, and S. Pahlman. 1987. Differentregulation of N- and c-myc expression during phorbol ester-induced maturation of human SH-SYSY neuroblastoma cells.Oncogene 2:73-77.

34. Jacobson, R. D., I. Virag, and J. H. P. Skene. 1986. A proteinassociated with axon growth, GAP-43, is widely distributed anddevelopmentally regulated in rat CNS. J. Neurosci. 6:1843-1855.

35. Karess, R. E., and H. Hanafusa. 1981. Viral and cellular srcgenes contribute to the structure of recovered avian sarcomavirus transforming protein. Cell 24:155-164.

36. Kmiecik, T. E., and D. Shalioway. 1987. Activation and suppres-sion of pp60c-src transforming ability by mutation of its primarysites of tyrosine phosphorylation. Cell 49:65-73.

37. Laemmli, U. K. 1970. Cleavage of structural proteins during theassembly of the head of bacteriophage T4. Nature (London)227:680-685.

38. LeBeau, J. M., 0. D. Wiestler, and G. Walter. 1987. An alteredform of pp60c-sr is expressed primarily in the central nervoussystem. Mol. Cell. Biol. 7:4115-4117.

39. Levy, B. T., L. K. Sorge, A. Meymandi, and P. F. Maness. 1984.pp60c-src is in chick and human embryonic tissues. Dev. Biol.104:9-17.

40. Levy, J. B., T. Dorai, L.-H. Wang, and J. S. Brugge. 1987. Thestructurally distinct form of pp60csrc detected in neuronal cellsis encoded by a unique c-src mRNA. Mol. Cell. Biol. 7:4142-4145.

41. Lipsich, L. A., A. J. Lewis, and J. S. Brugge. 1983. lsolation ofmonoclonal antibodies that recognize the transforming proteinsof avian sarcoma viruses. J. Virol. 48:352-360.

42. Lynch, S. A., J. S. Brugge, and J. M. Levine. 1986. Induction ofaltered c-src product during neural differentiation of embryonalcarcinoma cells. Science 234:873-876.

43. Maness, P., M. Aubry, C. G. Shores, L. Frame, and K. H.

VOL. 10, 1990

370 BJELFMAN ET AL.

Pfenniger. 1988. c-src gene product in developing brain isenriched in nerve growth cone membranes. Proc. Natl. Acad.Sci. USA 85:5001-5005.

44. Martinez, R., B. Mathey-Prevot, A. Bernards, and D. Baltimore.1987. Neuronal ppWc-src contains a six-amino acid insertionrelative to its non-neuronal counterpart. Science 237:411-415.

45. Mattsson, M. E. K., E. Enberg, A.-I. Ruusala, K. Hall, and S.Pahlman. 1986. Mitogenic response of human SH-SY5Y neuro-blastoma cells to insulin-like growth factors I and II is depen-dent on the stage of differentiation. J. Cell Biol. 102:1949-1954.

46. Mellstrom, K., C. Bjelfman, U. Hammerling, and S. Pahhman.1987. Expression of c-src in cultured human neuroblastoma andsmall-cell lung carcinoma cell lines correlates with neurocrinedifferentiation. Mol. Cell. Biol. 7:4178-4184.

47. Menko, A. S., and D. Boettiger. 1988. Inhibition of chickenembryo lens differentiation and lens junction formation in cul-ture by pp6fi-src. Mol. Cell. Biol. 8:1414-1420.

48. O'Shaughnessy, J., V. Deseau, S. Amini, N. Rosen, and J. B.Bolen. 1987. Analysis of the src gene product structure, abun-dance, and protein kinase activity in human neuroblastoma andglioblastoma cells. Oncogene Res. 2:1-18.

49. PAhlman, S., L. Odelstad, E. Larsson, G. Grotte, and K. Nilsson.1981. Phenotypic changes of human neuroblastoma cells inculture induced by 12-O-tetradecanoylphorbol-13-acetate. Int.J. Cancer 28:583-589.

50. Pahlman, S., A.-I. Ruusala, L. Abrahamsson, L. Odelstad, andK. Nilsson. 1983. Kinetics and concentration effects of TPA-induced differentiation of cultured human neuroblastoma cells.Cell Differ. 12:165-170.

51. Parsons, S. J., and C. E. Creutz. 1986. pp60C-src activity detectedin the chromaffin granule membrane. Biochem. Biophys. Res.Commun. 134:736-742.

52. Patschinsky, T., T. Hunter, and B. M. Sefton. 1986. Phosphor-ylation of the transforming protein of Rous sarcoma virus: directdemonstration of phosphorylation of serine 17 and identificationof an additional site of tyrosine phosphorylation in p60v-src ofPrague Rous sarcoma virus. J. Virol. 59:73-81.

53. Piwnica-Worms, H., K. B. Saunders, T. M. Roberts, A. E.Smith, and S. H. Cheng. 1987. Tyrosine phosphorylation regu-lates the biochemical and biological properties of pp60-src. Cell47:75-82.

54. Reynolds, A. B., J. Vila, T. J. Lansing, W. M. Potts, M. J.

Weber, and J. T. Parsons. 1987. Activation of the oncogenicpotential of the avian cellular src protein by specific structuralalteration of the carboxy terminus. EMBO J. 6:2359-2364.

55. Scott, I. G., K. E. 0. Akerman, J. E. Heikkila, and L. C.Andersson. 1986. Development of neural phenotype in differen-tiating ganglion cell-derived human neuroblastoma cells. J. Cell.Physiol. 128:285-292.

56. Skene, J. H. P., and M. Willard. 1981. Changes in axonallytransported proteins during axon regeneration in toad retinalganglion cells. J. Cell Biol. 89:86-95.

57. Sorge, L. K., B. T. Levy, and P. F. Maness. 1984. pp6OC-src isdevelopmentally regulated in the neural retina. Cell 36:249-257.

58. Swarup, G., S. Cohen, and D. L. Garbers. 1982. Inhibition ofmembrane phosphotyrosyl-protein phosphatase activity by van-adate. Biochem. Biophys. Res. Commun. 107:1104-1109.

59. Tamura, T., R. R. Friis, and H. Bauer. 1984. pp60-src is asubstrate for phosphorylation when cells are stimulated to entercycle. FEBS Lett. 177:151-156.

60. Tanaka, A., C. P. Gibbs, R. R. Arthur, S. K. Anderson, H.-J.Kung, and D. J. Fujita. 1987. DNA sequence encoding theamino-terminal region of the human c-src protein: implicationsof sequence divergence among src-type kinase oncogenes. Mol.Cell. Biol. 7:1978-1983.

61. Towbin, H., T. Staehelin, and J. Gordon. 1979. Electrophoretictransfer of proteins from polyacrylamide gels to nitrocellulose:procedure and some applications. Proc. Natl. Acad. Sci. USA76:4350-4354.

62. Vardimon, L., L. E. Fox, and A. A. Moscona. 1986. Accumula-tion of c-src mRNA is developmentally regulated in embryonicneural retina. Mol. Cell. Biol. 6:4109-4111.

63. Waalas, S. I., A. Lustig, P. Greengard, and J. S. Brugge. 1988.Widespread distribution of the c-src gene product in nerve cellsand axon terminals in the adult rat brain. Mol. Brain Res.3:215-222.

64. Warren, S. L., and W. J. Nelson. 1987. Nonmitogenic morpho-regulatory action of pp6O-src on multicellular epithelial struc-tures. Mol. Cell. Biol. 7:1326-1337.

65. Wiestler, 0. D., and G. Walter. 1988. Developmental expressionof two forms of pp60c-src in mouse brain. Mol. Cell. Biol.8:502-504.

66. Yang, X., and G. Walter. 1988. Specific kinase activity andphosphorylation state of pp6fc-src from neuroblastomas andfibroblasts. Oncogene 3:237-244.

MOL. CELL. BIOL.