Mapping of the p56lck-mediated phosphorylation of GAP and analysis of its influence on p21ras-GTPase activity in vitro

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Biochimica et BiophysicaActa 1222 (1994) 441-446

Mapping of the p56tck-mediated phosphorylation of GAP and analysis of its influence on p21ras-GTPase activity in vitro Kurt E. Amrein a,l,*, Baerbel Panholzer a, Juliette Molnos a, Nicholas A. Flint a, Julie Scheffler b Hans-Werner Lahm a, Willi Bannwarth a Paul Burn a a Department of Biology, Pharmaceutical Research - New Technologies, F. Hoffmann-La Roche Inc., CH-4002 Basel, Switzerland; b Department of Oncology, F. Hoffmann-La Roche Inc., Nutley, NJ 07110, USA

(Received 10 December 1993)

Abstract

The protein tyrosine kinase p56 tc~ and other members of the src family can transduce signals from activated cell-surface receptors. As we showed earlier the GTPase-activating protein (GAP), a regulator of p2V as, is a substrate of p56 lck. Here, tryptic peptides of p56tCk-phosphorylated GAP were generated and analyzed by two-dimensional thin layer chromatography and mass spectroscopy. Results revealed that p56 l~k phosphorylates GAP specifically on Tyr-460 in vitro and in vivo. The effect of tyrosine phosphorylation of GAP on its GTPase-activating activity versus p21 r~s was then tested using a p2VaS-dependent GTPase assay system. Our results demonstrate that p56tCk-mediated tyrosine phosphorylation of GAP is not sufficient to change directly its effect on the intrinsic GTPase activity of p2V a~. Key words: src kinase; Signal transduction; Tyrosine phosphorylation; Mass spectroscopy

1. Introduction lck encodes a membrane localized protein tyrosine kinase of the src family. It is predominantly expressed in cells of lymphoid origin [1]. In T-cells, p56/ck is found associated with the T-cell surface antigens CD4, CD8 [2-4] and with the IL-2 receptor [5]. In addition p56 tck is a proto-oncogene. Aberrant expression and the expression of mutant forms of p56 tck were shown to induce cellular transformation [6,7]. Several lines of evidence suggest that src-ldnases regulate signaling processes upstream of p21 r~s [8,9]. However, the molecular mechanisms by which src kinases influence p21 ras function is uncertain. The GTPase-activating protein (GAP) is a direct negative regulator of the p21 ra~ protein. It stimulates the conversion of active G T P - b o u n d p21 ra~ into inac-

* Corresponding author. Fax: + 1 (201) 235 8128. 1permanent address: Department of Metabolic Diseases, Hoffmann-La Roche Inc., 340 Kingsland Street, Nutley, NJ 071 10, USA. 0167-4889/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0167-4889(94)00003-W

tive GDP-bound p21 ras [10]. Furthermore, several lines of evidence indicate that GAP might also be the target or effector of p21 ras [11]. In cells expressing activated tyrosine kinases, G A P was shown to bind to two tyrosine phosphorylated proteins of 62 and 190 kDa (p62 and p190) [12]. Moreover, GAP itself has been shown to be a substrate of mitogenic receptor kinases and oncogenic cellular protein tyrosine kinases [12,13]. In particular, evidence has been provided that GAP is a substrate of p56 lck in vitro and in vivo [14,15]; however, the exact site(s) of tyrosine phosphorylation has not yet been identified. Our earlier studies indicated that phosphorylation of GAP might be important for its association with src kinases such as p56 lck. Alternatively, it was suggested by several groups that GAP phosphorylation might modulate its r a s G T P a s e activating activity. Such a mechanism would be of general importance, since signaling pathways controlled by various tyrosine kinases depend on p21 ras activity. In this report we identified, by peptide map analysis, Tyr-460 as the p56tck-mediated in vitro and in vivo phosphorylation site of GAP. Furthermore, recombinant purified GAP, p21 ras and p56 tck were used to set

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up an in vitro assay system to test the effect of p56 t~mediated phosphorylation of GAP on its ras-GTPaseactivating activity. 2. Materials and methods

Cell culture, biosynthetic labeling and immunoprecipitation of GAP. The establishment and growth of NIH3T3 fibroblasts expressing p56 tckFS°5 as well as the biosynthetic labeling of these cells with 32pi has been described previously [7]. For anti-GAP immunoprecipitation 32p-labeled cells were lysed by boiling in SDS lysis buffer and then diluted with 4 volumes of RIPA correction buffer [16]. Lysates were cleared by centrifugation at 10 000 × g for 30 min before they were incubated with 10 /xl of anti-GAP antiserum (Upstate Biotechnology, Lake Placid, USA) for 1 h at 4°C. Then 20 /xl of fixed Staphylococcus aureus were added and incubated for another hour. Immunocomplexes were washed three times with RIPA buffer (10 mM phosphate (pH 7.2), 0.15 M NaCI, 1% Trasylol, 1% sodium deoxycholate, 1% NP40, 0.1% SDS, 200/zM Vanadate, 2 mM EDTA, 2 mM DTT), once in TN (10 mM Tris-HCl (pH 7.2), 0.15 M NaCl) and then boiled in SDS sample buffer. Immunoprecipitated GAP was separated by electrophoresis on a 10% SDS polyacrylamide gel and transferred electrophoretically to nitrocellulose membranes [17]. Expression and purification of recombinant GAP, p21 ras and p56 tck. Human p56 lck and p21 ras were expressed in E. coli whereas human GAP was expressed in sf9 cells using the Bacculo virus system. All these proteins were expressed and purified using protocols described before [14,18,19]. CNBr and trypsin cleavage of in vitro labeled GAP. 2 /zg of GAP were phosphorylated in vitro for 10 min as described below in the presence of 10 /~Ci of [l'32p]ATP (instead of 50 /xM ATP). Phosphorylated GAP was separated by polyacrylamide gel electrophoresis (PAGE) and transferred to nitrocellulose filters. The band containing phosphorylated GAP was cut out and digested with CNBr or with trypsin [20]. The peptides generated by CNBr were analyzed by 22% PAGE analysis as described elsewhere [20] and tryptic peptides were analyzed by two-dimensional chromatography as described below. Two-dimensional tryptic peptide map analysis. After proteolytic digestion peptides were oxidized with performic acid and then analyzed by two-dimensional thin-layer chromatography [21] together with synthetic candidate phosphopeptides. The first dimension was an electrophoretic separation at pH 1.9 and the second dimension was a separation by ascending chromatography in phospho-chromatography buffer (n-butanol/ pyridine/acetic acid/deionized water (750: 500: 150: 600 v/v).

Mass spectroscopy of the tryptic GAP-phosphopeptide. 20 /xg of GAP were phosphorylated with p56 tck, digested with trypsin, and analyzed by two-dimensional thin-layer chromatography as described. The area containing the 32p-labeled peptide was isolated from the thin-layer chromatography plate and extracted twice with 0.5 ml of water. The recovered peptides were concentrated by lyophilization and desalted on a Poros R / H column. Mass spectrometric analyses were performed on a SCIEX API III mass spectrometer in positive-ion mode. Synthesis of (P)Tyr-containing synthetic peptides corresponding to predicted tryptic GAP peptides. Synthetic tyrosine-phosphorylated peptides were synthesized using a solid phase strategy [22]. The tyrosine building block was introduced without side-chain protection. Global phosphorylation of the peptide, deprotection and characterization were performed as described [22]. Two-dimensional phosphoamino acid analysis (PAA). Tryptic peptides were first resolved by phosphopeptide map analysis and then the areas containing the 32p_ labeled peptides were isolated from the nitrocellulose thin-layer chromatography plate, extracted into 1 ml of 50 mM ammonium carbonate and then lyophilized. The recovered phosphopeptides were analyzed by two-dimensional PAA as described elsewhere [21]. In vitro phosphorylation of GAP by p56 tck. 2 /zg (20 ~g) of purified recombinant GAP were phosphorylated by recombinant p56 t~k in 30 /xl (300 /xl) of reaction buffer (30 mM Hepes (pH 6.8), 10 mM MnCIz, and 50 /zM ATP) for 120 min at 30°C. To insure saturating phosphorylation of GAP, 100 ng (1 ~g) aliquots of

30

m

21.5 14.3

m

6.5

Fig. 1. PAGE analysis of in vitro phosphorylated, CNBr-cleaved GAP. Recombinant purified GAP was phosphorylated in vitro by recombinant purified p56 tck in the presence of [y-32p]ATP. The labeled protein was separated by PAGE and transferredto nitrocellulose. The piece of nitrocellulosecontainingphosphorylatedGAP was cut out, treated with CNBr and the released phosphopeptides were analyzed by PAGE. The marks on the left side represent 14C-labeled protein size markers.

K.E. Amrein et aL / Biochimica et Biophysica Acta 1222 (1994) 441-446

p56 tck were added at 0, 40 and 80 min of incubation time. GAP stimulation of p21raS-GTPase activity. First, 0.5 /zg of recombinant purified p21 r"s were equilibrated with [y-32p]GTP in 20 /zl of pre-binding buffer (100 mM NaPO 4 (pH 6.8), 0.5 mM DTT, 0.5 mM EDTA, 0.005% sodium cholate, 0.5 mg/ml bovine serum albumin (BSA) and 0.25 /~M [y-32p]GTP ( = 106 clam)). The pre-binding reaction mixture was then diluted 1:10 into TNM buffer (10 mM Tris (pH 7.0), 10 mM NaCI, 1 mM MgCI 2) containing 1% of BSA. 20-/zl aliquots of this diluted reaction buffer were incubated for 30 min with various amounts of purified phosphorylated or non-phosphorylated GAP protein. At the end of the incubation period the reaction mixtures were applied to a dot blot apparatus (BioRad) containing a nitrocellulose sheet prewashed with TNM buffer. After application of the different probes each well was washed twice with 150/~1 of TNM buffer. The nitrocellulose sheet was then removed from the apparatus and washed three times in 100 ml of TNM buffer. Non-hydrolyzed [3'-32p]GTP that was still bound to p21 ras, and therefore retained on the nitrocellulose filter, was analyzed by autoradiography. 3. Results

p120 GAP is phosphorylated on Tyr-460 in vitro and in vivo CNBr cleavage of GAP phosphorylated in vitro by purified recombinant p56 lck yields one major phosphorylated fragment with an apparent mass of approx. 18 kDa (Fig. 1). Based on the GAP protein sequence, this fragment corresponds most likely to the largest predicted CNBr fragment (amino acids 444-569) having a size of ~ 15 kDa. The small difference in the predicted size and the apparent size is not unexpected, since it is well known that phosphorylation can modify the migration properties of proteins analyzed by SDS-polyacrylamide gel electrophoresis. The ~ 15 kDa CNBr fragment is flanked by two CNBr fragments having a predicted size of ~ 4 kDa and ~ 12 kDa, which suggests that the three minor, slower migrating bands observed in the analysis of the CNBr digest may arise as the result of an incomplete cleavage (22 kDa = 18 + 4 kDa, ~ 29 kDa -'- 18 + 12 kDa, ~ 33 kDa = 18 + 4 + 12 kDa). Tryptic digestion of a peptide corresponding to amino acids 444-569, the ~ 15 kDa CNBr-fragment, is predicted to yield six tyrosine- containing peptides. To determine which of these peptides contained the tyrosine site phosphorylated by p56 lck, we synthesized (P)Tyr containing candidate peptides. The first two peptides synthesized corresponded to amino acids 479-483 and amino acids 458-464. Of these two peptides, the second co-migrated exactly with the tryptic

A

443

B

in vitro

in vivo

Fig. 2. Tryptic peptide map analysis of in vitro and in vivo labeled GAP. (A) GAP phosphorylated in vitro by p56 lck, and (B) GAP immunoprecipitated from 32pi-labeled NIH3T3 cells expressing lck F5°5 were separated by PAGE and transferred to nitrocellulose. Pieces of nitrocellulose containing phosphorylated GAP were cut out and treated with trypsin. Released peptides and a synthetic phosphopeptide containing (P)Tyr-460 (amino acids 458-464) were separated by two-dimensional thin-layer plate chromatography and then visualized either by fluorograpby or Ninhydrin staining. Circles mark the area of the Ninhydrin-stained synthetic peptide and the arrows point to the place of sample application.

phosphopeptide from in vitro labeled GAP when analyzed by two-dimensional thin-layer plate chromatography (Fig. 2A). Co-migration was observed both when the electrophoretic separation was performed at pH 1.9 (Fig. 2) and at pH 8.9 (data not shown). To provide further evidence that the 32p-containing spot contained the phosphorylated Tyr-460 peptide, the phosphopeptide was extracted and then analyzed by mass spectroscopy. A mass of 988.7 Da was found, which was in agreement with the calculated mass of the Tyr-460containing tryptic phosphopeptide, and the mass experimentally determined for the synthetic phosphopeptide corresponding to amino acids 458-464 (Table 1). Since none of the other possible tryptic phosphopeptides of GAP would have a similar mass, we concluded that p56 tck phosphorylates GAP specifically on Tyr-460. Recently it was shown that the expression of an activated mutant form of p56 lck, p56 lckFs°5, induces tyrosine phosphorylation of GAP, suggesting that GAP is also an in vivo substrate of p56 lck [15]. To analyze the in vivo phosphorylation sites of GAP we labeled NIH3T3 cells expressing Ick F5°5 biosynthetically with 32pi. GAP was then isolated by specific immunoprecipitation, separated by PAGE and transferred to nitrocellulose membrane. The area containing phosphorylated GAP was identified by fluorography, cut out, and treated with trypsin. The released peptides were then analyzed by two-dimensional peptide map analysis, together with the synthetic phosphopeptide corresponding to amino acids 458-464. The synthetic peptide, identified by Ninhydrin staining, co-migrated exactly

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Table 1 Comparison between the measured mass of the tryptic GAP phosphopeptide and the theoretical mass of candidate tryptic phosphopeptides of GAP GAP tryptic Theoretical Measured peptide mass (Da) mass (Da) GAP tryptic phosphopeptide Synthetic phosphopeptide a.a. 458-464 988.5 Candidate peptides within a.a. 458-464 988.5 the CNBr fragment a.a. 444-569 a.a. 469-473 722.7 a.a. 479-483 672.7 a.a. 492-511 2459.5 a.a. 518-537 2259.5 a.a. 538-570 4101.4

with o n e o f t h e t r y p t i c p h o s p h o p e p t i d e s o f in vivo l a b e l e d G A P (Fig. 2B, s p o t 1). Thus, w e c o n c l u d e d t h a t G A P c a n b e p h o s p h o r y l a t e d by p56 tck in vivo o n t h e s a m e site as in vitro. T h e t w o - d i m e n s i o n a l m a p s o f in vivo l a b e l e d G A P r e v e a l e d a s e c o n d s p o t c o r r e s p o n d i n g m o s t likely to

another potential G A P phosphorylation site. To further analyze this phosphorylation site the corresponding area was scraped off the plate, the phosphopeptide was extracted,hydrolyzed in 6 N HCI and the resulting phosphoamino acids were analyzed and compared to phosphoamino acid standards using two-dimensional thin-layer electrophoresis. Since the total recovered radioactivitywas too low for analysis by conventional fluorography, we analyzed the thin-layerelectrophoresis plate with a phosphoimager. Table 2 shows that the unidentified phosphopeptide(s) contained predominantly (P)Ser and (P)Thr, whereas the Tyr-460-containTable 2 Phosphoamino acid analysis of tryptic phosphopeptides of in vivo phosphorylated GAP Spot 1 Spot 2 334 128 2936

Ras, GAP Ras, GAP, p561ck

988.7 987.9

The phosphopeptide of GAP (see Fig. 2) and a synthetic phosphotyrosine peptide corresponding to amino acids (a.a.) 458-464 were analyzed by mass spectroscopy. The table includes the experimentally determined masses of these two peptides and the calculated masses of potentially tyrosine-phosphorylated tryptic peptides contained within the ~ 15 kDa CNBr fragment of GAP (amino acids 444-569, Fig, 1). Note that only the calculated mass of the tryptic peptide containing Tyr-460 is in agreement with the experimentally determined mass of the GAP-phosphopeptide. Moreover, none of the other potential, (P)Tyr-containing tryptic peptides of GAP would have a calculated mass close to 988 Da (data not shown).

P-Ser P-Thr P-Tyr

Ras

3767 3914 139

Tryptic phosphopeptides from in vivo labeled GAP (see Fig. 2B; spot 1 and 2) were isolated and phosphoamino acid analysis was performed. The resulting two-dimensional maps were analyzed using a phospho-imager. Values given represent arbitrary units of radioactivity present in the area of the Ninhydrin-stained phosphoamino acid markers.

Ras, GAP, p561ck, ATP 30

10

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0.4

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Fig. 3. Both GAP and tyrosine-phosphorylated GAP induce p21rasGTPase activity, p21ras was pre-loaded with ['y-32p]GTP and then incubated for 30 min with various amounts of GAP (lane 2), with GAP and p56 tck pre-incubated in kinase buffer in the absence of ATP (lane 3), and with GAP phosphorylated by p56 tc~ in vitro (lane 4). After the incubation samples were filtered through nitrocellulose using a dot blot apparatus and the filter was analyzed by autoradiography. The first lane represents three samples of untreated p21"as[y-32p]GTP complexes. Note that each preparation of GAP has the same potency to induce the intrinsic GTPase activity of p21ras, resulting in a loss of 32p-labeled p2VaS-GTP complexes retained on the nitrocellulose filter. ing tryptic p h o s p h o p e p t i d e , w h i c h was a n a l y z e d in p a r allel, c o n t a i n e d m a i n l y (P)Tyr. Tyrosine phosphorylation o f G A P does not change its G T P a s e activity in vitro T o test t h e effect o f t y r o s i n e p h o s p h o r y l a t i o n o f G A P on its G T P a s e - a c t i v a t i n g activity t o w a r d s p21 ras, an in vitro ras G T P a s e assay system was e s t a b l i s h e d . First, p u r i f i e d r e c o m b i n a n t h u m a n G A P was p h o s p h o r y l a t e d to s a t u r a t i o n w i t h p u r i f i e d r e c o m b i n a n t h u m a n p56 lck. In this t y p e o f assay G A P p r o v e d to b e a very g o o d s u b s t r a t e , having a K m o f 2 - 3 /zM, w h i c h is in t h e s a m e r a n g e as d e n a t u r a t e d e n o l a s e , o n e o f t h e b e s t in vitro s u b s t r a t e s k n o w n for src k i n a s e s [14]. K i n e t i c studies revealed that under the above conditions 408 0 % of G A P b e c a m e p h o s p h o r y l a t e d ( d a t a n o t shown). S e c o n d , p u r i f i e d r e c o m b i n a n t p21 ras was e q u i l i b r a t e d with [ 3'- 32 P ] G T P . S u b s e q u e n t l y t h e G T P a s e - a c t i v a t i n g activity of in vitro p h o s p h o r y l a t e d G A P t o w a r d s [3'3zp]GTP-p21ra~ c o m p l e x e s was c o m p a r e d to t h e G T P a s e - a c t i v a t i n g activity o f u n p h o s p h o r y l a t e d G A P . A s shown in Fig. 3, all p r e p a r a t i o n s o f G A P w e r e e q u a l l y p o t e n t in i n d u c i n g t h e intrinsic G T P a s e activity o f p21 ras. W e c o n c l u d e d t h a t G A P p h o s p h o r y l a t i o n at Tyr-460 is n o t sufficient to c h a n g e d i r e c t l y its p21 ra~G T P a s e - a c t i v a t i n g activity.

4. Discussion O u t o f 38 p o s s i b l e t y r o s i n e r e s i d u e s , p56 tck p h o s p h o r y l a t e d G A P in vitro only o n t h e t y r o s i n e r e s i d u e 460. T h e e q u i v a l e n t r e s i d u e o f G A P was p h o s p h o r y l a t e d in N I H 3 T 3 cells, e x p r e s s i n g a n a c t i v a t e d f o r m o f p56 tck. T o g e t h e r this i n d i c a t e s t h a t G A P is a d i r e c t

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substrate of p561ok in p561~k-controlled signaling pathways. Our finding that GAP can be phosphorylated on Tyr-460 is in agreement with a recent study demonstrating that the equivalent site of GAP is phosphorylated in EGF-activated and vanadate-treated mouse fibroblasts [23]. Thus, different types of protein tyrosine kinases can phosphorylate GAP specifically at the same residue. In addition to the major tyrosine phosphorylation site (Tyr-460), tryptic phosphopeptide-map analysis of in vivo labeled GAP resulted in the identification of another phosphopeptide. Phosphoamino acid analysis of this tryptic peptide(s) suggests that it contains mainly P-Ser and P-Thr. We cannot, however, discriminate whether one peptide with two different phosphorylation sites gives rise to the observed spot or alternatively whether this spot represents two phosphopeptides, one labeled on serine and the other on threonine. Nevertheless, this finding indicates that in addition to tyrosine kinases serine/threonine kinases may modify and regulate GAP function in vivo. Several groups reporting on tyrosine phosphorylation of GAP have suggested that this phosphorylation may link tyrosine protein kinases to the r a s - s i g n a l i n g pathway. Tyrosine phosphorylation might directly affect the p21raS-GTPase-activating activity of GAP [14,23-25]. Here we have tested this hypothesis using purified recombinant p56 l¢k, GAP and p21 ra~ in an in vitro assay system. Our results show that tyrosine phosphorylation of GAP alone is not sufficient to significantly change its effect on p2V a~. Together with the observation that the activation of receptor tyrosine kinases induces GAP phosphorylation only at low stoichiometry [23], it seems unlikely that tyrosine phosphorylation of GAP directly controls the transactivation of the p2VaS-GTPase activity. As an alternative option we and others have recently tested whether GAP phosphorylation might be important for mediating interactions with SH2-containing proteins. Indeed we have shown that GAP in its phosphorylated form can interact specifically with the /ck-SH2 domain [14,26]. Similarly it has been shown that phosphorylated GAP can interact with the SH2 domain of the src kinase p53/p56 Irn [27]. Together these findings suggest that tyrosine phosphorylation of GAP may induce binding to membrane-anchored proteins having an SH2 domain. In agreement with such a view is the finding that in v-src transformed cells, approx. 8% of GAP becomes associated with the particulate fraction and that it is this fraction of GAP which displays an increased tyrosine phosphorylation [24]. The importance of membrane binding for GAP function has been pointed out in a recent report which studied the effect of GAP protein targeted to the membrane via a palmitylation signal [28]. In summary, we have identified Tyr-460 of GAP as

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the site phosphorylated specifically by p561ok in vitro and in vivo. In addition, using an in vitro assay system we provide clear evidence that phosphorylation of GAP is not sufficient to directly change its GTPase-activating activity versus p21 ras. However, based on earlier publications [14,26,27], tyrosine phosphorylation of GAP may be important for translocation to the membrane by inducing binding to SH2-containing proteins such as p56 tck and p53/p56 lrn.

Acknowledgements We are most grateful to Francis Vilbois for mass spectral analysis and to Alex Wood, Martin Gassmann and Thomas Jascur for their interest and suggestions.

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