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[22] I d e n t i f i c a t i o n o f P h o s p h o r y l a t i o n Sites: U s e o f t h e Epidermal Growth Factor Receptor By GARY J. HEISERMANN and GORDON N. GILL

Introduction Phosphorylation is basic to our understanding of the action of many hormones and growth factors. One well-studied example is the epidermal growth factor (EGF) receptor which contains an intrinsic protein tyrosine kinase activity 1 essential to its ability to stimulate cellular growth. 2 The transforming protein product of the v-erbB oncogene resulted from truncation of the receptor ligand-binding domain, suggesting that this domain normally acts to restrain the tyrosine kinase activity of the receptor in the absence of ligand. 3 The tyrosine kinase activity of the EGF receptor appears to also be inhibited by several self-phosphorylation sites located near its C terminus. 4 Kinetic analysis indicates that these sites in their nonphosphorylated state act as competitive inhibitors to block phosphorylation of exogenous substrates. 5 The EGF receptor is also regulated by heterologous phosphorylation following treatment of cells with phorbol esters. Phorbol esters activate protein kinase C resulting in phosphorylation of EGF receptor TAr-654 and inhibition of EGF binding and signal transduction. 6'7 The EGF receptor is phosphorylated in vivo on several additional threonine and serine residues, 6'8 which may also act to regulate receptor activity. Identification of the phosphorylation sites is an important step toward understanding their function in EGF receptor signaling. Other growth factor receptors and regulatory enzymes are similarly phosphorylated, and identification of sites of phosphorylation is a necessary prerequisite to investigation of the regulatory function of these sites. H. Ushiro and S. Cohen, J. Biol. Chem. 255, 8363 (1980). 2 W. C. Chen, C. S. Lazar, M. Poenie, R. Y. Tsien, G. N. Gill, and M. G. Rosenfeld, Nature (London) 328, 820 (1987). 3 A. Wells and J. M. Bishop, Proe. NatL Acad. Sci. U.S.A. 85, 7597 (1988). 4 j. Downward, P. Parker, and M. D. Waterfield, Nature (London) 311, 483 (1987). 5 p. j. Bertics and G. N. Gill, J. Biol. Chem. 260, 14642 (1985). 6 C. Cochet, G. N. Gill, J. Meisenhelder, J. A. Cooper, and T. Hunter, J. Biol. Chem. 259, 2553 (1984). 7 T. Hunter, N. A. Ling, and J. A. Cooper, Nature (London) 311, 480 (1985). 8 T. Hunter and J. A. Cooper, Cell (Cambridge, Mass.) 24, 741 (1981).

METHODS IN ENZYMOLOGY. VOL. 198

Copyright © 1991 by Academic Press. Inc. All rights of reproduction in any form reserved.

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Principle The location of phosphorylation sites in the EGF receptor sequence is based on radioactive labeling of the receptor either in intact cells or using purified protein kinases. Receptor radiolabeled in vivo is isolated under conditions which preserve its native phosphorylation state. Phosphopeptides are generated by protease treatment and are purified by successive chromatography on two reversed-phase HPLC columns in different buffers. The purified peptides are sequenced by automated Edman degradation, and the phosphorylated residues are identified by one of three methods: (1) localization of the radiolabel to a peptide containing a single potential phosphorylation site, (2) release of 32p as inorganic phosphate during the sequencing cycle of the phosphorylated residue, and (3) loss of 32p-labeled peptide and an increase in 32po 4 bound to the sequencing filter during Edman degradation of the phosphorylated residue. Site-directed mutagenesis of the identified phosphorylation sites permits assessment of their role in EGF receptor signal transduction. Materials

Phosphopeptides are purified by HPLC on an LKB UltraChrom GTi Bioseparation System. The separations are performed on a Vydac C~8 reversed-phase column (218TP54) and a Brownlee C8 reversed-phase column (RP300). Mouse EGF and immune absorbent 528 IgG-agarose are prepared as previously described. 9 Cells. Human epidermoid carcinoma A431 cells, clone 29I, ~0are grown in a I : 1 mixture of Dulbecco's modified Eagle's medium (DMEM) and Ham's FI2 medium containing 5% calf serum. Mouse B82 L cells expressing transfected human EGF receptors are grown in DMEM containing 5% dialyzed fetal calf serum and 5/zM methotrexate. Buffers Buffer A: 200 mM potassium phosphate, pH 7.2, 150 mM NaCI, 2 mM EDTA, 1% (w/v) deoxycholic acid, 1% (v/v) Nonidet P-40 (NP40), 0.1% (w/v) sodium dodecyl sulfate (SDS), 4 mM benzamidine, 0.5 mM phenyimethylsulfonyl fluoride, 0.5 units/ml aprotinin, 0.04 mg/ml leupeptin, 6 mM 2-mercaptoethanol, 10/~M ammonium molybdate, 1 mM sodium vanadate, 50 mM sodium fluoride, 5 mM p-nitrophenyl phosphate 9 G. N. Gill and W. Weber, this series, Vol. 146, p. 82. i0 j. B. Santon, M. T. Cronin, C. L. M a c L e o d , J. Mendelsohn, H. Masui, and G. N. Gill, Cancer Res. 46, 4701 (1986).

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Buffer B: 20 mM HEPES, pH 7.4, 1 mM EDTA, 130 mM NaCI, 0.05% Triton X-100, 10% glycerol, 1 mM dithiothreitol (DTT) Low-phosphate DMEM: 95% phosphate-free DMEM (Irvine, Santa Ana, CA), 5% regular DMEM Methods

Labeling of Cells with 32p04. Cultured cells at 50% confluence are washed once in low-phosphate DMEM and incubated with l ml per 10-cm dish of low-phosphate DMEM containing 0.2-1.0 mCi 32po 4. Cells are rocked for 12-14 hr in a Plexiglas box at 37 ° in a humidified 10% CO2 atmosphere. Prior to harvesting, cells are transferred to an ice tray and are washed with 5 ml of ice-cold phosphate-buffered saline (PBS) containing 5 mM EGTA. Cells are scraped in PBS and collected by centrifugation (1 min, 800 g). The cell pellet is immediately resuspended in 3 volumes of buffer A, l0 /~l per ml of 100 mM PMSF in 2-propanol is added, and extracts are frozen on dry ice and stored at - 7 0 °. Purification of 32p-Labeled EGF Receptor. EGF receptor is purified by a modification of the immunoaffinity method described earlier in this series. 9 Frozen extracts are thawed and homogenized with 3-4 strokes of a Teflon-glass homogenizer. The homogenate is transferred to a plastic centrifuge tube, and the homogenizer is rinsed with 0.25 volume of buffer B, which is pooled with the sample. Cellular debris is removed by centrifugation (10 min, 4 °, 10,000 g), and the supernatant is filtered through glass wool into a tube containing 528 IgG-agarose beads. The beads and sample are mixed gently at 4 ° for 1-2 hr, and the beads are pelleted by a 5- to 10-sec spin in a microcentrifuge. The supernatant is removed with a disposable plastic Pasteur pipette, and the beads are washed with 5 volumes of buffer B (8 washes), buffer B containing 1 M NaCl (3 washes), buffer B (3 washes), again with buffer B containing 1 M NaC1 (3 washes), buffer B (3 washes), and buffer B containing 1 M urea (3 washes). The purified EGF receptor is eluted by three incubations with equal volumes of buffer C containing 8 M urea for 30 min at 22°. Phosphorylation of EGF Receptor in Vitro by Purified Protein Kinases. The EGF receptor may be phosphorylated in vitro by purified protein kinases, as has been reported using protein kinase C 6and cAMP-dependent protein kinase. ~ Analysis of in vitro phosphorylation data is complicated by the additional phosphopeptides resulting from extensive autophosphorylation of active solubilized EGF receptor. This also creates a need to separate the two protein kinases prior to proteolysis. One approach which tt W. R. Rackoff, R. A. Rubin, and H. S. Earp, Mol. Cell. Endocrinol. 34, 113 (1984).

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has been used to circumvent this problem is to self-phosphorylate the EGF receptor immobilized on an affinity matrix, first with nonradioactive ATP, followed by incubation with [y-aEp]ATP and the second protein kinase. 12 The components of the reaction mixture are readily washed away, and the phosphorylated EGF receptor is then eluted. Phosphorylation of inactive receptor preparations is problematic because denaturation may expose spurious phosphorylation sites unavailable in the native receptor structure. Proteolytic Digestion and Peptide Purification. The purified 32p-labeled EGF receptor is dialyzed against 100 mM NH4HCO 3 containing [ mM DTT and 0.01% Triton X-100. The purity of the dialyzed receptor is assessed by electrophoresis of an aliquot on a 10% SDS-polyacrylamide gel. The yield is determined by comparison of Coomassie blue or silverstained EGF receptor with bovine serum albumin. 13The dialyzed receptor is digested by addition of 0.5 mM CaCI2 and 1% (w/v) trypsin and incubation overnight at 37 °, followed by a second trypsin addition and incubation. The tryptic digests are concentrated under reduced pressure in a Savant Speed-Vac (Hicksville, NY). Purified receptor can alternatively be digested with V8 protease. 14 This protease recognizes different substrate sites than trypsin, 15 and it may be advantageous in the identification of phosphorylated residues located distant from sites of tryptic cleavage. Before protease treatment, the receptor may be reduced and its sulfhydryl groups alkylated 16to prevent secondary structure from impeding the proteolytic digestion. The concentrated tryptic peptides are mixed with an equal volume of freshly made 6 M guanidine hydrochloride and filtered through a Microfilterfuge tube (Rainin, Woburn, MA). The filtrate is applied to a C1~ reversed-phase HPLC column equilibrated in l0 mM potassium phosphate buffer (pH 6.0) and 3% acetonitrile. The column is washed for 10 min at a flow rate of 0.5 ml/min, and the peptides are eluted by a 3 to 53% acetonitrile gradient (0.5%/min). The absorbance at 220 nm is monitored to follow peptide elution, and phosphopeptides are detected by Cerenkov radiation. The phosphoamino acids present in the purified phosphopeptides are determined after hydrolysis in 0.1 ml of 6 N HCI for 1.5 hr at ! 10°. Samples are dried, washed twice in water, and resuspended in an aqueous solution of phosphoamino acid standards. The phosphoamino 12 j. Downward, M. D. Waterfield, and P. J. Parker, J. Biol. Chem. 260, 14538 (1985). 13 W. Weber, P. J. Bertics, and G. N. Gill, J. Biol. Chem. 259, 14631 (1984). t4 G. M. Walton, P. J. Bertics, L. G. Hudson, T. S. Vedvick, and G. N. Gill, Anal. Biochem. 161, 425 (1987). t5 G. R. Drapeau, this series, Vol. 47, p. 189. 16A. Henschen, in "Advanced Methods in Protein Microsequence Analysis" (B. WittmannLiebold, ed.), Springer-Verlag, Heidelberg, 1986.

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acids are separated by high-voltage electrophoresis at 1000 V for 2 hr at pH 3.517 and analyzed by standard procedures.~8 Once the pattern of phosphopeptides eluting from the Cla column at pH 6.0 is established, a larger pool of EGF receptor is prepared to generate peptides for sequencing. EGF receptor which is phosphorylated but not radiolabeled is obtained by treating and harvesting cells as described above except that 32po 4 is omitted from the low-phosphate DMEM. The inclusion of this nonradioactive EGF receptor increases the total amount of material available for sequencing and has the additional advantage of increasing the yield of 3zp-labeled phosphopeptides up to 2- to 3-fold. The nonradioactive and radioactive frozen cell extracts are pooled on thawing, and the EGF receptor is purified and digested with protease as described above. The peptides eluted from the Cm column in phosphate buffer are concentrated on the Speed-Vac to remove the acetonitrile. The peptides are then applied to a C8 reversed-phase HPLC column equilibrated in 0.05% trifluoroacetic acid (TFA) and 3% acetonitrile. The column is washed for l0 min at 0.2 ml/min, and the peptides are eluted with a 3 to 53% acetonitrile gradient (0.5%/min). The absorbance and Cerenkov radiation are monitored as above. The phosphopeptides eluted from the C8 column are concentrated on the Speed-Vac and are applied directly to a Polybrenecoated glass fiber filter for automated Edman degradation.19 The two-step peptide purification scheme described above is useful because of the large size of the EGF receptor and because of its multisite phosphorylation. 6"8This two-step purification is effective with tryptic peptide digests, permitting the separation of EGF receptor phosphopeptides of similar size which differ in negative charge or phosphorylation state. 17 These phosphopeptides elute from the C18 column in phosphate buffer at unique positions (Table I). When rechromatographed in 0.05% TFA the phosphopeptides become more hydrophobic because their negatively charged amino acids are protonated, and consequently they elute at higher acetonitrile concentrations. Pairs of peptides which eluted in distinct positions from the Cm column in phosphate buffer now elute at similar acetonitrile concentrations in TFA (Table I), emphasizing the value of this twostep purification procedure relative to a single HPLC separation in TFA. The relative elution positions of the positively charged peptides resulting from digestion with V8 protease would not be expected to vary as significantly between the phosphate buffer and TFA systems. A new strong 17 G. J. Heisermann and G. N. Gill, J. Biol. Chem. 263, 13152 (1988). m j. A. Cooper, B. M. Sefton, and T. Hunter, this series, Vol. 99, p. 387. 19 M. W. Hunkapiller, R. M. Hewick, W. J. Dreyer, and L. E. Hood, this series, Vol. 91, p. 399.

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TABLE I EPIDERMAL GROWTH FACTOR RECEPTOR TRYPTIC PHOSPHOPEPTIDE SEQUENCES AND ELUTION BEHAVIORa

Acetonitrile (%) Peptide

Sequence

pH 6.0

pH 2.3

12.2

21.2

14.2

21.2

P2

P P ELVEPLTPSGEAPNQALLR 681 P ELVEPLTPSGEAPNQALLR TM

P3

MHLPSPTDSNFYR 975

14.9

22.5

P4

ALMDEEDMDDVVDADEYLIPQQGFFSSPSTSR ~°°7 PP YSSDPTGALTEDSIDDTFLPVPEYINQSVPKR 1°76

15.7

30.0

18.1

30.6/31.3

P1

P5

EGF receptor tryptic phosphopeptides, purified by two sequential HPLC steps, were sequenced on an Applied Biosystems 470A gas-phase sequencer. The sequences of the EGF receptor tryptic peptides are shown in single-letter code. Superscript numbers refer to residues in the predicted EGF receptor sequence. A " P " above an amino acid designates a phosphorylation site identified by the methods described in the text. Potential phosphorylation sites as determined by phosphoamino acid analysis are indicated by asterisks. Also listed are the positions in the acetonitrile gradient where each peptide eluted in phosphate buffer (pH 6.0) and in TFA (pH 2.3). Peptide P5 has two adjacent tryptic cleavage sites at its C terminus and eluted as two closely spaced peaks when chromatographed in TFA.

cation-exchange (SCX) HPLC matrix has been reported which separates peptides on the basis of positive charge, 2° and SCX columns have been shown to efficiently separate peptides resulting from V8 digestionfl I Further purification by reversed-phase HPLC in TFA should result in homogeneous peptides even from a very large protein.

Identification of Phosphorylated Residues Localization by Inspection. If a purified phosphopeptide contains a single serine or threonine residue, then this residue must be the phosphorylation site. This was the case with EGF receptor phosphopeptides PI and P2 (Table I), which represent the same peptide sequence phosphorylated on Thr-669 and Ser-671, or on Thr-669 alone, respectively. The major 2o D. L. Crimmins, J. Gorka, R. S. Thoma, and B. D. Schwartz, J. Chromatogr. 443, 63 (1988). 2i D. L. Crimmins, R. S. Thoma, D. W. McCourt, and B. D. Schwartz, Anal. Biochem. 176, 255 (1989).

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limitation to identification of phosphorylation sites by this method is the rarity of candidate peptides containing a single serine or threonine. One caveat with the method is that the phosphopeptide must be pure; a small amount of phosphopeptide unresolved from a larger quantity of a nonphosphorylated peptide will make the latter appear to be phosphorylated. Although a sharp symmetrical peak on the final HPLC purification is suggestive of a pure peptide, the most stringent test of purity is the release of a single phenylthiohydantoin (PTH) amino acid during each sequencing cycle. Identification by 32p Release during Edman Degradation. The most common way to identify a phosphorylated residue is to measure the free 32p released during Edman degradation. The 32p is released from phosphoserine by /3-elimination under the acid conditions employed in Edman degradation, and two PTH-derivatized serine breakdown products are produced. 22 Analogous breakdown products and 32p release occur during the sequencing of peptides containing phosphothreonine and phosphotyrosine. Nearly all of the released 32p binds to the glass fiber filter as inorganic phosphate because of the nonpolar solvents used in the gas-phase sequencer. Typically only 1-2% of the total radioactivity accompanies the released PTH-amino acid, but this is often adequate. This method has been used most often to identify phosphorylated residues after in vitro phosphorylation, and in such cases it is not difficult to prepare a very " h o t " phosphopeptide. One major advantage of this technique is that it requires no extra sample manipulation. Prior to HPLC analysis of the PTH-amino acid derivative released during each sequencing cycle, an aliquot (typically 40-50%) is removed and counted for 32p after addition of scintillation fluid. When aliquots were taken and counted during the sequencing of EGF receptor phosphopeptide P2, a peak of radioactivity was observed in the seventh sequencing cycle, establishing Thr-669 as the phosphorylated amino acid (Table I, Ref. 17). Split-Filter Technique for Phosphorylation Site Identification. The split-filter method is based on the shift of sequencer filter-bound radioactivity from 32p-labeled peptide to inorganic 32po4 following Edman degradation of a phosphorylated residue. 23 The method involves splitting a sequencer filter into multiple sections after sample application. The sample is sequenced normally, and individual filter pieces are removed from the gas-phase sequencer in the cycles surrounding suspected phosphorylation sites. This requires prior knowledge of the peptide sequence, making this 22 G. Allen, "Sequencing of Proteins and Peptides." Elsevier, Amsterdam, 1989. 23 C. J. Fiol, A. M. Mahrenholz, Y. Wang, R. W. Roeske, and P. J. Roach, J. Biol. Chem. 262, 14042 (1987).

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a convenient technique to use if a phosphorylation site is not precisely located by 32p release. The pieces of sequencer filter removed in successive sequencing cycles are sonicated 3 times in 50% formic acid to extract both bound peptide and inorganic 32po4. These components of the extracted samples are separated by reversed-phase HPLC or thin-layer chromatography. Edman degradation of a phosphorylated residue results in a loss of radioactivity from the peptide and an increase in inorganic 32PO4. This method is several times more sensitive than simply measuring 32p release and is particularly useful in characterizing adjacent potential phosphorylation sites. The split-filter technique was employed to identify Ser-1046 and Ser-1047 as the phosphorylated residues in EGF receptor phosphopeptide P5 (Table I). Comments The ability to purify sufficient protein for characterization is a basic requirement for the identification of phosphorylation sites. This is easily accomplished with the EGF receptor because of the availability of cell lines such as A431 human epidermoid carcinoma cells, which contain an amplified EGF receptor gene and express the receptor at greatly elevated levelsJ 4 High level expression of the EGF receptor can also be achieved by selection after transfection of receptor cDNA into recipient cells. 25 An efficient purification method is required not only to generate pure EGF receptor but also to rapidly separate the phosphorylated receptor from cellular phosphatases. A variety of purification strategies for the EGF receptor have utilized the power of affinity chromatography, including use of immobilized EGF, 26 monoclonal anti-EGF receptor antibodies, 9 and monoclonal antiphosphotyrosine antibodies. 27 To retain the phosphorylation state of the EGF receptor during purification, cells are lysed in a denaturing buffer which contains several phosphatase inhibitors. Even with these precautions it is important to work quickly, and in certain situations it may be necessary to lyse cells on the plate rather than first scraping in PBS. A recently developed method promises to greatly increase the sensitivity of detection of phosphoserine residues. 28 This method entails modification of purified phosphopeptides with ethanethiol, which 24 C. M. Stoscheck and G. Carpenter, J. Cell. Biochem. 23, 191 (1983). 25 C. R.Lin, W. S. Chen, C. S. Lazar, C. D. Carpenter, G. N. Gill, R. M. Evans, and M. G. Rosenfeld, Cell (Cambridge, Mass.) 44, 839 (1986). 26 S. Cohen, this series, Vol. 99, p. 379. 27 p. B. Wedegaertner and G. N. Gill, J. Biol. Chem. 264, 11346 (1989). 28 H. E. Meyer, E. Hoffmann-Posorske, H. Korte, and M. G. Heilmeyer, Jr., FEBS Lett. 204, 61 (1986).

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reacts specifically with phosphoserine residues. On Edman degradation a unique PTH derivative is quantitatively produced. This circumvents problems of identification and yield of PTH-serine and avoids the need to follow radioactivity through the automated sequencer. It is necessary to characterize a phosphorylation site in vivo to establish its physiological significance. In certain cases it may be easier to phosphorylate a protein in vitro, but the results should always be correlated with in vivo data. This is critical because of the tendency of protein kinases to phosphorylate many more proteins as well as additional sites on established substrates, when assayed in vitro. The range of in vitro studies is restricted because of the limited number of purified protein kinases available. Correlation of in vitro and in vivo phosphorylation data is not always possible because of the inability to specifically activate certain protein kinases in intact cells. It is nevertheless possible to qualitatively compare an identified in vitro phosphorylation site with in vivo phosphopeptide results, especially if the phosphopeptides do not contain multiple serine or threonine residues. Once a phosphorylation site has been established, its role in regulation of protein function can be studied by specific mutation of the phosphorylated residue. The function of a mutant EGF receptor was studied after transfection into a cell line which does not normally express the receptor, mouse B82 L cells. 25 To create the mutant receptor, a fragment of the EGF receptor cDNA was inserted into M 13 single-stranded phage to serve as a template for mutagenesis. 29 A mutagenic oligonucleotide encoding the desired amino acid alteration served as a primer for generating the replicative form of the phage containing the mutation. The EGF receptor cDNA fragment containing the desired mutation was excised and placed into an expression vector containing the remaining portions of the EGF receptor cDNA linked to the SV40 promoter. The expression vector was transfected into recipient cells, and high levels of EGF receptor expression were achieved by selection in methotrexate. Cells expressing the mutant EGF receptor were then used in studies of EGF-induced receptor internalization, tyrosine phosphorylation, and mitogenesis. 25'3° Acknowledgments We wouldlike to thank Dr. Deborah L. Cadenafor helpfuldiscussions.Studiesfromthe authors' laboratory were supported by a grant from The Council for Tobacco Research--U,S.A., Inc. (1622) and by The MarkeyCharitable Trust. 29 M. J. Zoller and M. Smith, this series, Vol. 100, p. 468. 3o G. J. Heisermann, H. S. Wiley, B. J. Walsh, H. A. lngraham, C. J. Fiol, and G. N. Gill, J. Biol. Chem. 265, 12820 (1990).

Identification of phosphorylation sites: use of the epidermal growth factor receptor.

[22] PHOSPHORYLATION SITE IDENTIFICATION 233 [22] I d e n t i f i c a t i o n o f P h o s p h o r y l a t i o n Sites: U s e o f t h e Epidermal G...
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