Proc. Natl. Acad. Sci. USAVol. 90, pp. 5176-5180, June 1993Cell Biology

Phosphorylation of Ser-42 and Ser-59 in the N-terminal region ofthe tyrosine kinase p56lck

(serine phosphorylation/phorbol ester/mitogen-activated protein kinase/protein kinase C)

DAVID G. WINKLER*t, INDAL PARK*, TAEUE KIM*, NICOLA S. PAYNE*t, CHRISTOPHER T. WALSHt,JACK L. STROMINGER*, AND JAEKYOON SHIN**Division of Tumor Virology, Dana-Farber Cancer Institute, and tDepartment of Biological Chemistry and Molecular Pharmacology, Harvard Medical School,44 Binney Street, Boston, MA 02115

Contributed by Jack L. Strominger, February 25, 1993

ABSTRACT Ser-42 and Ser-59 in the N-terminal regionhave been identified as the major phorbol ester-induced phos-phorylation sites of p56kk. Phosphorylation of Ser-59 results ina gel shift from 56 kDa to 61 kDa. Simultaneous phosphory-lation of Ser-42 and Ser-59 results in a further gel shift to 63kDa. In vitro kinase assays show that Ser-59 can be uniquelyphosphorylated by mitogen-activated protein kinase and thatSer-42 can be phosphorylated by either protein kinase A orprotein kinase C.

Like other members of the src family of cytoplasmic tyrosinekinases, p561ck has an N-terminal myristoylation site, tworegulatory domains termed the src homology regions (theSH3 and SH2 domains) (1), and a large C-terminal catalyticdomain with conserved regulatory tyrosine phosphorylationsites (2, 3). With the exception of the N-myristoylation signalat the extreme N terminus, the first 67 amino acids of p56Ickare unrelated in amino acid sequence to those in othermembers of the src family (2). A cysteine pair (cysteines 20and 23) in this region of p56lck, in concert with conservedcysteine pairs in the cytoplasmic domains of both CD4 andCD8, is essential for complex formation between p561ck andCD4 or p56lck and CD8 (4). While the association of p561ckwith CD4 through this unique N-terminal region is thought tostimulate the p56lck kinase activity (5), little else is knownabout the regulatory function(s) of this region.Treatment of T cells with phorbol esters induces dissocia-

tion of p561ck from CD4 and internalization and down-modulation of the CD4 complex, events which are accom-panied by phosphorylation of both C.D4 and p56lck (6-8). Theextensive phosphorylation of p56lck induced by phorbol estertreatment has been localized approximately to the N-terminalhalf of the protein, and it has been characterized by thesubstantial phosphorylation-induced retardation of the pro-tein mobility on SDS/PAGE (9, 10). N-terminal phosphory-lation and the accompanying gel retardation have also beenobserved upon treatment ofT cells with a number of differentagents including interleukin 2, CD4-T-cell receptor crosslink-ing agents, and calcium ionophore (10-14). The appearanceof these gel-shifted forms of p56lck in response to thesemitogenic and activating stimuli has led to speculation thatN-terminal phosphorylation could be involved in regulationof the kinase.

In this study the phorbol ester-inducible phosphorylationsites of p56lck in the N-terminal region have been identifiedand protein kinase C (PKC) [or protein kinase A (PKA)] andmitogen-activated protein kinase (MAP kinase) have beenimplicated in separate phosphorylation processes.

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.

MATERIALS AND METHODSSite-Directed Mutagenesis and Expression. The p56lck cDNA

was subcloned in the EcoRI site ofM13mpl8 and site-directedmutagenesis was performed on uracil-containing phage DNA(15) by using the M13 Muta-Gene kit (Bio-Rad). All mutantsequences were confirmed by single-strand sequencing. TheEcoRI fragments of the wild-type and mutant cDNAs werethen subcloned in the pCDNA-1 expression vector (Invitro-gen, San Diego). HeLa and CD4+ HeLa cells (16) weremaintained in Dulbecco's modified Eagle's medium supple-mented with 10%o fetal bovine serum. Cells were then trans-fected with 20 Ag ofcDNA per 10-cm plate, using the 2-[bis(2-hydroxyethyl)amino]ethanesulfonic acid (Bes)-buffered sa-line-based calcium phosphate precipitation method (16).

Immunoprecipitation and Western Blot Analysis. All assayswere performed between 48 and 72 hr after transfection. Cells(1 x 107) were lysed in 1 ml of lysis buffer (10 mM Tris HCl,pH 7.4/150 mM NaCl/1% Triton X-100, supplemented with1 mM phenylmethylsulfonyl fluoride, leupeptin at 1 Ag/ml, 1mM sodium orthophosphate, and 1 mM NaF). Insolublematerials were removed by centrifugation for 30 min at 4°Cin a Microfuge (16,000 x g). For immunoprecipitation,lysates were precleared by incubation with staphylococcalprotein A-Sepharose (Boehringer Mannheim) for 1 hr, andthe desired proteins were precipitated with antibodies andprotein A-Sepharose. Monoclonal antibody OKT4 and poly-clonal anti-p56lck antiserum (a generous gift of J. Travilian,University of Texas, or purchased from UBI, Lake Placid,NY) were used for precipitations of CD4 and p561ck, respec-tively. Samples were analyzed by electrophoresis onSDS/8% polyacrylamide gels or by Western blotting after theprotein bands had been transferred to a nitrocellulose mem-brane. Proteins on Western blots were visualized by 1251-labeled protein A (NEN).In Vivo 32P Labeling, Two-Dimensional Mapping, and Phos-

phoamino Acid Analysis. HeLa cells (1 x 107) expressingwild-type and mutant p56Ick were starved of phosphate inphosphate-free buffer (16) for 1 hr and further incubated foranother hour in 3 ml of prewarmed phosphate-free buffercontaining 5 mCi (1 mCi = 37 MBq) of [32P]orthophosphate.Cells were then treated with phorbol 12-myristate 13-acetate(PMA) at 50 ng/ml for 15 min and lysed, and proteins wereimmunoprecipitated by using anti-p56lck antiserum and pro-tein A-Sepharose. Phosphorylated p561ck was separated byelectrophoresis on SDS/8% polyacrylamide gels, transferredto nitrocellulose, and visualized by autoradiography. Radio-active bands were excised from the dried nitrocellulosemembranes and digested with CNBr as described (17), andthe products were analyzed by electrophoresis on SDS/18%

Abbreviations: MAP kinase, mitogen-activated protein kinase;PKA, protein kinase A; PKC, protein kinase C; PMA, phorbol12-myristate 13-acetate; GST, glutathione S-transferase.

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polyacrylamide gels. The CNBr digestion products weretransferred to Immobilon-P (Millipore), excised from themembrane, and digested with the Asp-N protease (Boehring-er Mannheim). The resulting peptides were analyzed bythin-layer electrophoresis at pH 1.9 in the first dimension andby chromatography in the second dimension (17). Phospho-peptide spots were analyzed by using a Phosphorlmager(Molecular Dynamics, Sunnyvale, CA) with 24-48 hr ofexposure. Phosphoamino acid analysis of CNBr-digestedphosphopeptides was done as described elsewhere (18, 19).

In Vitro Kinase Assays. cDNAs encoding the first 77 aminoacids of p561ck were subcloned in PGEX-3Xb, expressed inEscherichia coli, and purified as described (20). The PKCassays were performed with a fusion protein concentration of0.3 mg/ml in 20 mM Tris HCl, pH 7.5/5 mM Mg(OAc)2/100,uM CaCl2 containing 0.25,g of phosphatidylserine, 0.025 ,ugof 1,3-diolein (Sigma), 10,uCi of [y-32P]ATP, 1,uM ATP, andpurified PKC (UBI) in a total volume of 15 ul (6). For MAPkinase and PKA the glutathione S-transferase (GST) fusionproteins were incubated with purified MAP kinase (UBI) or

the catalytic subunit ofPKA (Sigma) in kinase buffer (50 mMHepes, pH 7.2/10 mM MgCl2/33 ,uM ATP/1 ,uCi of [y_32p]-ATP. After 15 min at 25°C the reactions were stopped by theaddition of SDS sample buffer and boiling, and the assays

were analyzed by SDS/PAGE and autoradiography.For immunodepletion of the Ser-59 kinase activity, lysates

from 2 x 109 Jurkat cells that were treated with PMA at 50ng/ml and 100 nM ionomycin for 10 min were partially purifiedon a Q-Sepharose column (21). A 260- to 350-mM NaClfraction from the Q-Sepharose column containing Ser-59 pep-

tide (DPLVTYEGSNPPA_PLQ) kinase activity was diluted1:20 into Hepes-buffered saline (50 mM Hepes, pH 7.2/150mM NaCl) and preincubated with 50 Al of heat-denaturedprotein A-agarose beads for 1 hr at 4°C. The supernatant was

incubated with 10 ,ul of either anti-MAP kinase antiserum or

preimmune serum, and the antigen-antibody complex was

removed by incubation with protein A-agarose beads for 2 hr.The resulting supernatants were assayed for Ser-59 kinaseactivity as described (19) and used for further immunodeple-tion. Supernatants from these serial immunodepletions were

analyzed for MAP kinase content by Western blotting.

RESULTSPMA-Induced N-Terminal Serine Phosphorylation Sites.

Human p56Ick has five serines (residues 6, 7, 42, 54, and 59)and two threonines (residues 35 and 50) within its N-terminalregion (Met-1 to Ile-67) (Fig. 1A). PMA-induced Ser/Thrphosphorylation sites of p56lck have been localized to theN-terminal CNBr fragment (Glu-15 to Met-261) (9), implicat-ing serines 42, 54, and 59 and threonines 35 and 50 as

candidates for PMA-induced phosphorylation sites. Thesepotential phosphorylation sites were changed to alanines bysite-directed mutagenesis, and the resulting mutants were

subcloned in the pcDNA-1 expression vector. These mutantswere then transfected into HeLa cells.PMA treatment of T cells (10-14) or p56lck cDNA-

transfected HeLa cells results in the appearance of two

slower-migrating forms of p56lck at 61 and 63 kDa (Fig. 1B).Serine-to-alanine mutants of p56lck were expressed in HeLacells and examined for their ability to produce this gel shiftupon PMA treatment. Only the alanine mutants at Ser-42 andSer-59 (S42A and S59A mutants, respectively) producedchanges in the gel shift patterns of p56lck after PMA treatment(Fig. 1B). The S42A mutant produced a shift to the 61-kDaform of p56lck but had no 63-kDa form. On the other hand, theS59A mutant migrated as 56 kDa, without a detectable shifteven after PMA treatment for 60 min. These data suggest thatphosphorylation of Ser-42 is responsible for the shift from 61to 63 kDa and that phosphorylation of Ser-59 is responsiblefor the shift from 56 to 61 kDa.

ASH3 SH2 Kinase

~~~~~~Y Y/ ~~~~~~~~~y y1 10 20 30( P0MGCGCS SHPEDDW MEN I DV CEN CH Y PI VPL

40 50 60IDGKGTLLIRNGSEVRIDPLVTYEGSNPPASPLQ1DNLVI

Bp561ck: WT T35A S42A T50A S54A S59A

i

PMA: -+ -+ -+ -+--+

63 _-,w61-56-

FIG. 1. Effects of serine/threonine mutations of p561ck on gelmobility. (A) Schematic diagram of p561ck and amino acid sequenceof the unique N-terminal region. The src homology regions and thekinase subdomain are represented by filled boxes. The deducedphosphorylation sites at serines 42 and 59 and the peptide productsof the Asp-N protease containing these sites are shown by the boxedsequences. (B) Effect of serine/threonine mutations on PMA-induced gel mobility change of p561ck from cDNA-transfected HeLacells. Immunoblotting analysis using anti-p561ck antiserum and 125I-labeled protein A shows the PMA-induced gel-shift patterns of wildtype (WT), Thr-35 to Ala mutant (T35A), Ser-42 to Ala (S42A),Thr-50 to Ala (T50A), Ser-54 to Ala (S54A), and Ser-59 to Ala (S59A).Numbers at the left are molecular mass in kDa. The differences inintensities of the bands in the various mutants are due to differencesin expression levels after transfection.

It is not clear, however, whether Ser-42 and Ser-59 are theonly phosphorylation sites and why the S59A mutant lackeda 2-kDa shift induced by Ser-42 phosphorylation. Two pos-sibilities are (i) sequential phosphorylation-Ser-42 is phos-phorylated only after Ser-59 phosphorylation-or (ii) con-certed gel shift-the gel shift seen from phosphorylation onSer-42 occurs only when Ser-59 is phosphorylated as well. Totest these possibilities and to confirm that Ser-42 and Ser-59are the PMA-inducible phosphorylation sites, the 56-, 61-,and 63-kDa bands were analyzed by phosphopeptide map-ping. HeLa cells transfected with cDNAs encoding wild typeand mutant p56lcks were labeled with [32P]orthophosphateand treated with PMA for 15 min (Fig. 2A). Wild-type p56lckproduced three phosphorylated bands (56, 61, and 63 kDa-bands 1, 2, and 3, respectively). The S42A mutant producedtwo phosphorylated bands (56 and 61 kDa-bands 4 and 5)and the S59A mutant produced only one 56-kDa phospho-protein band (band 6) (Fig. 2A). The 32P-labeled bands weretransferred to nitrocellulose, subjected to CNBr digestion,and analyzed by SDS/PAGE. The N-terminal CNBr frag-ments had relative migration patterns similar to those of theundigested proteins. Bands 1 and 6 (the 56-kDa bands ofwildtype and S59A) produced a 32-kDa phosphopeptide band;bands 2 and 5 (the 61-kDa bands of wild type and S42A)produced a 35-kDa phosphopeptide band; and band 3 (the63-kDa band of wild type) produced a 36-kDa fragment (Fig.2B). Phosphoserine was the only detectable phosphoaminoacid in these 32- to 36-kDa phosphopeptides (Fig. 2C, lanes1-6). In addition to the 32- to 36-kDa fragments, all sixphosphoprotein bands in Fig. 2A yielded a 4-kDa phospho-peptide band upon CNBr digestion. This 4-kDa fragment wasfound to contain only phosphotyrosine residues upon phos-

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Ap561ck: WT S42A S59A

_-- 63.- 61:-w-56

B1 2 3 4 5 6

- 68-- 43

*- 32

The relationships between serine phosphorylation and theobserved gel shifts were further analyzed by subsequenttwo-dimensional phosphopeptide mapping. The N-terminalCNBr fragments were isolated and digested with Asp-Nprotease. The resulting peptides were separated on a cellu-lose plate by electrophoresis and thin-layer chromatography.Either one or both of two major phosphopeptide spots (PPS-1and PPS-2, Fig. 3A) were apparent in the two-dimensionalmaps of all the isolated bands. The 36-kDa CNBr fragmentfrom band 3 (wild type, 63 kDa) generated both PPS-1 andPPS-2. The 35-kDa bands from band 2 (wild type, 61 kDa) andband 5 (S42A mutant, 61 kDa) produced PPS-1, while the32-kDa bands from bands 1 and 6 (wild type and S59A

A4

_- 22

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_ S-Tyr

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_; t ... : K ... s ,*.... .. t ,..:: ..

6

1 2 3 4 5 6 7

FIG. 2. In vivo 32P-phosphorylation, CNBr digestion, and phos-phoamino acid analysis of wild-type, S42A mutant, and S59A mutantp5o6ck in HeLa cells. (A) HeLa cells expressing wild-type and twodifferent serine mutants of p561ck were loaded with [32P]orthophos-phate and phosphorylation of p561ck was induced by treatment withPMA at 50 ng/ml for 15 min. Immunoprecipitates of p561ck wereseparated by electrophoresis on SDS/8% polyacrylamide gels, trans-ferred to nitrocellulose, and autoradiographed. Molecular masses ofthree distinctly separated bands are marked in kDa. Individual bandsare identified by number: bands 1, 2, and 3 are, respectively, the 56-,61-, and 63-kDa species of wild-type pS61ck; bands 4 and 5 are,respectively, the 56- and 61-kDa species of the S42A mutant p561ck;and band 6 is the 56-kDa form of the S59A mutant p561ck. (B)32P-labeled bands were excised, digested with CNBr, separated byelectrophoresis on SDS/18% polyacrylamide gels, transferred toImmobilon-P, and autoradiographed. Lane numbers correspond tothe band numbers ofA. (C) Phosphoamino acid analysis ofthe CNBrdigestion products. Lanes 1-6 correspond to the 32-kDa regionfragments from lanes 1-6 in B. Lane 7 is the acid-hydrolyzed 4-kDaband from lane 1 in A.

phoamino acid analysis (Fig. 2C, lane 7), and it has beenidentified as the CNBr peptide containing the Tyr-505 phos-phorylation site (Arg473 to Pro-509) (9). These results suggestthat the wild type and the serine mutants ofp561ck are similarlyphosphorylated at its negative regulatory tyrosine phosphor-ylation site and that the gel shifts observed after PMA treat-ment are due to N-terminal serine phosphorylations.

..... .. .

O'

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-- elecirophoresis

FiG. 3. Two-dimensional proteolytic mapping ofthe wild-type andmutant p561ck N-terminal regions. (A) The 32-kDa regions from CNBrdigestion of the wild-type and mutant p56ICk (Fig. 2B) were excisedfrom Immobilon, digested with the Asp-N protease, and separated bysequential electrophoresis and chromatography on a thin-layer cellu-lose plate. Panel numbers 1-6 correspond to lane numbers of Fig. 2B.The origins in panels 1, 3, and 6 are marked with 35S. Imaging wasfacilitated by the use of a Molecular Dynamics PhosphorImager with24 hr of exposure. (B) Diagrammatic representation ofthe Asp-N mapof the 32-kDa CNBr products of p56Ck showing the phosphopeptidescontaining Ser-42 (PPS-2) and Ser-59 (PPS-1).

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mutant, respectively) generated PPS-2. The weak PPS-2signal in the band 2 digest is most likely contamination fromband 3 during excision of the bands from the nitrocellulosemembrane. Synthetic peptides containing the Ser-59 andSer-42 phosphorylation sites (Fig. 1A) migrated identically toPPS-1 and PPS-2, respectively, upon in vitro phosphorylation(data not shown). These data confirm that Ser-42 and Ser-59are two major PMA-induced phosphorylation sites, and thatPPS-1 and PPS-2 represent peptides containing phospho-serine-59 and phosphoserine-42, respectively (Fig. 3B).

Since Ser-42 and Ser-59 are the only PMA-inducible phos-phorylation sites, Ser-59 would be the only intact phosphor-ylation site in the S42A mutant p56lck. Upon CNBr digestion,band 4 (the 56-kDa band of the S42A mutant) produced the4-kDa phosphopeptide but not the 32-kDa phosphopeptideband, while band 5 (61 kDa, S42A mutant) generated both the35- and 4-kDa phosphopeptides. The 56- and 61-kDa forms ofthe S42A mutant of p56lck, therefore, probably represent lckproteins without and with Ser-59 phosphorylation, respec-tively. These data also suggest that phosphorylation at Ser-59causes the 5-kDa gel shift of p56lck from 56 to 61 kDa onSDS/PAGE.Although the S59A mutant migrated as only a single 56-kDa

phosphoprotein band (band 6), digestion of this band withCNBr produced both the 4- and the 32-kDa phosphopeptides.Since this mutant p56Ick possesses an intact Ser-42 phosphor-ylation site, the 56-kDa phosphoprotein band (band 6) mayrepresent a mixture of mutant p56lck proteins that either haveor have not been phosphorylated on Ser-42. This result alsosuggests that Ser-42 phosphorylation alone may not induce agel shift. The phosphorylation of Ser-42 occurs in the S59Amutant, and phosphorylation of Ser-59 occurs in the S42Amutant, indicating that independent, rather than sequential,phosphorylation ofthese serine residues occurs. The absenceof a gel shift by Ser-42 phosphorylation in the S59A mutant,and the 63-kDa band seen upon phosphorylation of bothSer-42 and Ser-59 in wild-type p56Ick (band 3, Fig. 2A),suggests that phosphorylation at Ser-42 can induce a gel shiftonly if Ser-59 is already phosphorylated.

Phosphorylation of Ser-42 and Ser-59 by Different ProteinKinases. The sequences surrounding the Ser-42 and Ser-59phosphorylation sites match the substrate consensus se-quences of different protein kinases. Three residues N-ter-minal to Ser-42 is an arginine (Arg-Asn-Gly-Ser; Fig. 1A), anarrangement that is seen in many PKA or PKC phosphory-lation sites (22). On the other hand, the sequence around theSer-59 phosphorylation site (Pro-Pro-Ala-Ser-Pro; Fig. 1A)resembles the substrate site of a proline-directed kinase suchas those found in the cdk or MAP kinase families.Fusion proteins containing the 77 N-terminal amino acids

of p561ck fused to GST were expressed in E. coli and affinitypurified on glutathione-agarose. The GST-77(WT), GST-77(SA42), and GST-77(SA59) fusion proteins were used inkinase reactions in vitro with purified protein kinases. TheGST-77(WT) and the GST-77(SA42), but not the GST-77(SA59), fusion proteins served as good substrates forpurified MAP kinase (Fig. 4A). These results indicate thatp561ck can be phosphorylated by MAP kinase and that the siteof this event is Ser-59. Furthermore, a Ser-59 peptide kinaseactivity (using as a substrate a synthetic peptide identical tothe predicted Asp-N protease peptide product containing theSer-59 phosphorylation site; Fig. 1A) partially purified fromPMA- and ionomycin-activated Jurkat cells can be removedby immunodepletion using MAP kinase antibodies (Fig. 4B).This result indicates that active MAP kinase from an appro-priate in vivo source can phosphorylate this sequence.

Since PMA is a direct activator of PKC the ability of thiskinase to phosphorylate Ser-42 in vitro was examined. TheGST-77(WT) and the GST-77(SA59), but not the GST-77(SA42), fusion proteins proved to be good substrates for

A r GST-77 1

MAPK

PKC * *

PKA S

B100 In

X 80- a)o ° 60-0 '

40EO.E 40-

-L 20-

--0::- p44- p42

number of immunodepletions

FIG. 4. In vitro phosphorylation of Ser-42 and Ser-59 by purifiedprotein kinases and the immunodepletion of Ser-59 kinase with MAPkinase antibodies. (A) GST fusion proteins containing the N-terminal77 amino acids of wild type and the serine-to-alanine mutations ofSer-42 and,Ser-59 of p561ck [GST-77(WT), GST-77(SA42), and GST-77(SA59), respectively] were used as substrates for purified MAPkinase, PKC, and PKA. The faint phosphorylation ofGST-77(SA42)by PKC is due to a low level ofGST phosphorylation by PKC (datanot shown). (B) PMA- and ionomycin-activated Jurkat cells werefractionated on a Q-Sepharose column. A 260-350 mM NaCl peakcontaining Ser-59 peptide kinase activity was serially immunode-pleted with MAP kinase antibodies or with preimmune serum. TheSer-59 peptide kinase activity in the supernatants after each succes-sive immunodepletion was measured by HPLC. The same superna-tant fractions from the serial immunodepletions were analyzed forMAP kinase content by Western blotting.

purified PKC (Fig. 4A). This preference for Ser42 as aphosphorylation site for purified PKC, and the reciprocalpreference ofMAP kinase for Ser-59, was also seen in kinaseassays in vitro using synthetic peptides containing the Ser-42and Ser-59 phosphorylation sites (Fig. 5). As PKC and MAPkinase are appropriately active during PMA-induced phos-phorylation of Ser42 and Ser-59, respectively, they are thepresumptive candidates for p561ck kinases. However, PMAtreatment affects the activities of many signal transducingproteins. For this reason, use of PKC and MAP kinase in invitro kinase assays for p561ck is not necessarily sufficient toidentify the enzymes responsible for in vivo phosphorylationof Ser42 and Ser-59. For example, PKA phosphorylates theGST-77(WT) and GST-77(SA59) proteins, indicating that PKAas well as PKC could be the Ser-42 kinase (Fig. 4A). Thedetermination of the kinases actually responsible for thesephosphorylation events in vivo awaits further analysis.

DISCUSSIONThe involvement of different kinases, most likely PKC andMAP kinase, in the phosphorylation of p56ck at Ser,42 and

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Ser-42 Ser-59Peptide (1 mM)

FIG. 5. Phosphorylations of synthetic peptides containing Ser-42and Ser-59. Synthetic peptides designed to match the predicteddigestion products of the Asp-N protease (Fig. 1A) were phosphor-ylated in vitro by purified PKC or partially purified MAP kinase (peak1 kinase, see Materials and Methods). 32p incorporation into thesepeptides was assayed by HPLC and quantified by the use ofan in-lineradioactivity detector and an integrator.

Ser-59, respectively, correlates with data on in vivo phos-phorylation of the p56lck N terminus. PKC may be activatedby a Ca2+-dependent pathway seen upon treatment with Ca2+ionophore A23187 (10), a treatment that induces N-terminalserine phosphorylation in the absence of a gel shift. MAPkinase may be activated by signaling from the T-cell receptorand the interleukin-2 receptor and result in the 61-kDa p561ckisoform. The 63-kDa p56lck gel shift is observed in T cellsupon phorbol ester treatment but not after the other mitoge-nic stimuli described above (10-14), suggesting that, in thephysiological T-cell environment, two different subpopula-tions of p56fck, restricted to phosphorylation on either Ser-42or Ser-59, but not both, may occur. Further analysis of serinephosphorylation of p561ck in T cells induced by differentialtreatment of T cells will help clarify the signaling pathwaysinvolving ps6lck.

Serine/threonine phosphorylation of the N-terminal regionof the p60c-src tyrosine kinase has been observed (23-28).Ser-12 and Ser-48 of p60c-src are phosphorylated by PKC, andSer-72 of p6Oc-src, by cdc2 in a mitosis-specific and activation-correlated manner (23-28). Both Ser-72 of p6Oc-src and Ser-59of p56Ick are localized at the border of the N-terminal uniqueregion and the SH3 domain and are phosphorylated by serinekinases directed to a proline motif (cdc2 and MAP kinase,respectively). These similarities in N-terminal phosphoryla-tion suggest that regulation by phosphorylation in the uniqueN-terminal regions ofthese kinases may be homologous eventhough the sequences within these regions are not conserved.

We thank Dr. J. Travillian for the anti-p561ck antisera and Dr. J.

Blenis for the MAP kinase antibody. These studies were supportedby National Institutes of Health Research Grants CA47554 (J.L.S.),A120182 (J.L.S.), and GM48961 (J.S.), and by Hoffmann-La Roche(C.T.W.).

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