Feb 4, 1994 - The protein tyrosine kinase expressed by the proto- oncogene c-fgr is phosphorylated and down-regulated in vitro by the c-Src kinase. (CSK).
Vol. 269, No. 22, Issue of June 3, pp. 15885-15891, 1994 Printed i n U.S.A.
JOURNAL OF Brolacrc~~ CHEMlsmY 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc. THE
Regulation of c-Fgr Protein Kinase byc-Src Kinase (CSK) and by Polycationic Effectors* (Received forpublication, February 4, 1994)
Maria Ruzzene, Peter James$,Anna Maria Brunati, Arianna Donella-Deana, and Lorenzo A. Pinnal From the Dipartimento di Chimica Biologica, Uniuersita di Padoua, and Centro per lo Studio della Fisiologia Mitocondriale del Consiglio Nazionale delle Ricerche, 35121 Padoua, Italy and the wrotein Chemistry Laboratory, ETH Zentrum, Zurich, Switzerland
from the catalytic domain. This includes an highly conserved The protein tyrosine kinase expressed by the protoin autophosphorylation site(Tyr-416 in c-Src), whose phosphoryloncogene c-fgr is phosphorylated and down-regulated vitro by the c-Src kinase (CSK).CSK catalyzed phospho- ation is generally believed to correlate with up-regulation of rylation affectsTyr-511 of c-Fgr, homologous to Tyr-527 catalytic activity (Cooper and Howell, 1993). In the cellular of c-Src and it prevents the autophosphorylation nor- members ofthe Src family, but not in the viral ones, the catalytic mally occurring at c-Fgr Tyr-400, homologous to c-Src domain is followed by a short regulatory C-terminal tail includTyr-416.Polylysine, histonesH1 and H2A and other poly- ing a tyrosyl residue (T)n”527in c-Src), whose phosphorylation cationic proteins on the other hand stimulate c-Fgr acin c-Src and Lck was unambiguously shown to inhibit the catativity while promoting enhanced autophosphorylation lytic activity of these kinases (Marth et al., 1988; Cooper and of both Tyr-400 and Tyr-511. Once phosphorylated Tyr- at King, 1986; Courtneidge, 1985; Kmiecik and Shalloway, 1987; 511 and down-regulated by CSK,c-Fgr is no more sus- Piwnica-Worms et al., 1987; Cartwright etal., 1987).A protein ceptible to polylysine stimulation. Previous autophos1988) phorylation (at Tyr-400) reduces c-Fgr susceptibility to tyrosine kinasehas been isolated (Okada and Nakagawa, down-regulation by CSK,although Tyr-511 can be still and cloned (Nada etal., 1991)which is capable to phosphorylate phosphorylated byit. If a more exhaustive autophospho- this Tyr-527 residue and down-regulate c-Src (Okada and Narylation (of both Tyr-400 and Tyr-511) is performed in kagawa, 1989).The mechanismby which such a negative regulation occurs involves a n interaction between the phosphorylthe presence of polylysine, c-Fgr becomes totally insenand theSH2 domain of the kinaseitself (Cantley ated ”527 sitive to CSK down-regulation. These data support the concept that down-regulation et al., 1991;Superti-Furgaet al.,1993;Cooper and Howell, 1993) of c-Fgr by Tyr-511 phosphorylation is prevented if Tyr- giving rise to an inactive conformation of the enzyme. Besides c-Src, other membersof the Srcfamily, namely Fyn,Lyn (Okada 400 is also phosphorylated and they are consistent with an outcompetition of phospho-Tyr-511 from the Src ho- et al., 1991) and Lck (Bergmann etal., 1992) have been shown mology 2 domain by phospho-Tyr-400, which, in c-Fgr, is to undergophosphorylation and down-regulation by CSK. Consurrounded by an amino acid sequence divergent from versely nothing was known about the regulation of c-Fgr, a memthat of the other Src-related protein tyrosine kinases. ber of the Srcfamily expressed in normal and malignant B lymphocytes infected with Epstein-Barrvirus (Cheah etal., 1986), as well as in normal peripheralblood monocytes and granuloSrc-related protein tyrosine kinases make up a family of cytes and inalveolar and splenic macrophages(Ley et al., 1989). nonreceptorial enzymes termed after the product of the proton- The biochemical properties of c-Fgr, whose autophosphorylation cogene c-src, the cellular counterpart of v-SI%, whose product, site sharesan only limited homology with thatof the othermemv-Src, was the first protein tyrosine kinase be to detected (Col- bers of the family (Katamine et al., 19881, have hardly been let and Erikson, 1978; Hunter and Sefton, 1980). Nowadays, investigated. We have recently shown that a protein tyrosine nine membersof the srcfamily are known, namely c-Src, c-Yes, kinase previously isolated from rat spleen and conventionally Lyn, Fyn, Lck, Hck, c-Fgr, Blk, and Yrk (Cooper, 1990; Cooper termed tyrosine protein kinase-I11 (Brunati and Pinna, 1988), once submitted to MonoQ fast protein liquid chromatography and Howell, 1993). All of these displaya remarkable homology and sharea com- gives rise to a predominant peak of activity (tyrosine protein mon structure, consistingof a N-terminal, poorly conserved seg- kinase-I11 61) due to an enzyme that wasidentified as c-Fgr, ment, followed by an SH3l anda n SH2 domains, both upstream based on both immunochemical and partial sequencing evi1993).Here we report a biochemical study dences (Brunati et al., on rat spleen tyrosine protein Ql, henceforth termed * This work was supported by Associazione Italiana per la Ricerca SUI c-Fgr, and in particularon itskinase-I11 regulation by CSK and by polyCancro, Italian Minister0 dell’ Universitae della Ricerca Scientifica e shown to stimulate the activity Tecnologica and Consiglio Nazionale delle Ricerche (target Projects on cationic proteins that have been Biotechnology and Bioinstrumentation andApplicazioni Cliniche della of other protein tyrosine kinases (Gatica et al., 1987; FujitaRicerca Oncologica). The costs of publication of this article were de- Yamaguchi et al., 1989;Abdel-Ghany et al., 1990;Donella-Deana frayed in part by the payment of page charges. This article must thereet al., 1992). fore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. EXPERIMENTAL PROCEDURES § T o whom correspondence should be addressed:Dipartimento di Chimica Biologica, Universita di Padova e Centro per lo Studio della Polylysine (average molecular mass of 46 kDa) was purchased from Fisiologia Mitocondriale del CNR, Via Trieste 75, 35121 Padova, Italy. Sigma, [y-32PlATPfrom Amersham Corp. The peptide EDNEYTA was Tel.: 39-49-8286410; Fax: 39-49-8073310. The abbreviationsused are: SH3, Src homology 3; SH2, Src homology 2; CSK,c-Src kinase; PAGE, polyacrylamide gel electrophoresis; nylbenzoyl 5’-adenosine;MS/MS,two mass scanning deviceslinked HPLC, high performace liquid chromatography; FSBA, p-fluorosulfotogether for fragmentation studiesto determine peptide sequences.
15885
Regulation of Tyrosine Kinase Protein c-Fgr
15886
synthesized by a manual synthesizer (model Biolinx 4175, LKB), as CSK - + - + - + - + + detailed by Marin et al. (1991); the peptides KDDEYNPA and + + Polylysine + + EPQYQPA were synthesizedby an automated synthesizerfrom Applied FSBA-c-Fgr c-Fgr Biosystem (model 431-A) (Brunati et al., 1992). I c-Fgr tyrosine protein kinase was purified from the particulate fraction of rat spleen, according to the procedureelsewherereported (Brunati et al., 1993).Its Tyr(P) content was negligible a s judged by lack koa of reactivity with anti-TydP) antibodies (monoclonal Py20, from ICN Biochemical). CSK was partially purified from rat spleen cytosol, a s described by Brunati et al. (1992). - 22 - 22 c-Fgr inactivation by FSBA was performedby 1h incubationa t 30 "C with 1mM FSBA in thepresence of 5 mM MgCI,; the residualFSBA was 14 - 14 quenched by addition of 5 mM 2-mercaptoethanol (Okada and Naka12 - 12 10 u gawa, 1989). c-Fgr autophosphorylation or phosphorylation by CSK were per(I,formed a t 30 "C, in 50 pl of a medium containing 50 mM Tris-HCI, pH 5 20-* -3 7.5,5 mM MgCI,, 5 mM MnCl,, 10 PM [y-'*PIATP (specific activity 2000-3 5000 cpm/pmol), 0.02% (vh) Triton X-100, 1 mM 2-mercaptoethanol, 6 7 a 9 1 2 3 4 5 50-200 ng c-Fgr, in thepresence or in theabsence of CSK (100-500 ng). For the evaluation of szP incorporated, the reactions were stopped by addition of 2% SDS, and the samples were subjected11% to SDS-PAGE, followed by autoradiography. 0 15 0 Preincubation time (min) ForCNBrdigestion, the phosphorylationreactionswerestopped 5 5 20 Incubation lime(min) with formic acid (70% final concentration) and the cleavage was performed essentially according to Luo et al. (1991). Briefly, CNBr was + + CSK added to a final concentrationof 50 mg/ml, the reaction was allowed to continue for 1 h a t room temperature in the dark, and stopped by dilution to 1:lO and lyophilization. The samples, resuspended in gel electrophoresis buffer containing2% SDS, were subjected to20% SDSkDa PAGE (Laemmli, 1970), followed by autoradiography. Phosphorylation of the peptides was performed in the same medium described for autophosphorylation reaction, inthe presence of the sub22 strate peptide at the concentration indicated in the legend of the fig" 20ures. The reaction was stoppedby adding acetic acid (final concentra14 12 tion 30% vh), and ',P incorporated into the peptide was evaluated by -@ 10combining ion exchange and isobutanoVtoluene extraction, as detailed previously (Cola et al., 1989). 5 e dY K., and V,,, values were determinedby double-reciprocal plots, constructed from initial-rate measurements fitted to the Michaelis-Menten -3 equation. The values reported are the means of at least three separated 10 11 12 experiments; the S.E. value was less than15%. FIG.1.CNBr S2P-peptidemaps of c-Fgrphosphorylated under Peptide microsequencing was performed a s follows: autophosphorylated c-Fgr was reduced in 20 plof a buffer containing 2mM dithio- different conditions. A, c-Fgr (150 ng) either native (lanes 1 4 ) or threitol, 8 M urea, 0.1 M Tris, pH 8.5, for 15 min at 37"C. Iodoacetic acid previously inactivated by FSBA (lanes 6-9) was incubated a t 30 "C for 20 min in the autophosphorylation medium (described under"Experiwas added to a final concentrationof 3 mM and the pH adjusted to 8.5 mental Procedures") either alone(lanes 1 and 6 ) or with the following with Tris-base, and incubated for 2 h at 37 "C before quenching the additions: CSK, 300 ng (lanes2 and 7); polylysine, 10 pg/ml (lanes 3 and reaction with dithiothreitol to a final concentration of 5 mM. The solu- 8);CSK plus polylysine (lane 9). In the experiment of lane 4, polylysine tion was diluted5-fold and trypsin added (1:100, w/w) and thedigestion (10pg/ml) was added after 20 min of incubation with CSK, and the carried out for 12 h a t 37 "C. The digestion was terminated by acidifi- incubation was continued for 10 minmore. In theexperiment of lane 5, cation to pH 2.0. Peptides were separatedon a Brownlee (Applied Bio- CSK alone was incubated a t 30 "C for 20 min in the same autophossystems, Foster City, CA) C-18, 300 A, 1 x 100-mm reversed phase phorylation medium as for c-Fgr. B, c-Fgr was incubated as inPanel A column. Fractions werecollected manually. Fractions containing radio- either alonefor 20 min (lane 12). or with CSK, for 5 min (lane 1 0 ) . In activity were analyzed by Edman degradation on an A B 1 475 protein the experiment of lane 11 CSK was addedafter 15 min of preincubation, sequenator with on linephenylthiohydantoindetectionandalso by and the incubation was continued for 5 min more. At the end of incuelectrospray ionization mass spectrometer. Chymotryptic subdigestion bation, each samplewas digested withCNBr and the radiolabeledfragwas carried out (1:25, w/w) and the peptides were separated as de- ments were resolved by 20% SDS-PAGE and evaluated by autoradiography. The positions of molecular mass markers (in kDa) are indicated scribed above. Sucrose density gradient analysis was performed with a linear gra- on the right. dient (5 ml) of sucrose 5-20%, w/w in 50 mM Hepes, pH 7.0, containing rylated in thepresence of polylysine; in thiscase, however, the 10 mM 2-mercaptoethanol, 1mM EDTA and 0.1 mM phenylmethylsulfonyl fluoride. Unphosphorylated orpreviously autophosphorylated c-Fgr other two bands, far from disappearing, become more intense (150 ng) was loaded on top of the gradient and centrifuged a t 48,000 (lane 3). Increased radiolabeling of the 20- and 10-kDa bands rpm in a Kontron TST60 rotor for 15 h a t 4 "C. 0.2-ml fractions were and appearance of the 5-kDa band were also observed by recollected and monitored for enzymatic activity. placing polylysine with histones H1 and H2A and protamine,
-
-
@
-
e
"
9.
-
-
but not with spermine (not shown). The stimulatory effect of polylysine on c-Fgr autophosphorylation almost disappears if Phosphorylation of c-Fgr under Different Conditions-The c-Fgr is previously incubated with CSK, in this case only the radiolabeled CNBr fragments of c-Fgr phosphorylated by 5-kDa band is evident, while the 20- and 10-kDa bands are as [y-32PlATPunder various conditions wereisolated by SDS- faint as when CSK alone is added (lane 4 ) . Previous inactivaPAGE and detected by autoradiography (Fig. lA).Autophos- tion of c-Fgr with FSBA does not prevent itsphosphorylation by phorylation under basal conditions gives rise to two radiola- CSK at the site(s)included in the5-kDa band (lane7),while, as beled bands of apparent 20,000 and 10,000 M, (lane I ) . The expected, autophosphorylation is prevented either in the abintensity of both these bands decreased while a new radiola- sence or in the presence of polylysine (lanes 6 and 8, respecbeled band of 5 kDa became prominent if the phosphorylation tively). Polylysine has no effect onCSK catalyzed phosphowas performed in the presence of CSK (lane 2). The 5-kDa rylation of FSBA-inactivated c-Fgr (lane 9),consistent withthe radiolabeled band iseven more evidentif c-Fg-r is autophospho- concept that CSK itself is insensitive to polylysine. RESULTS
15887
Regulation of c-Fgr Tyrosine Protein Kinase TABLEI Summary of phosphopeptides obtained from CNBr and trypsin digestion followed by either SDS-PAGE or HPLC, respectively Phosphorylation degree is expressedas radioactivity incorporated in percentage of the totalfor each sample. Polylysine concentration (where present) was 10 pgml. Phosphopeptides isolated by SDS-PAGE are indicated accordingt o their apparent Mr (see 21, Fig.while phosphopeptides purified by reversed phase HPLC C-18 column are referred to as A and B according to their elution order (see 2). Fig. Other experimental conditions are detailed under “Experimental Procedures.”
O.’OO~
n
B
Radioactivity incorporated Peptides
Procedure
c-Fgr+polylysine c-Fgr
w 20-kDa CNBrPAGE 10-kDa 5-kDa A
TrypsinlHPLC
45 55
