260
EGF
RECEPTOR
AND RELATED
RECEPTORS
[25]
[25] P h o s p h o r y l a t i o n o f L i p o c o r t i n - 1 b y t h e E p i d e r m a l Growth Factor Receptor By R. BLAKE PEPINSKY
Introduction The lipocortins are a recently characterized family of Ca 2+- and phospholipid-binding proteins which differ from conventional Ca2+-binding proteins in that they lack an EF-hand type Ca2+-binding loop and they inhibit phospholipase Az in in vitro assay systems. Eight related proteins have been identified to date, all sharing extensive sequence similarity (for references, see Refs. I and 2). Most conserved are a series of 70 amino acid repeat units that are organized into a core structure that binds Ca z+ and phospholipid. The 35- to 45-kDa proteins contain four copies of the repeat and the 70-kDa form, eight copies. Each protein also contains a short amino-terminal segment that is distinct from the core structure and unique to each protein. While the repeat units directly bind Ca z+ and phospholipid, the amino terminus regulates the binding affinities and thus may provide functional identity to the individual family members, s-5 Lipocortin-like proteins have been implicated in diverse processes, ranging from intracellular events where they affect membrane-cytoskeleton interactions and signal transduction to extracellular events where they affect inflammation, the immune response, blood coagulation, growth, and differentiation (for references, see Ref. 6). Of the eight proteins lipocortins-1 and -2 have been of particular interest as major substrates for oncogene and receptor protein kinases. In 1984, Fava and Cohen characterized a 39-kDa CaZ+-binding protein from A431 cells that was a substrate of the epidermal growth factor (EGF) receptor/kinase in vitro using receptor-rich cell membranes as a source of i R. B. Pepinsky, R. Tizard, R. J. Mattaliano, L. K. Sinclair, G. T. Miller, J. L. Browning, E. P. Chow, C. Burne, K.-S. Huang, D. Pratt, L. Wachter, C. Hession, A. Z. Frey, and B. P. Wallner, J. Biol. Chem. 263, 10799 (1988). M. J. Crumpton and J. R. Dedman, Nature (London) 345, 212 (1990). 3 y. Ando, S. Imamura, Y.-M. Hong, M. K. Owada, T. Kakunaga, and R. Kannag, J. Biol. Chem. 264, 6948 (1989). 4 D. S. Drust and C. E. Creutz, Nature (London) 331, 88 (1988). 5 j. Glenney and L. Zokas, Biochemistry 27, 2069 (1988). 6 R. B. Pepinsky, L. K. Sinclair, E. P. Chow, and B. Greco-O'brine, Biochem. J. 263, 97 (1989).
METHODS IN ENZYMOLOGY, VOL. 198
Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
[25]
EGF-DEPENDENT PHOSPHORYLATION OF LIPOCORTIN
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the kinase. 7 The phosphorylation was EGF-dependent and occurred on tyrosine. Subsequent studies revealed that the 39-kDa protein was phosphorylated in intact cells and, excluding the receptor itself, represented the first known physiological substrate of the EGF receptor. 8 The extent of phosphorylation was growth cycle-dependent and accounted for as much as 25% of the protein. In studying lipocortin-like proteins, we observed that lipocortin-I also was a substrate of the EGF receptor and demonstrated that it and the 39-kDa band were the same protein. 9 Because we had already cloned and expressed lipocortin-1 ,~0 this finding placed us in an unusual position to evaluate the activities and properties of the protein. The following sections describe some of these studies, focusing on those that pertain to the phosphorylation of lipocortin by the EGF receptor and characterization of the phosphorylated product. Materials and Methods
Labeling and Immune Precipitation Procedures The A431 cell line, derived from a human epidermal carcinoma, is obtained from the American Type Culture Collection (CRL 1555, Rockville, MD). The cells are grown as monolayers at 36° in a tissue culture incubator in Dulbecco's modified Eagle's medium supplemented with 10% calf serum, 50 units/ml penicillin, and 50/zg/ml streptomycin. Cells are maintained in T75 flasks and trypsinized and passaged at a 1 : 5 split ratio every third day. Cells for metabolic labeling studies are split at the same ratio into 6-well culture dishes. After 48 hr, the growth medium is discarded and the wells washed twice with 2.5 ml serum-free medium that is deficient either in methionine or phosphate (Flow Laboratories, McLean, VA). Parallel cultures are labeled for 4 hr with 100 t~Ci/well [35S]methionine (900 Ci/mmol) or 1 mCi/well [32p]phosphate (1200 Ci/mol) in either the presence or absence of 200 ng/ml EGF. The labeling medium is discarded, and the cells are scraped into 1 ml of ice-cold lysis buffer (50 mM NaCI, 20 mM Tris-HC1, pH 7.3, 0.5% Nonidet P-40, 0.5% sodium deoxycholate, 4 mM iodoacetic acid, 1 mM ammonium vanadate). The lysates are subjected to vortex mixing for 30 sec and clarified in an Eppendorf centrifuge (5 min, 10,000 rpm, 4°). 7 R. A. Fava and S. Cohen, J. Biol. Chem. 259, 2636 (1984). 8 S. T. Sawyer and S. Cohen, J. BioL Chem. 260, 8233 (1985). 9 R. B. Pepinsky and L. K. Sinclair, Nature (London) 321, 81 (1986). 10 B. P. Wallner, R. J. Mattaliano, C. Hession, R. L. Cate, R. Tizard, L. K. Sinclair, C. Foeller, E. P. Chow, J. L. Browning, K. L. Ramachandran, and R. B. Pepinsky, Nature (London) 320, 77 (1986).
262
E G F RECEPTOR AND RELATED RECEPTORS
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For immune precipitations the labeled lysates are split into two aliquots, one receiving 2 p,l of preimmune serum and the other receiving 2/,d of antilipocortin-1 antiserum (lipocortin antiserum is developed in rabbits using recombinant protein produced in Escherichia coli as immunogen; see Ref. 11). The samples are incubated on ice for 2 hr, and then the immune complexes are collected by adsorption to protein A-Sepharose (Sigma Chemical Company, St. Louis, MO) for 1 hr at 4 ° with continuous mixing (25 p~l of packed beads per sample). The adsorbed complexes are washed 4 times, each with 0.5 ml of lysis buffer, suspended in 50/zl of electrophoresis sample buffer [2% sodium dodecyl sulfate (SDS), 50 mM Tris-HCl, pH 6.8, 12.5% glycerol, 1.5% 2-mercaptoethanol, 0.1% bromophenol blue], and heated at 65 ° for 10 min. The protein A beads are pelleted in an Eppendorf centrifuge (1 min, 10,000 rpm, 4°). Supernatants are transferred to new tubes and stored at - 2 0 ° for subsequent analysis. Aliquots (10/zl) are subjected to SDS-PAGE in minigels (7 x 7 x 0.14 cm) using the Laemmli system. Separating gels contain 12.5% acrylamide, 0.1% methylene bisacrylamide and stacking gels, 7.6% acrylamide, 0.21% methylene bisacrylamide. Gels are dried under reduced pressure for 1.5 hr at 80° and radioactive bands visualized by autoradiography. Generally, an overnight exposure is needed to visualize the immune precipitates. In Vitro Phosphorylation Reactions
A431 cells are split at a 1 : 5 ratio into T75 flasks. After 48 hr the cells are washed with serum-free growth medium and incubated for 1 hr at 37 ° in 7 ml of the same medium without or with 200 ng/ml EGF, Monolayers are rinsed with 2.5 ml of ice-cold hypotonic buffer (10 mM HEPES, pH 7.5, 0.5 mM MgC1z) and the cells scraped into 1.5 ml of the same buffer. After 30 min on ice, the cells are lysed with a Dounce homogenizer (20 strokes, Type A pestle) and clarified of large debris by centrifugation for 5 min at 2000 g. Cell membranes are pelleted by centrifugation at 10,000 g for 10 rain and suspended in 300/xl of phosphorylation buffer (20 mM HEPES, pH 7.5, 2 mM MgCI 2, I0/zM ammonium vanadate) plus 2/~g/ ml EGF for EGF-treated samples. For in vitro phosphorylations, 60-/xl reactions, each containing 30 txl of the membrane fraction plus 30/~Ci of [T-3ZP]ATP and the indicated amount of lipocortin in 30 tzl of the same buffer, are held on ice for 10 min. Reactions are stopped by adding 20/zl of 5 x electrophoresis sample buffer. Samples are heated at 65 ° for l0 min, u K.-S. Huang, B. P. Wallner, R. J. Mattaliano, R. Tizard, C. Burne, A. Frey, C. Hession, P. McGray, L. Sinclair, E. P. Chow, J. L. Browning, K. L. Ramachandran, J. Tang, J. E. Smart, and R. B. Pepinsky, Cell (Cambridge, Mass.) 46, 191 (1986).
[25]
E G F - D E P E N D E N T PHOSPHORYLATION OF LIPOCORTIN
263
and 5-td aliquots are subjected to SDS gel analysis. Gels are dried under reduced pressure and exposed to X-ray film for 1 hr at 23 °. Epidermal Growth Factor-Dependent Phosphorylation of Lipocortin- 1 in A431 Cells The mitogenic or in some instances inhibitory effects of E G F on target cells are mediated through its receptor, a 170-kDa transmembrane protein with an extracellular E G F binding site and an intracellular tyrosine kinase domain. Of multiple immediate changes in target cells that are initiated on binding of E G F to the receptor, the earliest is the activation of the kinase, producing a cascade of phosphorylation reactions presumed to be crucial for transduction of the E G F signal. Two prominent phosphorylation products of the kinase are the receptor itself and lipocortin-1. 7,9'12 Recent immunohistochemical studies with lung tissue have revealed a striking correlation between the cellular distribution of lipocortin-I and the EGF receptor, supporting a role for the protein in receptor function.13 In A431 cells the E G F receptor is overproduced, TM leading to the frequent use of the cell line as a source of the kinase. A431 cells also produce large amounts of lipocortin-1 ( - 0 . 5 % of the total protein) and therefore provide a simple, accessible system for studying the phosphorylation of lipocortin-I by the E G F receptor. 7-9'15 Figure I shows results from two sets of experiments where EGF-dependent phosphorylation of lipocortin-1 was studied in intact cells (Fig. 1, lanes i-p) and in v i t r o using A431 cell membranes as a source of the kinase (Fig. 1, lanes a-h). To assess phosphorylation in intact cells, A431 cells or EGF-treated A431 cells are metabolically labeled with [32p]phosphate. Lipocortin-I is selectively precipitated from the lysates with lipocortin-l-specific antiserum and analyzed for [32p]phosphate by SDS-PAGE. From control cultures labeled with [35S]methionine, lipocortin-I is the only band detected by the gel analysis (Fig. 1, lanes i-I) with E G F having no effect on the labeling. In contrast, from [32p]phosphate-labeled cells (lanes m - p , Fig. 1), lipocortin- 1 is only observed in extracts from cells that are first treated with EGF. Typically, about a 5-fold increase in incorporation of phosphate into lipocortin-1 was observed in the EGF-treated samples. Phosphoamino acid 12B. K. De, K. S. Misono, T. J. Lukas, B. Mroczkowski,and S. Cohen, J. Biol. Chem. 261, 13784 (1986). 13M. D. Johnson, M. E. Gray, G. Carpenter, R. B. Pepinsky, H. Sundell, and M. T. Stahlman, Pediatr. Res. 25, 535 (1989). 14R. N. Fabricant, J. E. DeLarco, and G. J. Todaro, Proc. Natl. Acad. Sci. U.S.A. 74, 565 (1977). 15D. D. Schlaepfer and H. T. Haigler, J. Biol. Chem. 262, 6931 (1987).
264
EGF RECEPTORAND RELATEDRECEPTORS a b c d
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FIG. 1. EGF-dependent phosphorylation of lipocortin-l. In vitro phosphorylation reactions using isolated A431 cell membranes (lanes a-h) and immune precipitations from metabolically labeled cells ([35Slmethionine, lanes i-l; [3Zp]phosphate, lanes m-p) were analyzed by SDS-PAGE and radioactive products visualized by autoradiography. Arrowheads denote the positions of the EGF receptor. (170 kDa) and of lipocortin-1 (39 kDa). Specific variables are indicated (Pre, preimmune serum; lm, immune serum). Lanes c and d each received 2 t~g lipocortin. Lanes e-h show phosphorylationresults obtained with membranes from EGFtreated cells that were incubated with 0.2, 2, 20, and 200/zg/ml of lipocortin, respectively. [Reprinted with permission from R. B. Pepinsky and L. K. Sinclair, Nature (London) 321, 81 (1989). Copyright © Macmillan Magazines Ltd.]
analysis revealed that the increase in label was predominantly on tyrosine. A series of bands were detected in the samples from phosphate-labeled cells, but of these only lipocortin-1 and an 18K band presumed to be a fragment of lipocortin showed E G F - d e p e n d e n t phosphorylation. While some o f the other proteins may be immunologically related to lipocortin,6' 16 many probably result from nonspecific binding to the protein A-Sepharose beads, since the appearance of the precipitations in subsequent analysis was improved by including SDS in the lysis buffer. 17Perhaps the cleanest results to date were generated by S a w y e r and Cohen, who partially purified the phosphorylated lipocortin prior to the immune precipitation step. 8 They obtained a 15-fold increase in labeled lipocortin from EGF-treated A431 cells. Phosphate incorporation increased steadily over the labeling 16A. Karasik, R. B. Pepinsky, and R. C. Kahn, J. Biol. Chem. 263, 11862(1988). 17A. Karasik, R. B. Pepinsky, S. E. Shoelson, and R. C. Kahn, J. Biol. Chem. 263, 18558 (1988).
[25]
EGF-DEPENDENT PHOSPHORYLATION OF LIPOCORTIN
265
period (4 hr), indicating that the phosphorylated adduct was relatively long lived. In working with A431 cells, we routinely use cells at around 48 hr after plating. At this time EGF induces a dramatic morphologic change, marked by a rapid movement of the cells from a confluent monolayer into a weblike network where the cells aggregate to form the cables of the web. If the cells are used too early, namely, within the first 24 hr after plating, the EGF treatment causes them to round up and detach, thus being lost to the analysis. By 72 hr growth is no longer logarithmic, and the cells are not as responsive to EGF.
Phosphorylation of Lipocortin-1 by Epidermal Growth Factor Receptor The phosphorylation of lipocortin- 1 by the EGF receptor is particularly dramatic in cell-free systems using A431 cell membranes as a source of the kinase (see Fig. 1, lanes a-d). The reconstituted preparation provides a simple system for evaluating the various parameters affecting phosphorylation and for testing structural variants of the protein as substrates or inhibitors of the kinase. Whereas lipocortin was barely detected in the absence of EGF (Fig. 1, lane c), it was the major labeled band in the EGFtreated preparations (lane d, Fig. 1). Phosphate incorporation varied as a function of the concentration of added protein (lanes e-h, Fig. 1) and coincided with a decrease in phosphorylation of the receptor. When similar reactions were performed without added protein (lanes a and b, Fig. I), endogenous lipocortin-1 was phosphorylated in an EGF-dependent manner. The identity of this band as lipocortin was previously verified by peptide mapping. 9 EGF-dependent phosphorylation of lipocortin-I is a Ca2÷-dependent reaction. Since the membranes for most phosphorylation studies are isolated in the presence of EDTA, Ca 2÷ must be added to the reaction cocktails in order to obtain phosphorylated protein. However, since our protocol is run in the absence of EDTA, there is no Ca 2÷ requirement, relying on residual Ca 2÷ in the preparation to promote phosphorylation. While micromolar Ca 2÷ may be necessary for quantitative phosphorylations, Ca 2+ induces proteolytic clipping of the amino-terminal segment, and thus its addition can be problematic. Care should be taken in monitoring this competing reaction, since the phosphorylated tail region is particularly sensitive to Ca2+-dependent proteolysis.18
18 H. T. Haigler, D. D. Schlaepfer, and W. H. Burgess, J. Biol. Chem. 262, 6921 (1987).
266
EGF RECEPTORAND RELATEDRECEPTORS
[25]
Purification of Lipocortin-1 Most purification strategies used for lipocortin-like proteins rely on Ca 2÷ and/or phospholipid binding as an affinity step. 1H8-2° Typically, a particulate subcellular fraction composed either of membranes or detergent-insoluble complexes is produced in the presence of Ca 2+, washed, and the proteins of interest are selectively eluted from the insoluble fraction with EDTA. The Ca2+-binding proteins, which are enriched in the EDTA extract, are then fractionated by a combination of cation- and anion-exchange chromatography steps and gel filtration. Using such a strategy, we can recover about l0 mg of lipocortin-1 from a single human placenta. 6 A similar approach was developed for purifying the recombinant protein. 9 Of the tissues that express lipocortin-l, lung and placenta are particularly rich sources of the protein and thus frequently used for its purification. Since milligram quantities of lipocortin-I can be purified from natural sources, many groups have purified the protein; however, in few instances has the protein been analyzed biochemically. Lack of rigor in characterizing the final product, particularly with respect to the state of the amino terminus, can lead to erroneous conclusions that result from differences in protein preparations. Routine gel analysis, while useful for evaluating purity, generally is insensitive to minor sequence differences and is particularly inadequate for lipocortin-l, which is susceptible to proteolytic clipping at multiple sites near the amino terminus. While results from binding studies have correlated specific clipping patterns with changes in Ca 2÷binding affinities, 3 experiments comparing the effects of clipped adducts on phosphorylation have not been performed. In the one set of experiments where phosphorylated lipocortin was directly sequenced, the protein was found to be a des-12 variant, t2 In producing recombinant lipocortin, by chance we obtained a des-9 preparation of the protein. Although the truncated and full-length proteins both were phosphorylated in an EGF-dependent manner, the label in the des-9 protein was tyrosine-specific whereas the majority of the label in the full-length protein was on threonine. The nature of the serine(threonine) protein kinase is unknown but the finding suggests that a second kinase may be associated with the E G F receptor. 21 Because most lipocortin preparations are a mixture of full-length and clipped species, we decided 19p. j. Shadle, V. Gerke, and K. Weber, J. Biol. Chem. 260, 16354 (1985). 20C. E. Creutz, W. J. Zaks, H. C. Hamman, S. Crane, W. H. Martin, K. L. Gould, K. M. Oddie, and S. J. Parsons, J. Biol. Chem. 262, 1860(1987). 21M. Abdel-Ghany,H. K. Kole, M. A. Saad, and E. Racker, Proe. Natl. Acad. Sci. U.S.A. 86, 6072 (1989).
[25]
EGF-DEPENDENT PHOSPHORYLATION OF LIPOCORTIN
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to mix the full-length and des-9 protein and test the effect of the heterogeneous mixture on phosphorylation. Surprisingly, phosphorylation was almost exclusively on tyrosine, indicating that the clipped moiety was a better substrate for the receptor. Localization of Phosphorylation Site by Peptide Mapping To identify a specific site of interest within a protein, it is useful to have at hand structural tools that allow the molecule to be rapidly dissected into smaller and smaller segments. While construction of defined peptide maps can be laborious, once established they serve as a blueprint for the protein, which can be referred to repeatedly. For analyzing lipocortin-lspecific modifications we have developed a two-step process, first relying on CNBr mapping to define the specificity of the modification and to produce a crude ffactionation and then using tryptic digests to provide a more detailed analysis. This two-step process was particularly important when studying phosphorylated [ipocortin since only a small fraction of the protein was phosphorylated and the perturbation due to label altered the chromatographic mobility of the phosphorylated fragments. While a description of the construction of the maps is beyond the limits of this chapter, the details have been laid out previously 22'23 and applied specifically to lipocortin-1 to identify sites of phosphorylation by pp60C-~",24 insulin receptor kinase, 17protein kinases A and C, ~-4and to characterize a dimeric form of lipocortin-I which we had purified from placenta. 6
Cyanogen Bromide Mapping Figure 2 summarizes the CNBr mapping data. CNBr cleaves lipocortin[ into eight fragments with masses of 6.2, 7.8, 13.3, 4.2, 1.5, 0.9, l. 1, and 3.1 kDa from the amino to carboxy termini respectively. The positioning of the fragments with respect to the lipocortin-I sequence is shown at the top of the panel. Under limiting conditions, digestion with CNBr should produce 36 (28 partial and 8 complete) cleavage fragments, where partials represent fragments that contain 2 or more cleavage products. Of the 36 products, 23 are detected on silver-stained SDS gels (land a, Fig.2). Of the missing cleavage products, 10 are simply too small to be detected by SDS-PAGE, and the other 3 were undetected because they comigrate 22 R. B. Pepinsky, J. Biol. Chem. 258, 11229 (1983). 23 K-S. Huang, P. McGray, R. J. Mattaliano, C. Burne, E. P. Chow, L. K. Sinclair, and R. B. Pepinsky, J. Biol. Chem. 262, 7639 (1987). 24 L. Varticovski, S. B. Chahwala, M. Whitman, L. Cantley, D. Schindler, E. P. Chow, L. K. Sinclair, and R. B. Pepinsky, Biochemistry 27, 3682 (1988).
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[25]
EGF-DEPENDENT PHOSPHORYLATION OF LIPOCORTIN
269
with other partials. All of the observed cleavage products have been characterized by CNBr mapping, and thus the pattern of bands can be used to distinguish between each of the CNBr fragments. 24 The schematic shown at the bottom right of Fig. 2 summarizes the mapping results. Profiles representing the total digest (lane T, Fig. 2) and specific subsets of the digest (lanes 1-8, Fig. 2) are indicated. Each CNBr fragment is represented by a distinct subset of products, which in turn serves as a hallmark for its identification. Lane c (Fig. 2) shows CNBr mapping results for lipocortin that had been phosphorylated on tyrosine by the EGF receptor; six fragments were labeled. The pattern of labeled products indicates that CNBr fragment l is phosphorylated, since a modification in fragment l is the only type that would produce such a profile (compare lane c with lanes 1-8 in Fig. 2) and thus localizes the site of phosphorylation within the first 55 amino acids of lipocortin. The CNBr mapping data were confirmed by epitope mapping, using a CNBr fragment l-specific monoclonal antibody to directly monitor the fragment I-containing cleavage products. When cleavage products were subjected to Western blotting and probed with the fragment l-specific antibody (lane b, Fig. 2), the five fragment l-containing partials were detected. Except for fragment 1 itself, which is not detected by the antibody on Western blots, the profiles of the fragment l-containing cleavage products (lane b, Fig. 2) and phosphorylated fragments (lane c, Fig. 2) were identical.
Tryptic Peptide Mapping To localize the phosphorylation site further, lipocortin-1 was subjected to tryptic peptide mapping, using reversed-phase HPLC to separate cleavage products. Previously, all of the peaks from the lipocortin-1 tryptic map were sequenced, which covered approximately 85% of the primary structure, z3 Most of the missing fragments were small and eluted in the flow-through. Figure 3 summarizes the sequence data, where fragment
(Bottom) CNBr mapping results. Lanes a - c show lipocortin-I cleavage products that were visualized by silver staining, by Western blotting with CNBr fragment I-specific antiserum, and by autoradiography, respectively. Lanes 1-8 and T show schematic diagrams summarizing mapping data for lipocortin-i. Numbers at right reflect fragment compositions, where 1 refers to the N-terminal CNBr fragment and 8 to the C-terminal fragment. Lane T represents a profile of the total digest. Apparent molecular weights of specific fragments are indicated at left. [Portions reprinted with permission from L. Varticovski, S. B. Chahwala, M. Whitman, L. Cantley, D. Schindler, E. P. Chow, L. K. Sinclair, and R. B. Pepinsky, Biochemistry 27, 3682 (1988). Copyright © American Chemical Society.]
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[25]
EGF-DEPENDENT PHOSPHORYLATION OF LIPOCORTIN
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designations correspond to peak numbers listed in the tryptic map shown at the top. All of the tryptic fragments from CNBr fragment 1 have been identified, and thus the map can be used directly for characterizing fragment 1-specific modifications. When [32p]phosphate-labeled protein that had been phosphorylated by the EGF receptor was digested with trypsin and the labeled fragments analyzed by HPLC, the peptide map shown in the middle was obtained. Two major peaks at fractions 102 and 106 were observed. Although the fragments are shifted in their chromatographic mobility due to the added phosphate group, the pattern is consistent with phosphorylation within peptide 9-26, since this is the only peptide from within CNBr fragment 1 that would produce such a profile. Subsequent sequencing of the peptide verified its identity. The observed site, Tyr-21, is a canonical phosphorylation site that is rich in glutamic acids. Tyr-21 was confirmed as the only phosphorylation site for the EGF receptor by two independent approaches, first using site-directed mutagenesis to convert the tyrosine into phenylalanine and second using limited proteolysis with plasmin to release selectively the phosphorylated tail region from the remainder of the protein. 24 The amino-terminal domain of lipocortin-like proteins, by regulating Ca 2+- and phospholipid-binding affinities, provides a mechanism for increasing or decreasing the affinity of the proteins for membranes. Tyr-21 falls within this region and through its phosphorylation has created a pathway whereby EGF can impact function. Although in vitro studies have demonstrated that tyrosine-specific phosphorylation of lipocortin alters the Ca 2÷-/phospholipid-binding affinities and enhances the suscepti-
trifluoroacetic acid at !.4 ml/min (0.5-min fractions were collected). The column eluate was monitored simultaneously at 280 and 214 nm. For mapping phosphorylated fragments, 1 nmol of lipocortin-I was spiked with 400,000 counts/min (cpm) of labeled protein that had been gel purified and then processed as described above. Column fractions were analyzed for absorbance at 214 and 280 nm and by scintillation counting. (Top) Elution profile monitored at 214 nm. Numbered peaks were subjected to amino acid and sequence analysis. These results are summarized with respect to the lipocortin sequence shown below. (Middle) Profile of phosphorylated fragments (10 = 76,000 cpm). (Bottom) Summary of sequence data. Potential tryptic peptides are represented with boxes. Large numbers refer to peak numbers in the top graph. Peaks 1, 8, 10, and 26 produced multiple sequences. Sequences that were derived from partial cleavage products are indicated above the junction between boxes. In two instances indicated by dashed lines, partials were generated because of a trypsin-resistant Lys-Pro sequence. Several peaks contain structural variants of the amino-terminal fragment, consistent with the known amino-terminal heterogeneity of this particular batch of E. coliderived protein. [Peptide sequences reprinted with permission from K.-S. Huang, P. McGray, R. J. Mattaliano, C. Burne, E. P. Chow, L. K. Sinclair, and R. B. Pepinsky, J. BioL Chem. 262, 7639 (1987). Copyright © American Society for Biochemistry and Molecular Biology.]
272
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[26]
bility of the tail region to proteolysis, ~5'~8 the significance of the event awaits a clearer understanding of the physiological roles of the protein. The phosphorylation of lipocortin-I in intact cells as a tag for activation of the EGF receptor provides a simple method for monitoring the activation of the transduction pathway.
[26] C l o n i n g , E x p r e s s i o n , a n d B i o l o g i c a l E f f e c t s o f erbB-2/neu G e n e in M a m m a l i a n Cells B y PIER PAOLO D I FIORE, ORESTE SEGATTO, and STUART A . AARONSON
Independent Approaches Identifying e r b B - 2 / n e u
Oncogene
Human tumors often contain genes (referred to as oncogenes) able to confer a dominant transformed phenotype when transfected into murine fibroblasts. J The majority of oncogenes encode altered versions of one of three closely related members of the r a s gene family. I Oncogenes different from r a s have also been detected. I One, termed n e u , was first identified in rat neuroblastomas induced with the chemical carcinogen ethylnitrosourea (ENU). z Sera obtained from mice bearing tumors induced with neuroblastoma transfectants reacted with a 185-kDa phosphoprotein specifically induced by the neuroblastoma transforming sequence, 3 thus identifying the n e u oncogene as distinct from the r a s family. Further studies revealed that n e u was distinct from but related to the epidermal growth factor receptor (EGFR). 4 Independent efforts aimed at detection of human oncogenes were based on evidence that the EGFR gene was amplified in certain human tumors. Using a v - e r b B probe in moderate stringency conditions, King e t al. analyzed alterations affecting EGFR-related genes in a series of human mammary tumors? DNA prepared from a human mammary carcinoma, MAC 117, showed a pattern of hybridization differing from that observed i p. Kahn and T. Graf, (eds.), "Oncogenes and Growth Control." Springer-Verlag, New York, 1986. 2 C. Shih, L. C. Padhy, M. Murray, and R. A. Weinberg, Nature (London) 290, 261 (1981). 3 L. C. Padhy, C. Shih, D. Cowing, R. Finkelstein, and R. A. Weinberg, Cell (Cambridge, Mass.) 28, 865 (1982). 4 A. L. Schechter, D. F. Stern, L. Vaidyanathan, S. J. Decker, J. A. Drebin, M. I. Greene, and R. A. Weinberg, Nature (London) 312, 513 (1984). 5 C. R. King, M. H. Kraus, and S. A. Aaronson, Science 229, 974 (1985).
METHODS IN ENZYMOLOGY, VOL. 198
EGF
RECEPTOR
AND RELATED
RECEPTORS
[25]
[25] P h o s p h o r y l a t i o n o f L i p o c o r t i n - 1 b y t h e E p i d e r m a l Growth Factor Receptor By R. BLAKE PEPINSKY
Introduction The lipocortins are a recently characterized family of Ca 2+- and phospholipid-binding proteins which differ from conventional Ca2+-binding proteins in that they lack an EF-hand type Ca2+-binding loop and they inhibit phospholipase Az in in vitro assay systems. Eight related proteins have been identified to date, all sharing extensive sequence similarity (for references, see Refs. I and 2). Most conserved are a series of 70 amino acid repeat units that are organized into a core structure that binds Ca z+ and phospholipid. The 35- to 45-kDa proteins contain four copies of the repeat and the 70-kDa form, eight copies. Each protein also contains a short amino-terminal segment that is distinct from the core structure and unique to each protein. While the repeat units directly bind Ca z+ and phospholipid, the amino terminus regulates the binding affinities and thus may provide functional identity to the individual family members, s-5 Lipocortin-like proteins have been implicated in diverse processes, ranging from intracellular events where they affect membrane-cytoskeleton interactions and signal transduction to extracellular events where they affect inflammation, the immune response, blood coagulation, growth, and differentiation (for references, see Ref. 6). Of the eight proteins lipocortins-1 and -2 have been of particular interest as major substrates for oncogene and receptor protein kinases. In 1984, Fava and Cohen characterized a 39-kDa CaZ+-binding protein from A431 cells that was a substrate of the epidermal growth factor (EGF) receptor/kinase in vitro using receptor-rich cell membranes as a source of i R. B. Pepinsky, R. Tizard, R. J. Mattaliano, L. K. Sinclair, G. T. Miller, J. L. Browning, E. P. Chow, C. Burne, K.-S. Huang, D. Pratt, L. Wachter, C. Hession, A. Z. Frey, and B. P. Wallner, J. Biol. Chem. 263, 10799 (1988). M. J. Crumpton and J. R. Dedman, Nature (London) 345, 212 (1990). 3 y. Ando, S. Imamura, Y.-M. Hong, M. K. Owada, T. Kakunaga, and R. Kannag, J. Biol. Chem. 264, 6948 (1989). 4 D. S. Drust and C. E. Creutz, Nature (London) 331, 88 (1988). 5 j. Glenney and L. Zokas, Biochemistry 27, 2069 (1988). 6 R. B. Pepinsky, L. K. Sinclair, E. P. Chow, and B. Greco-O'brine, Biochem. J. 263, 97 (1989).
METHODS IN ENZYMOLOGY, VOL. 198
Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
[25]
EGF-DEPENDENT PHOSPHORYLATION OF LIPOCORTIN
261
the kinase. 7 The phosphorylation was EGF-dependent and occurred on tyrosine. Subsequent studies revealed that the 39-kDa protein was phosphorylated in intact cells and, excluding the receptor itself, represented the first known physiological substrate of the EGF receptor. 8 The extent of phosphorylation was growth cycle-dependent and accounted for as much as 25% of the protein. In studying lipocortin-like proteins, we observed that lipocortin-I also was a substrate of the EGF receptor and demonstrated that it and the 39-kDa band were the same protein. 9 Because we had already cloned and expressed lipocortin-1 ,~0 this finding placed us in an unusual position to evaluate the activities and properties of the protein. The following sections describe some of these studies, focusing on those that pertain to the phosphorylation of lipocortin by the EGF receptor and characterization of the phosphorylated product. Materials and Methods
Labeling and Immune Precipitation Procedures The A431 cell line, derived from a human epidermal carcinoma, is obtained from the American Type Culture Collection (CRL 1555, Rockville, MD). The cells are grown as monolayers at 36° in a tissue culture incubator in Dulbecco's modified Eagle's medium supplemented with 10% calf serum, 50 units/ml penicillin, and 50/zg/ml streptomycin. Cells are maintained in T75 flasks and trypsinized and passaged at a 1 : 5 split ratio every third day. Cells for metabolic labeling studies are split at the same ratio into 6-well culture dishes. After 48 hr, the growth medium is discarded and the wells washed twice with 2.5 ml serum-free medium that is deficient either in methionine or phosphate (Flow Laboratories, McLean, VA). Parallel cultures are labeled for 4 hr with 100 t~Ci/well [35S]methionine (900 Ci/mmol) or 1 mCi/well [32p]phosphate (1200 Ci/mol) in either the presence or absence of 200 ng/ml EGF. The labeling medium is discarded, and the cells are scraped into 1 ml of ice-cold lysis buffer (50 mM NaCI, 20 mM Tris-HC1, pH 7.3, 0.5% Nonidet P-40, 0.5% sodium deoxycholate, 4 mM iodoacetic acid, 1 mM ammonium vanadate). The lysates are subjected to vortex mixing for 30 sec and clarified in an Eppendorf centrifuge (5 min, 10,000 rpm, 4°). 7 R. A. Fava and S. Cohen, J. Biol. Chem. 259, 2636 (1984). 8 S. T. Sawyer and S. Cohen, J. BioL Chem. 260, 8233 (1985). 9 R. B. Pepinsky and L. K. Sinclair, Nature (London) 321, 81 (1986). 10 B. P. Wallner, R. J. Mattaliano, C. Hession, R. L. Cate, R. Tizard, L. K. Sinclair, C. Foeller, E. P. Chow, J. L. Browning, K. L. Ramachandran, and R. B. Pepinsky, Nature (London) 320, 77 (1986).
262
E G F RECEPTOR AND RELATED RECEPTORS
[25]
For immune precipitations the labeled lysates are split into two aliquots, one receiving 2 p,l of preimmune serum and the other receiving 2/,d of antilipocortin-1 antiserum (lipocortin antiserum is developed in rabbits using recombinant protein produced in Escherichia coli as immunogen; see Ref. 11). The samples are incubated on ice for 2 hr, and then the immune complexes are collected by adsorption to protein A-Sepharose (Sigma Chemical Company, St. Louis, MO) for 1 hr at 4 ° with continuous mixing (25 p~l of packed beads per sample). The adsorbed complexes are washed 4 times, each with 0.5 ml of lysis buffer, suspended in 50/zl of electrophoresis sample buffer [2% sodium dodecyl sulfate (SDS), 50 mM Tris-HCl, pH 6.8, 12.5% glycerol, 1.5% 2-mercaptoethanol, 0.1% bromophenol blue], and heated at 65 ° for 10 min. The protein A beads are pelleted in an Eppendorf centrifuge (1 min, 10,000 rpm, 4°). Supernatants are transferred to new tubes and stored at - 2 0 ° for subsequent analysis. Aliquots (10/zl) are subjected to SDS-PAGE in minigels (7 x 7 x 0.14 cm) using the Laemmli system. Separating gels contain 12.5% acrylamide, 0.1% methylene bisacrylamide and stacking gels, 7.6% acrylamide, 0.21% methylene bisacrylamide. Gels are dried under reduced pressure for 1.5 hr at 80° and radioactive bands visualized by autoradiography. Generally, an overnight exposure is needed to visualize the immune precipitates. In Vitro Phosphorylation Reactions
A431 cells are split at a 1 : 5 ratio into T75 flasks. After 48 hr the cells are washed with serum-free growth medium and incubated for 1 hr at 37 ° in 7 ml of the same medium without or with 200 ng/ml EGF, Monolayers are rinsed with 2.5 ml of ice-cold hypotonic buffer (10 mM HEPES, pH 7.5, 0.5 mM MgC1z) and the cells scraped into 1.5 ml of the same buffer. After 30 min on ice, the cells are lysed with a Dounce homogenizer (20 strokes, Type A pestle) and clarified of large debris by centrifugation for 5 min at 2000 g. Cell membranes are pelleted by centrifugation at 10,000 g for 10 rain and suspended in 300/xl of phosphorylation buffer (20 mM HEPES, pH 7.5, 2 mM MgCI 2, I0/zM ammonium vanadate) plus 2/~g/ ml EGF for EGF-treated samples. For in vitro phosphorylations, 60-/xl reactions, each containing 30 txl of the membrane fraction plus 30/~Ci of [T-3ZP]ATP and the indicated amount of lipocortin in 30 tzl of the same buffer, are held on ice for 10 min. Reactions are stopped by adding 20/zl of 5 x electrophoresis sample buffer. Samples are heated at 65 ° for l0 min, u K.-S. Huang, B. P. Wallner, R. J. Mattaliano, R. Tizard, C. Burne, A. Frey, C. Hession, P. McGray, L. Sinclair, E. P. Chow, J. L. Browning, K. L. Ramachandran, J. Tang, J. E. Smart, and R. B. Pepinsky, Cell (Cambridge, Mass.) 46, 191 (1986).
[25]
E G F - D E P E N D E N T PHOSPHORYLATION OF LIPOCORTIN
263
and 5-td aliquots are subjected to SDS gel analysis. Gels are dried under reduced pressure and exposed to X-ray film for 1 hr at 23 °. Epidermal Growth Factor-Dependent Phosphorylation of Lipocortin- 1 in A431 Cells The mitogenic or in some instances inhibitory effects of E G F on target cells are mediated through its receptor, a 170-kDa transmembrane protein with an extracellular E G F binding site and an intracellular tyrosine kinase domain. Of multiple immediate changes in target cells that are initiated on binding of E G F to the receptor, the earliest is the activation of the kinase, producing a cascade of phosphorylation reactions presumed to be crucial for transduction of the E G F signal. Two prominent phosphorylation products of the kinase are the receptor itself and lipocortin-1. 7,9'12 Recent immunohistochemical studies with lung tissue have revealed a striking correlation between the cellular distribution of lipocortin-I and the EGF receptor, supporting a role for the protein in receptor function.13 In A431 cells the E G F receptor is overproduced, TM leading to the frequent use of the cell line as a source of the kinase. A431 cells also produce large amounts of lipocortin-1 ( - 0 . 5 % of the total protein) and therefore provide a simple, accessible system for studying the phosphorylation of lipocortin-I by the E G F receptor. 7-9'15 Figure I shows results from two sets of experiments where EGF-dependent phosphorylation of lipocortin-1 was studied in intact cells (Fig. 1, lanes i-p) and in v i t r o using A431 cell membranes as a source of the kinase (Fig. 1, lanes a-h). To assess phosphorylation in intact cells, A431 cells or EGF-treated A431 cells are metabolically labeled with [32p]phosphate. Lipocortin-I is selectively precipitated from the lysates with lipocortin-l-specific antiserum and analyzed for [32p]phosphate by SDS-PAGE. From control cultures labeled with [35S]methionine, lipocortin-I is the only band detected by the gel analysis (Fig. 1, lanes i-I) with E G F having no effect on the labeling. In contrast, from [32p]phosphate-labeled cells (lanes m - p , Fig. 1), lipocortin- 1 is only observed in extracts from cells that are first treated with EGF. Typically, about a 5-fold increase in incorporation of phosphate into lipocortin-1 was observed in the EGF-treated samples. Phosphoamino acid 12B. K. De, K. S. Misono, T. J. Lukas, B. Mroczkowski,and S. Cohen, J. Biol. Chem. 261, 13784 (1986). 13M. D. Johnson, M. E. Gray, G. Carpenter, R. B. Pepinsky, H. Sundell, and M. T. Stahlman, Pediatr. Res. 25, 535 (1989). 14R. N. Fabricant, J. E. DeLarco, and G. J. Todaro, Proc. Natl. Acad. Sci. U.S.A. 74, 565 (1977). 15D. D. Schlaepfer and H. T. Haigler, J. Biol. Chem. 262, 6931 (1987).
264
EGF RECEPTORAND RELATEDRECEPTORS a b c d
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FIG. 1. EGF-dependent phosphorylation of lipocortin-l. In vitro phosphorylation reactions using isolated A431 cell membranes (lanes a-h) and immune precipitations from metabolically labeled cells ([35Slmethionine, lanes i-l; [3Zp]phosphate, lanes m-p) were analyzed by SDS-PAGE and radioactive products visualized by autoradiography. Arrowheads denote the positions of the EGF receptor. (170 kDa) and of lipocortin-1 (39 kDa). Specific variables are indicated (Pre, preimmune serum; lm, immune serum). Lanes c and d each received 2 t~g lipocortin. Lanes e-h show phosphorylationresults obtained with membranes from EGFtreated cells that were incubated with 0.2, 2, 20, and 200/zg/ml of lipocortin, respectively. [Reprinted with permission from R. B. Pepinsky and L. K. Sinclair, Nature (London) 321, 81 (1989). Copyright © Macmillan Magazines Ltd.]
analysis revealed that the increase in label was predominantly on tyrosine. A series of bands were detected in the samples from phosphate-labeled cells, but of these only lipocortin-1 and an 18K band presumed to be a fragment of lipocortin showed E G F - d e p e n d e n t phosphorylation. While some o f the other proteins may be immunologically related to lipocortin,6' 16 many probably result from nonspecific binding to the protein A-Sepharose beads, since the appearance of the precipitations in subsequent analysis was improved by including SDS in the lysis buffer. 17Perhaps the cleanest results to date were generated by S a w y e r and Cohen, who partially purified the phosphorylated lipocortin prior to the immune precipitation step. 8 They obtained a 15-fold increase in labeled lipocortin from EGF-treated A431 cells. Phosphate incorporation increased steadily over the labeling 16A. Karasik, R. B. Pepinsky, and R. C. Kahn, J. Biol. Chem. 263, 11862(1988). 17A. Karasik, R. B. Pepinsky, S. E. Shoelson, and R. C. Kahn, J. Biol. Chem. 263, 18558 (1988).
[25]
EGF-DEPENDENT PHOSPHORYLATION OF LIPOCORTIN
265
period (4 hr), indicating that the phosphorylated adduct was relatively long lived. In working with A431 cells, we routinely use cells at around 48 hr after plating. At this time EGF induces a dramatic morphologic change, marked by a rapid movement of the cells from a confluent monolayer into a weblike network where the cells aggregate to form the cables of the web. If the cells are used too early, namely, within the first 24 hr after plating, the EGF treatment causes them to round up and detach, thus being lost to the analysis. By 72 hr growth is no longer logarithmic, and the cells are not as responsive to EGF.
Phosphorylation of Lipocortin-1 by Epidermal Growth Factor Receptor The phosphorylation of lipocortin- 1 by the EGF receptor is particularly dramatic in cell-free systems using A431 cell membranes as a source of the kinase (see Fig. 1, lanes a-d). The reconstituted preparation provides a simple system for evaluating the various parameters affecting phosphorylation and for testing structural variants of the protein as substrates or inhibitors of the kinase. Whereas lipocortin was barely detected in the absence of EGF (Fig. 1, lane c), it was the major labeled band in the EGFtreated preparations (lane d, Fig. 1). Phosphate incorporation varied as a function of the concentration of added protein (lanes e-h, Fig. 1) and coincided with a decrease in phosphorylation of the receptor. When similar reactions were performed without added protein (lanes a and b, Fig. I), endogenous lipocortin-1 was phosphorylated in an EGF-dependent manner. The identity of this band as lipocortin was previously verified by peptide mapping. 9 EGF-dependent phosphorylation of lipocortin-I is a Ca2÷-dependent reaction. Since the membranes for most phosphorylation studies are isolated in the presence of EDTA, Ca 2÷ must be added to the reaction cocktails in order to obtain phosphorylated protein. However, since our protocol is run in the absence of EDTA, there is no Ca 2÷ requirement, relying on residual Ca 2÷ in the preparation to promote phosphorylation. While micromolar Ca 2÷ may be necessary for quantitative phosphorylations, Ca 2+ induces proteolytic clipping of the amino-terminal segment, and thus its addition can be problematic. Care should be taken in monitoring this competing reaction, since the phosphorylated tail region is particularly sensitive to Ca2+-dependent proteolysis.18
18 H. T. Haigler, D. D. Schlaepfer, and W. H. Burgess, J. Biol. Chem. 262, 6921 (1987).
266
EGF RECEPTORAND RELATEDRECEPTORS
[25]
Purification of Lipocortin-1 Most purification strategies used for lipocortin-like proteins rely on Ca 2÷ and/or phospholipid binding as an affinity step. 1H8-2° Typically, a particulate subcellular fraction composed either of membranes or detergent-insoluble complexes is produced in the presence of Ca 2+, washed, and the proteins of interest are selectively eluted from the insoluble fraction with EDTA. The Ca2+-binding proteins, which are enriched in the EDTA extract, are then fractionated by a combination of cation- and anion-exchange chromatography steps and gel filtration. Using such a strategy, we can recover about l0 mg of lipocortin-1 from a single human placenta. 6 A similar approach was developed for purifying the recombinant protein. 9 Of the tissues that express lipocortin-l, lung and placenta are particularly rich sources of the protein and thus frequently used for its purification. Since milligram quantities of lipocortin-I can be purified from natural sources, many groups have purified the protein; however, in few instances has the protein been analyzed biochemically. Lack of rigor in characterizing the final product, particularly with respect to the state of the amino terminus, can lead to erroneous conclusions that result from differences in protein preparations. Routine gel analysis, while useful for evaluating purity, generally is insensitive to minor sequence differences and is particularly inadequate for lipocortin-l, which is susceptible to proteolytic clipping at multiple sites near the amino terminus. While results from binding studies have correlated specific clipping patterns with changes in Ca 2÷binding affinities, 3 experiments comparing the effects of clipped adducts on phosphorylation have not been performed. In the one set of experiments where phosphorylated lipocortin was directly sequenced, the protein was found to be a des-12 variant, t2 In producing recombinant lipocortin, by chance we obtained a des-9 preparation of the protein. Although the truncated and full-length proteins both were phosphorylated in an EGF-dependent manner, the label in the des-9 protein was tyrosine-specific whereas the majority of the label in the full-length protein was on threonine. The nature of the serine(threonine) protein kinase is unknown but the finding suggests that a second kinase may be associated with the E G F receptor. 21 Because most lipocortin preparations are a mixture of full-length and clipped species, we decided 19p. j. Shadle, V. Gerke, and K. Weber, J. Biol. Chem. 260, 16354 (1985). 20C. E. Creutz, W. J. Zaks, H. C. Hamman, S. Crane, W. H. Martin, K. L. Gould, K. M. Oddie, and S. J. Parsons, J. Biol. Chem. 262, 1860(1987). 21M. Abdel-Ghany,H. K. Kole, M. A. Saad, and E. Racker, Proe. Natl. Acad. Sci. U.S.A. 86, 6072 (1989).
[25]
EGF-DEPENDENT PHOSPHORYLATION OF LIPOCORTIN
267
to mix the full-length and des-9 protein and test the effect of the heterogeneous mixture on phosphorylation. Surprisingly, phosphorylation was almost exclusively on tyrosine, indicating that the clipped moiety was a better substrate for the receptor. Localization of Phosphorylation Site by Peptide Mapping To identify a specific site of interest within a protein, it is useful to have at hand structural tools that allow the molecule to be rapidly dissected into smaller and smaller segments. While construction of defined peptide maps can be laborious, once established they serve as a blueprint for the protein, which can be referred to repeatedly. For analyzing lipocortin-lspecific modifications we have developed a two-step process, first relying on CNBr mapping to define the specificity of the modification and to produce a crude ffactionation and then using tryptic digests to provide a more detailed analysis. This two-step process was particularly important when studying phosphorylated [ipocortin since only a small fraction of the protein was phosphorylated and the perturbation due to label altered the chromatographic mobility of the phosphorylated fragments. While a description of the construction of the maps is beyond the limits of this chapter, the details have been laid out previously 22'23 and applied specifically to lipocortin-1 to identify sites of phosphorylation by pp60C-~",24 insulin receptor kinase, 17protein kinases A and C, ~-4and to characterize a dimeric form of lipocortin-I which we had purified from placenta. 6
Cyanogen Bromide Mapping Figure 2 summarizes the CNBr mapping data. CNBr cleaves lipocortin[ into eight fragments with masses of 6.2, 7.8, 13.3, 4.2, 1.5, 0.9, l. 1, and 3.1 kDa from the amino to carboxy termini respectively. The positioning of the fragments with respect to the lipocortin-I sequence is shown at the top of the panel. Under limiting conditions, digestion with CNBr should produce 36 (28 partial and 8 complete) cleavage fragments, where partials represent fragments that contain 2 or more cleavage products. Of the 36 products, 23 are detected on silver-stained SDS gels (land a, Fig.2). Of the missing cleavage products, 10 are simply too small to be detected by SDS-PAGE, and the other 3 were undetected because they comigrate 22 R. B. Pepinsky, J. Biol. Chem. 258, 11229 (1983). 23 K-S. Huang, P. McGray, R. J. Mattaliano, C. Burne, E. P. Chow, L. K. Sinclair, and R. B. Pepinsky, J. Biol. Chem. 262, 7639 (1987). 24 L. Varticovski, S. B. Chahwala, M. Whitman, L. Cantley, D. Schindler, E. P. Chow, L. K. Sinclair, and R. B. Pepinsky, Biochemistry 27, 3682 (1988).
268 1
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[25]
EGF-DEPENDENT PHOSPHORYLATION OF LIPOCORTIN
269
with other partials. All of the observed cleavage products have been characterized by CNBr mapping, and thus the pattern of bands can be used to distinguish between each of the CNBr fragments. 24 The schematic shown at the bottom right of Fig. 2 summarizes the mapping results. Profiles representing the total digest (lane T, Fig. 2) and specific subsets of the digest (lanes 1-8, Fig. 2) are indicated. Each CNBr fragment is represented by a distinct subset of products, which in turn serves as a hallmark for its identification. Lane c (Fig. 2) shows CNBr mapping results for lipocortin that had been phosphorylated on tyrosine by the EGF receptor; six fragments were labeled. The pattern of labeled products indicates that CNBr fragment l is phosphorylated, since a modification in fragment l is the only type that would produce such a profile (compare lane c with lanes 1-8 in Fig. 2) and thus localizes the site of phosphorylation within the first 55 amino acids of lipocortin. The CNBr mapping data were confirmed by epitope mapping, using a CNBr fragment l-specific monoclonal antibody to directly monitor the fragment I-containing cleavage products. When cleavage products were subjected to Western blotting and probed with the fragment l-specific antibody (lane b, Fig. 2), the five fragment l-containing partials were detected. Except for fragment 1 itself, which is not detected by the antibody on Western blots, the profiles of the fragment l-containing cleavage products (lane b, Fig. 2) and phosphorylated fragments (lane c, Fig. 2) were identical.
Tryptic Peptide Mapping To localize the phosphorylation site further, lipocortin-1 was subjected to tryptic peptide mapping, using reversed-phase HPLC to separate cleavage products. Previously, all of the peaks from the lipocortin-1 tryptic map were sequenced, which covered approximately 85% of the primary structure, z3 Most of the missing fragments were small and eluted in the flow-through. Figure 3 summarizes the sequence data, where fragment
(Bottom) CNBr mapping results. Lanes a - c show lipocortin-I cleavage products that were visualized by silver staining, by Western blotting with CNBr fragment I-specific antiserum, and by autoradiography, respectively. Lanes 1-8 and T show schematic diagrams summarizing mapping data for lipocortin-i. Numbers at right reflect fragment compositions, where 1 refers to the N-terminal CNBr fragment and 8 to the C-terminal fragment. Lane T represents a profile of the total digest. Apparent molecular weights of specific fragments are indicated at left. [Portions reprinted with permission from L. Varticovski, S. B. Chahwala, M. Whitman, L. Cantley, D. Schindler, E. P. Chow, L. K. Sinclair, and R. B. Pepinsky, Biochemistry 27, 3682 (1988). Copyright © American Chemical Society.]
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[25]
EGF-DEPENDENT PHOSPHORYLATION OF LIPOCORTIN
271
designations correspond to peak numbers listed in the tryptic map shown at the top. All of the tryptic fragments from CNBr fragment 1 have been identified, and thus the map can be used directly for characterizing fragment 1-specific modifications. When [32p]phosphate-labeled protein that had been phosphorylated by the EGF receptor was digested with trypsin and the labeled fragments analyzed by HPLC, the peptide map shown in the middle was obtained. Two major peaks at fractions 102 and 106 were observed. Although the fragments are shifted in their chromatographic mobility due to the added phosphate group, the pattern is consistent with phosphorylation within peptide 9-26, since this is the only peptide from within CNBr fragment 1 that would produce such a profile. Subsequent sequencing of the peptide verified its identity. The observed site, Tyr-21, is a canonical phosphorylation site that is rich in glutamic acids. Tyr-21 was confirmed as the only phosphorylation site for the EGF receptor by two independent approaches, first using site-directed mutagenesis to convert the tyrosine into phenylalanine and second using limited proteolysis with plasmin to release selectively the phosphorylated tail region from the remainder of the protein. 24 The amino-terminal domain of lipocortin-like proteins, by regulating Ca 2+- and phospholipid-binding affinities, provides a mechanism for increasing or decreasing the affinity of the proteins for membranes. Tyr-21 falls within this region and through its phosphorylation has created a pathway whereby EGF can impact function. Although in vitro studies have demonstrated that tyrosine-specific phosphorylation of lipocortin alters the Ca 2÷-/phospholipid-binding affinities and enhances the suscepti-
trifluoroacetic acid at !.4 ml/min (0.5-min fractions were collected). The column eluate was monitored simultaneously at 280 and 214 nm. For mapping phosphorylated fragments, 1 nmol of lipocortin-I was spiked with 400,000 counts/min (cpm) of labeled protein that had been gel purified and then processed as described above. Column fractions were analyzed for absorbance at 214 and 280 nm and by scintillation counting. (Top) Elution profile monitored at 214 nm. Numbered peaks were subjected to amino acid and sequence analysis. These results are summarized with respect to the lipocortin sequence shown below. (Middle) Profile of phosphorylated fragments (10 = 76,000 cpm). (Bottom) Summary of sequence data. Potential tryptic peptides are represented with boxes. Large numbers refer to peak numbers in the top graph. Peaks 1, 8, 10, and 26 produced multiple sequences. Sequences that were derived from partial cleavage products are indicated above the junction between boxes. In two instances indicated by dashed lines, partials were generated because of a trypsin-resistant Lys-Pro sequence. Several peaks contain structural variants of the amino-terminal fragment, consistent with the known amino-terminal heterogeneity of this particular batch of E. coliderived protein. [Peptide sequences reprinted with permission from K.-S. Huang, P. McGray, R. J. Mattaliano, C. Burne, E. P. Chow, L. K. Sinclair, and R. B. Pepinsky, J. BioL Chem. 262, 7639 (1987). Copyright © American Society for Biochemistry and Molecular Biology.]
272
EGF
RECEPTOR AND RELATED RECEPTORS
[26]
bility of the tail region to proteolysis, ~5'~8 the significance of the event awaits a clearer understanding of the physiological roles of the protein. The phosphorylation of lipocortin-I in intact cells as a tag for activation of the EGF receptor provides a simple method for monitoring the activation of the transduction pathway.
[26] C l o n i n g , E x p r e s s i o n , a n d B i o l o g i c a l E f f e c t s o f erbB-2/neu G e n e in M a m m a l i a n Cells B y PIER PAOLO D I FIORE, ORESTE SEGATTO, and STUART A . AARONSON
Independent Approaches Identifying e r b B - 2 / n e u
Oncogene
Human tumors often contain genes (referred to as oncogenes) able to confer a dominant transformed phenotype when transfected into murine fibroblasts. J The majority of oncogenes encode altered versions of one of three closely related members of the r a s gene family. I Oncogenes different from r a s have also been detected. I One, termed n e u , was first identified in rat neuroblastomas induced with the chemical carcinogen ethylnitrosourea (ENU). z Sera obtained from mice bearing tumors induced with neuroblastoma transfectants reacted with a 185-kDa phosphoprotein specifically induced by the neuroblastoma transforming sequence, 3 thus identifying the n e u oncogene as distinct from the r a s family. Further studies revealed that n e u was distinct from but related to the epidermal growth factor receptor (EGFR). 4 Independent efforts aimed at detection of human oncogenes were based on evidence that the EGFR gene was amplified in certain human tumors. Using a v - e r b B probe in moderate stringency conditions, King e t al. analyzed alterations affecting EGFR-related genes in a series of human mammary tumors? DNA prepared from a human mammary carcinoma, MAC 117, showed a pattern of hybridization differing from that observed i p. Kahn and T. Graf, (eds.), "Oncogenes and Growth Control." Springer-Verlag, New York, 1986. 2 C. Shih, L. C. Padhy, M. Murray, and R. A. Weinberg, Nature (London) 290, 261 (1981). 3 L. C. Padhy, C. Shih, D. Cowing, R. Finkelstein, and R. A. Weinberg, Cell (Cambridge, Mass.) 28, 865 (1982). 4 A. L. Schechter, D. F. Stern, L. Vaidyanathan, S. J. Decker, J. A. Drebin, M. I. Greene, and R. A. Weinberg, Nature (London) 312, 513 (1984). 5 C. R. King, M. H. Kraus, and S. A. Aaronson, Science 229, 974 (1985).
METHODS IN ENZYMOLOGY, VOL. 198
