ARCHIVES

OF BIOCHEMISTRY

AND

BIOPHYSICS

Vol. 292, No. 1, January, pp. 273-280, 1992

Effect of Carboxyi Terminal Truncation on the Tyrosine Kinase Activity of the Epidermal Growth Factor Receptor’ Philip B. Wedegaertner”

and Gordon N. Gill?*’

*Department of Chemistry and TDepartment of Medicine, 9500 Gilman Drive, La Jolla, California 92093-0650

University

of California

at San Diego,

Received September 6, 1991

The carboxyl terminal domain of the epidermal growth factor receptor (EGFR) is an important regulatory region in mediating the tyrosine kinase-dependent biological effects of EGF. The effect of a 164-amino-acid carboxyl deletion of the EGFR or the EGFR cytoplasmic kinase domain on in vitro tyrosine kinase activity was assessed. C’-terminal truncation of the EGFR resulted in dependence on Mn2+ for full activity. The EGFR kinase domain (kd EGFR) and the C’-terminally truncated kinase domain (kd ~‘1022 EGFR) also exhibited a strong preference for Mn2+ compared to Mg’+, with kd ~‘1022 EGFR being completely inactive in the presence of Mg2+ alone. Sphingosine or ammonium sulfate specifically activated both kd EGFR and kd ~‘1022 EGFR. EGFR and ~‘1022 EGFR displayed similar EGF-stimulated in vitro tyrosine kinase activities; however, kd EGFR was 5- to lo-fold more active in vitro than kd ~‘1022 EGFR. Thus, the regulatory contribution of the C’-terminus is most evident when the EGFR ligand binding domain is removed. These results indicate that an intact EGFR C’-terminus is necessary for the protein to assume a fully active conformation. @ 1992 Academic Press, Ihc.

Ligand binding to the epidermal growth factor receptor (EGFR)’ activates its intrinsic protein tyrosine kinase ‘These studies were supported by NIH Grant DK 13149 and the Lucille P. Markey Charitable Trust. P.B.W. was supported by NIH Training Grant 2T32 CA 09523. 2 Abbreviations used: EGFR, epidermal growth factor receptor; ~‘1022 EGFR, EGFR C-terminally truncated at residue 1022; kd EGFR, kinase domain of EGFR, kd ~‘1022 EGFR, kinase domain of the EGFR c’terminally truncated at residue 1022; EGTA, ethylene glycol bis(&aminoethyl ether) N,N’-tetraacetic acid; EDTA, ethylenediaminetetraacetate; Hepes, 4-(2.hydroxyethyl)-l-piperazineethanesulfonic acid, SDSPAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; AII, angiotensin II; IgG, immunoglobulin G; FPLC, fast protein liquid chromatography. 0003.9861/92

$3.00

Copyright 0 1992 by Academic Press, [nc. All rights of reproduction in any form Ireserved.

activity, resulting in self-phosphorylation and phosphorylation of numerous cellular proteins. The tyrosine kinase activity is required to mediate all known biological effects of EGF (1, 2). Although binding of EGF to the receptor is the dominant regulatory mechanism for controlling the tyrosine kinase activity, other mechanisms to modulate EGFR function exist. The large, approximately 230-amino-acid, carboxyl terminus of the EGFR has been implicated as an important regulatory region. This domain, located distal to the tyrosine kinase domain, contains all identified sites of tyrosine autophosphorylation (3-5) and several sites of heterologous serine phosphorylation (6). Tyrosine-phosphorylated regions of the EGFR serve as binding sites for src homology 2 domain substrate proteins, such as phospholipase C-y and the GTPase-activating protein, GAP (7). The (Y-terminus also contains sequences mediating ligand-induced endocytosis (8), further demonstrating the biological importance of this domain. Analyses of the function of the (Y-terminus of the EGFR using C-terminal mutations and deletions have yielded conflicting results. Progressive deletions from the C’-terminus led to progressive decreases in biological activity as measured by transforming ability (9-11). The furthest truncation of 123 amino acids almost abolished transforming ability, and a 2040% reduction in tyrosine phosphorylation of exogenous substrates was observed using immunoprecipitated receptors (11). Changing the three most C’-terminal autophosphorylation sites to phenylalanine also decreased biological activity. In contrast, others have shown that removal of 202 amino acids from the C’terminus of the EGF receptor enhanced fibroblast transformation (12), and various C’-terminal deletions increased in vivo EGF-dependent tyrosine kinase activity and mitogenesis (5,8, 13). The C’-terminus of the EGFR is not strictly required for biological activity because C’-terminal truncation does not abolish the mitogenic ac273

274

WEDEGAERTNER

tion of EGF (13,14). When the extracellular ligand binding domain of the EGF receptor is deleted, similar to v-erbB, C-terminal deletions enhance transforming ability (10, 12). Studies of C’-terminal sequence removal from the homologous c-erbB-2 protein have also yielded conflicting results. DiFiore et al. report greatly reduced transforming activity (15), whereas Akiyama et al. report that C’-terminal truncation of c-erbB-2 increased transforming activity (16). C’-terminal deletion combined with an N-terminal truncation is reported to enhance transformation (17). Kinetic evidence indicates that the autophosphorylation sites act as alternative substrates competing with exogenous substrates as a means of regulating tyrosine kinase activity (18). Such substrate motifs are a common regulatory feature of many members of the protein kinase gene family (19). Autophosphorylation is proposed to remove an inhibitory constraint, thereby increasing EGFR tyrosine kinase activity (18, 20); however, other studies have reported that autophosphorylation has little effect on EGF receptor activity in vitro (21-23). Because the role of the C-terminus of the EGFR and related proteins remains uncertain, the present studies were undertaken to directly assess the effect of removal of C’terminal sequences on EGFR and EGFR cytoplasmic domain in vitro tyrosine kinase activities. The C’-terminally truncated cytoplasmic domain provides a less complex protein to analyze, allowing the separation of ligand binding activating effects from C-terminal regulatory domain effects on tyrosine kinase activity. EXPERIMENTAL

PROCEDURES

Reagents. Restriction enzymes, T4 DNA ligase, and XmaI DNA linkers were purchased from New England Biolabs. The Klenow fragment of DNA polymerase I and T4 polynucleotide kinase were purchased from Stratagene. [y-32P)ATP was supplied by ICN. Excel1 400 Sf9 insect cell media were purchased from JRH Biosciences. Other reagents were of the highest grade available and were from Sigma. Sf9 insect cells, AcNPV, and pAc360 were generously provided by Dr. Max Summers, Texas A&M University. AcNPV DNA was isolated and provided by Dr. Deborah Cadena, University of California at San Diego. A peptide (RRKGSTAENAEYLRV) corresponding to the major EGFR selfphosphorylation site at residue 1173 (1173 peptide) was synthesized at the UCSD Peptide Synthesis Facility. Oligonucleotide synthesis was performed on an Applied Biosystems 380B DNA synthesizer. Antiphosphotyrosine monoclonal antibody PY-20 coupled to Affigel beads (Bio-Rad) was the generous gift of Dr. John Glenney, University of Kentucky. N-Acetyl sphingosine was the gift of Dr. Robert Bell, Duke University. The Construction and isolation of Ac360X-KD and Ac360X-KD1022. pAc360 baculovirus vector was modified to pAc360X by changing the BamHI cloning site to XmaI. pAc360 was digested with BamHI endonuclease and treated with the Klenow fragment of DNA polymerase I to yield blunt ends. SmaI linkers (5’.CCCCGGGG-3’) were ligated to the vector and digested with XmaI following standard procedures (24). Ligation of the plasmid created pAc360X, which was confirmed by DNA sequencing using the oligonucleotide (B-CAATATATAGTTGCTGATATCATGGAG-3’). Digestion of pAc401-NH (25) with XmaI allowed the isolation of a 1.9-kilobase cDNA fragment corresponding to the

AND

GILL

EGFR cytoplasmic domain. This XmaI fragment was ligated into pAc360X to create pAc360X-KD. The vector pXT1022 (8) was digested with NarI and Hind111 endonucleases to isolate a l.l-kilobase cDNA fragment coding for a C’-terminal truncated cytoplasmic domain. Bluntend formation by the Klenow fragment of DNA polymerase I was followed by ligation of SmaI linkers (5’.CCCCGGGG-3’). This fragment was digested with XmaI and cloned into the XmaI site of pAc360X to create pAc360X-KD1022. DNA sequencing confirmed that the correct translational reading frame was maintained. Homologous recombination in St9 cells after contransfection of AcNPV DNA and pAc360X-KD or pAc360X.KD1022 provided recombinant baculovirus. Three rounds of visual screening and dot-blot hybridization (26) allowed identification and purification of Ac360X-KD and Ac360X-KD1022. of holo EGFR, cl1022 EGFR, kd EGFR, and kd cl1022 Purification EGFR. Ho10 EGFR protein was purified from A431 cells, and cl1022 EGFR protein was purified from B82 cells using immunoaffinity chromatography on 528 IgG-Sepharose with competitive elution with EGF, as described previously (5, 27). This provided a highly purified preparation of ligand-activated receptors. kd EGFR and kd ~‘1022 EGFR were purified using a modification of a previously described procedure (25). Sf9 cells (2 X 10s cells/ml) were cultured in Excel1 400 serum-free media in l-liter spinner flasks containing 500 ml of cell suspension. The cells were infected with Ac360XKD or Ac360X-KD1022 at a multiplicity of infection of 10. At 65-72 h postinfection, the infected cells were harvested by centrifugation at 1OOOg at 4°C for 10 min and resuspended in l/10 original cell suspension volume of 50 mM Hepes, pH 7.4, 10 mM NaCl, 4 mM EGTA, 4 mM benzamidine, 2 lg/ml leupeptin, 2 *g/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride, 1% glycerol, 1% Triton X-100, 10 FM ammonium molybdate, and 100 ).LMNalVO,. Homogenization (10 strokes of a glass/ glass Dounce homogenizer) was followed by centrifugation at 100,OOOg for 1 h. The supernatant was collected, and kd EGFR or kd ~‘1022 EGFR was maximally autophosphorylated by addition of 10 mM MnClz and 1 mM ATP. After 30 min at O”C, 5 mM EDTA was added, and the cytosol was loaded onto an anti-phosphotyrosine monoclonal antibody column (PY-20 Affigel) at a flow rate of 50 ml/h and recycled three times. The column was washed with greater than 10 column volumes each of buffers containing 10 mM Hepes, pH 7.4, 100 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% glycerol, and 1% Triton X-100, followed by 10 mM Hepes, pH 7.4, 50 mM NaCl, and 1% glycerol. Protein was competitively eluted by addition of 50 mM phenylphosphate in 10 mM Hepes, pH 7.4,50 mM NaCl, and 1% glycerol. Phenylphosphate was subsequently removed by extensive dialysis. To further purify kd ~‘1022 EGFR, the phenylphosphate elution was dialyzed into 10 mM Tris-HCl, pH 7.4, 25 mM NaCl, and 1% glycerol, and loaded onto a Mono Q FPLC column (HR 5/5 Pharmacia). Proteins were eluted with an increasing NaCl gradient, and kd ~‘1022 EGFR was eluted at 200 mM NaCI. Fractions exhibiting tyrosine kinase activity were pooled and 1 mg/ml bovine serum albumin was added to maintain activity. kd EGFR was quantitated by the method of Bradford (28). kd ~‘1022 EGFR was quantitated by Bradford analysis and by densitometric measurement of silver staining of varying amounts of protein on SDS-PAGE relative to known amounts of kd EGFR.

Tyrosine kinase assay. Phosphate incorporation into the various peptide substrates was determined using the phosphocellulose paper binding assay (18, 25). Assay conditions are described in the figure legends. Sphingosine stimulation was measured using 5 and 10 PM sphingosine for kd EGFR and kd ~‘1022 EGFR, respectively. Ammonium sulfate stimulation of substrate phosphorylation was assayed with 1 and 1.5 M ammonium sulfate added to kd EGFR and kd ~‘1022 EGFR, respectively. Kinetic data were analyzed in terms of Michaelis-Menten enzyme kinetics using the Enzfitter program (Elsevier-BIOSOFT). Each kinetic data point was performed in duplicate, and each value presented represents the average for three experiments. Immunoblotting. Samples were separated by 9.0% SDS-PAGE and electrotransferred to Immobilon-P membranes (Millipore). The membranes were placed in a blocking solution of PBS/0.05% Tween 20 and 2.5% bovine serum albumin for 1 h at 22°C. Phosphotyrosine-containing

CARBOXYL TRUNCATION OF THE EPIDERMAL GROWTH FACTOR RECEPTOR proteins were detected by incubation with iz51-labeled PY-20 monoclonal anti-phosphotyrosine antibody ((29) for 2 h at 22”C, followed by washing with PBS/O.OS% Tween 20, drying of the membrane, and autoradiography. The proteins kd EGFR and kd ~‘1022 EGFR were detected by incubation with the EGFR cytoplasmic domain specific polyclonal antibody 1964 for 2 h at 22’C. After washing with PBS/O.O5% Tween 20, the membrane was incubated with goat anti-rabbit IgG conjugated with alkaline phosphatase (Promega) for 1 h at 22°C. After further washing with PBS/0.05% Tween 20, imlmunoreactive bands were visualized by addition of nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate. Polyclonal rabbit antibody 19864was prepared using a bacterially produced EGFR cytoplasmic domain. Production of the EGFR cytoplasmic domain in Escherichia coli using the T7 expression vector PET-& (30) resulted in insoluble protein. The insoluble EGFR cytoplasmic domain pH 8.0, was purified by centrifugation, washed with 50 mM Tris-HCl, 1 mM EDTA, 1% Triton X-100, 1% deoxycholate, and 1 mM urea, and solubilized in 50 mM Tris-HCl, pH 8.0, 1 mM EDTA, and 0.2% SDS. The protein was injected into rabbits, and the serum was collected by Bethyl Laboratories, Inc. (Montgomery, TX).

RESULTS

Kinetic comparison of ho10 EGF receptor and cl1022 EGF receptor. To compare in vitro tyrosine kinase activity, holo EGF receptor and an EGFR G-terminally truncated at amino acid 1022 were purified to homogeneity using immunoaffinity chromatography (5,27). A kinetic analysis of phosphorylation of two peptide substrates, angiotensin II (AII) and a 15-amino-acid peptide representing the major site of EGFR autophosphorylation, was performed. Because the receptors were purified by elution from the affinity column with EGF, the enzymes were saturated with ligand. AI1 was phosphorylated by holo EGFR with a V,,, of 1.38 mol of phosphate incorporated min-’ mall’ EGFR and a K, of 489 PM, while a I’,,,, of 0.862 mol of phosphate incorporated min-’ molll ~‘1022 EGFR and a K, of 875 PM was obtained using ~‘1022 EGFR (Fig. 1A). The V,,, and K, for phosphorylation of the 1173 peptide by holo EGFR were 2.77 mol phosphate incorporated min-’ mall 1 EGFR and 63.4 PM, respectively, and 2.17 mol phosphate incorporated min-’ mall’ ~‘1022 EGFR and 51.5 PM, respectively, using ~‘1022 EGFR (Fig. 1B). The holo EGFR and ~‘1022 EGFR were thus similar with respect to phosphorylation of the two peptide substrates, with the holo EGFR exhibiting slightly higher activity. The K,,, of AI1 for phosphorylation by ~‘1022 EGFR was 1.8-fold higher than that for phosphorylation by holo EGFR, but the K,s for phosphorylation of the better substrate, 1173 peptide, were similar. The observed enhanced in vivo tyrosine kinase activity of cl022 EGFR compared to holo EGFR (5,8) is not retained when purified receptors are analyzed in vitro. Further in vitro analysis revealed a significant difference in the divalent meta. ion requirement of the two forms of EGF receptor. ThLe holo EGFR is fully active in the presence of either Mn2+ or Mg2+ with Mn2+ the more potent of the two (Fig. 2A), as previously reported (31). In contrast, the ~‘1022 EGFR displayed a strong preference for Mn*+. When assayed with Mg2+ concentrations

275

up to 50 mM, ~‘1022 EGFR displayed tyrosine kinase activity of less than 40% of that observed with 1 mM Mn*’ (Fig. 2B). C!‘-terminal truncation thus renders the enzyme Mn*+ dependent for full activity. Baculovirus expression and purification of kd EGFR and kd ~‘1022 EGFR. To further analyze the effect of C’terminal deletion on EGFR tyrosine kinase activity the cytoplasmic protein tyrosine kinase domain of the EGFR and a C’-terminal truncation of the cytoplasmic kinase domain were studied, allowing physical separation of enzymatic activity from ligand binding regulatory activity. For this study, expression in Sf9 insect cells and subsequent purification of a modified kinase domain was enhanced compared to a previous report (25), allowing recovery of 5 mg purified kd EGFR/liter of Sf9 cells. In addition, an intracellular kinase domain truncated at amino acid 1022 of the human EGFR sequence was produced. A modified pAc360 baculovirus expression vector (26) (pAc360X) was used to express the two proteins. pAc360X-KD and pAc360X-KD1022 encoded for fusion proteins containing the sequence NH,-MPDYSYRPTIGPDPPG followed by EGFR sequences 647-1186 (kd EGFR) and 647-1022 (kd ~‘1022 EGFR), respectively. Expression of kd EGFR and kd ~‘1022 EGFR in Sf9 cells, infected with recombinant virus Ac360X-KD or Ac360X-KD1022, was detected by immunoblotting of total cellular proteins. A monoclonal anti-phosphotyrosine antibody revealed similar levels of phosphotyrosine in kd EGFR (Fig. 3A, lane 2) and kd ~‘1022 EGFR (Figure 3A, lane 3). The kinase domain specific polyclonal antibody 1964 detected similar levels of the two proteins (Fig. 3A, lanes 5 and 6) in Sf9 cells. Although some degradation of kd EGFR exists in a whole cell lysate (Fig. 3A, lane 5), purification removes the proteolytic fragments (Fig. 3B, lane 7). No immunoreactive protein was detected in uninfected cells (Fig. 3A, lanes 1 and 4). The observation of more than one band corresponding to kd EGFR or kd ~‘1022 on the kinase domain specific immunoblot (Fig. 3A, lanes 5 and 6) and the anti-phosphotyrosine immunoblot (Fig. 3A, lanes 2 and 3) indicates that both proteins are incompletely autophosphorylated in uiuo. Four of the five identified sites of EGFR autophosphorylation are deleted in kd ~‘1022 EGFR, including tyrosine 1173, the major in uiuo site (3-5). This suggested in uiuo protein tyrosine kinase activity of kd ~‘1022 EGFR was equivalent to or greater than the in uivo activity of kd EGFR. To more accurately compare kd EGFR and kd ~‘1022 EGFR an analysis of purified proteins was necessary. Purification of the two proteins utilized absorption of autophosphorylated protein to a monoclonal anti-phosphotyrosine antibody column followed by competitive elution with phenylphosphate. Column adsorption was preceded by incubation of the cytosol fraction with ATP to fully autophosphorylate kd EGFR and kd ~‘1022 EGFR. This resulted in a highly pure preparation of kd EGFR (Fig.

276

WEDEGAERTNER

A c E b k “; 6 E -i .g E a 3 6 E 7 -

12.0 lO.O-8.0 --

/

/

6.0 --

,

/

/

/

A

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4.0 -2.0~0.0, -4

I -2

0

2

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4

8

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2.5-

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1.0 0.5 -0.01 -20

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30

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1/[1173 peptide] mM-l

~/[AII~ mM_l

FIG. 1. Kinetic analysis of purified holo EGFR and ~‘1022 EGFR. (A) Purified holo EGFR (0.31 pmol) (A) or ~‘1022 EGFR (0.38 pmol) (A) was incubated for 10 min at 22°C with 37.5 mM Hepes, pH 7.4,5 mM MgCl,, 2 mM MnCl*, 10 PM (y-s’P]ATP (2 X lo4 cpm/pmol), 50 @M Na3V04, and the indicated concentrations of angiotensin II in a reaction volume of 40 ~1. (B) Ho10 EGFR (0.31 pmol) (A) or ~‘1022 EGFR (0.38 pmol) (A) was incubated under the conditions described above and the indicated concentrations of 1173 peptide. The incorporation of 32P into each peptide was determined using the phosphocellulose paper binding assay. The data are presented as double-reciprocal plots of reaction velocity versus substrate concentration. Similar results were obtained in three different experiments, and the V,,,., and K, values presented in the text are the average of three experiments.

3B, lane 7). Anti-phosphotyrosine antibody column purified kd ~‘1022 EGFR was subjected to a further purification step of FPLC Mono Q chromatography (Fig. 3B, lane 8). The identity of the two purified proteins was confirmed by immunoblotting (Fig. 3B, lanes 9 and 10). Antiphosphotyrosine purified and Mono Q purified kd cl1022 showed identical activity per mole of kd ~‘1022, and both preparations were used in subsequent experiments. kd EGFR has an apparent molecular weight of 67,000 on SDS-PAGE, and kd ~‘1022 EGFR migrates with an apparent molecular weight of 46,000. Kinetic comparison of kd EGFR and kd ~‘1022 EGFR. The kinetic parameters, K,,, and V,,,, were de-

termined for ATP and three peptide substrates, AI1 (ERVYIHPF), a peptide corresponding to tyrosine 416 in p60”‘” (src peptide, RRLIEDAEYAARG), and 1173 peptide (RRKGSTAENAEYLRV) (Table I). K, values for each substrate tested were similar when kd ~‘1022 EGFR was compared to kd EGFR. The K, values for peptide substrates for both forms of the kinase domain were larger than those for the corresponding EGFRs. A comparison of VIn,, values revealed much lower tyrosine kinase activity for kd ~‘1022 EGFR than for kd EGFR. kd ~‘1022 EGFR V,,, values were consistently 3- to lo-fold lower, depending on the peptide substrate. Although in vivo observations of self-phosphorylation (Fig. 3A) suggested

c ~'1022 EGFR

\ c

ao

5 Q

60

a ; x

4o 20

0 I 0

5

10 [Me2+]

15 mM

20

25

10

20

30

[Me2+]

40

50

mM

FIG. 2. Divalent metal ion requirements of holo EGFR and ~‘1022 EGFR. (A) Purified holo EGFR (0.31 pmol) was incubated for 10 min at 22’C with 37.5 mM Hepes, pH 7.4, 10 PM [y-32P]ATP (1.8 X 10’ cpm/pmol), 50 PM NasVO,, 2 mM AH, and the indicated concentrations of Mn*+ (0) or Mg’+ (0) in a reaction volume of 40 ~1. The incorporation of 32P into AI1 was measured as described previously. (B) ~‘1022 EGFR (0.38 pmol) was assayed as described above using the indicated concentrations of Mn2+ (0) or Mg ‘+ (0). A value of 100% was given to the highest measured activity in each experiment. Each point is the average of duplicate points, and similar results were obtained in three separate experiments for each receptor.

CARBOXYL

TRUNCATION

OF THE

EPIDERMAL

GROWTH

FACTOR

TABLE (Mr x 10-3) 97 -

kd ~‘1022 EGFR

66-

46-

45-

*-

Substrate

V max

Kl

30-

mol phosphate min-’ mall’ kd ~‘1022 EGFR

mM

29 7 123

I

Kinetic Constants

(Mrx 10-3) 11697 /-

69-

277

RECEPTOR

‘a

456

FIG. 3. Detection in St9 cells and purification of kd EGFR and kd ~‘1022 EGFR. (A) Uninfected (lanes 1 and 4), Ac360X-KD-infected (lanes 2 and 5), or Ac360X-KD1022-infected (lanes 3 and 6) St9 cells (2.5 X 105) were solubilized with Laemmli sample buffer at 65 h postinfection. The samples (2.5% of total) were resolved by SDS-PAGE (9.0%) and electrotransferredto Immobilon. Phosphotyrosine-containing proteins were detected with ‘ZSI-labeled anti-phosphotyrosine monoclonal antibody PY-20 (lanes l-3). An autoradiogram (15 h exposure at -7O’C with intensifying screen) is shown. kd EGFR and kd ~‘1022 EGFR proteins were detected by incubation with 1964 antisera followed by goat anti-rabbit IgG conjugated with alkaline phosphatase (lanes 4-6). (B) Purified kd EGFR (3.8 rg) (lane 7) and purified kd ~‘1022 EGFR (1.8 gg) (lane 8) were resolved by 9.0% SDS-PAGE and stained with Coomassie blue. Similar aliquots of kd EGFR (50 ng) (lane 9) and kd ~‘1022 EGFR (50 ng) (lane 10) were subjected to 9.0% SDS-PAGE, electrotransferred to Immobilon, immunoblotted with 1964 antisera, and visualized as described.

equivalent or enhanced activity of kd ~‘1022 EGFR relative to kd EGFR, removal of the C’-terminus resulted in decreased kd ~‘1022 EGF:R catalyzed phosphorylation of peptide substrates in uitro relative to kd EGFR. Interestingly, the 1173 peptide is the best substrate, in terms of vmax, for both kd ~‘1022 EGFR and kd EGFR. The specific activity of kd EGFR toward 1173 peptide is 5fold higher than that for AI1 or the src peptide. Using the 1173 peptide substrate, the V,,, of kd EGFR is 1.09 mol phosphate mini’ malll enzyme compared to 2.77 mol phosphate mini’ molll enzyme for the holo EGFR. This close similarity contrasts with the 5- to lo-fold greater activity of the holo EGFR using other peptide substrates (25). Effecters of kd cl1022 EGFR and kd EGFR tyrosine kinase actiuity. kd EGFR displays a strong preference for Mn2+ over Mg 2+to satisfy the divalent metal ion requirement (25), and kd ~‘1022 exhibits a similar metal ion profile (Fig. 4). For both proteins, 10 mM Mn2+ is the optimal metal ion concentration :for substrate phosphorylation. kd ~‘1022 EGFR displays even less ability to use Mg2+. Whereas almost 20% of the maximal kd EGFR activity is obtained with 10 mM Mg’+, no more than 5% of the maximal kd ~‘1022 EGFR activity is detected with 10 mM activity obI%‘+, similar to the lack of Mg2+-stimulated served with a 42-kDa tyrosine kinase domain produced by proteolytic cleavage of EGFR (32). Increasing Mg2+ to 80 mM showed the same low level of kd ~‘1022 EGFR activity (data not shown).

1173 peptide src peptide Angiotensin II ATP

0.408 0.360 1.32 0.00962

f f 2 f

0.086 0.12 0.54 0.00083

0.141 0.0228 0.0417 0.07960

I 0.04 + 0.012 + 0.018 3~0.011

kd EGFR Substrate

V max

Kit

mol phosphate mini kd EGFR

mM

1173 peptide src peptide Angiotensin II ATP

0.436 0.25 1.8 0.0112

’ Values determined

+ 0.16 310.1” k 0.05” * 0.0019”

previously

1.09 0.21 0.17 0.19

mall’

f 0.025 f 0.06” ?z 0.09” f 0.01”

(25).

Sphingosine and ammonium sulfate are specific activators of the kinase domain (25). The effect of these activating molecules on kd ~‘1022 EGFR substrate phosphorylation was examined and compared to kd EGFR

lOO--

.

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--

/

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bid+

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_ o-

- - -o-

h4g2+ - - - - - - _ _ _ _ _

-0 0

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10

15

[Me2+]

20

# 25

mM

FIG. 4. Divalent metal ion requirements of kd EGFR and kd ~‘1022 EGFR. Purified kd ~‘1022 EGFR (1.6 pmol) was incubated for 10 min at 22°C with 37.5 mM Hepes, pH 7.4, 10 FM [y-a*P]ATP (3 X lo4 cpm/ pmol), 50 FM Na,VO,, 2 mM AII, and the indicated concentrations of Mn2+ (m) or Mg2+ (0) in a reaction volume of 40 ~1. Purified kd EGFR (0.33 pmol) was assayed under similar conditions using the indicated concentrations of Mn2+ (0) or Mg*+ (0). The incorporation of “P into AI1 was measured and a value of 100% was given to the activity measured at 10 mM Mnzf. This equaled 0.36 and 0.033 mol phosphate mini mol-’ enzyme for kd EGFR and kd ~‘1022 EGFR, respectively. Each point is the mean of duplicate points, and similar results were obtained in three separate experiments for each kinase.

278

WEDEGAERTNER

AND

GILL

B E L

l

z : 4 > g Ik ? a

: 0 0 s ‘; cr E ‘; .c E

P 0

5

10 15 CONCENTRATION

20 (194)

25

0.25

l-

0 control [ZZI + sphingosine

0.20

t 0.15

0.10

0.05

.5

src peptide

I1

73

peptide

FIG. 5. Effect of sphingosine on kd cl022 EGFR activity. (A) Purified kd ~‘1022 EGFR (1.6 pmol) was incubated for 10 min at 22°C with 37.5 mM Hepes, pH 7.4, 10 mM MnCl,, 20 pM [y-s*P]ATP (1.7 X lo4 cpm/pmoI), 50 pM Na3V04, 2 mM AII, and the indicated concentrations of sphingosine and N-acetyl sphingosine in a reaction volume of 40 pl. Each point is the mean of duplicate points, and similar results were obtained in two separate experiments. (B) Purified kd ~‘1022 EGFR (1.6 pmol) was incubated for 10 min at 22°C with 37.5 mM Hepes, pH 7.4, 10 mM MnCl,, 20 pM [y-32P]ATP (1.4 X lo4 cpm/pmoI), 50 pM Na3V04, with or without 10 pM sphingosine, and 2 mM AII, 0.5 mM src peptide, or 1 mM 1173 peptide in a reaction volume of 40 ~1. The incorporation of 32P into each peptide was determined as previously described.

catalyzed phosphorylation. Sphingosine stimulated the phosphorylation of AI1 by kd cl1022 EGFR greater than fivefold at an optimal concentration of 10 PM (Fig. 5A). The addition of N-acetyl sphingosine provided no enhancement of kd ~‘1022 EGFR activity. kd ~‘1022 EGFR phosphorylation of three different peptide substrates was stimulated by 10 PM sphingosine (Fig. 5B). The substrate with the highest rate of unstimulated kd ~‘1022 EGFR phosphorylation, 1173 peptide, was the least affected by sphingosine, showing less than twofold stimulation. Sphingosine activation of kd ~‘1022 EGFR substrate phosphorylation was directly compared to activation of kd EGFR substrate phosphorylation (Table II). The phosphorylation of AI1 and src peptide was strongly stimulated by sphingosine in the case of both kd ~‘1022 EGFR

and kd EGFR. Phosphorylation of 1173 peptide by kd ~‘1022 EGFR was increased by sphingosine; however, the phosphorylation of 1173 peptide by kd EGFR was slightly inhibited by the addition of sphingosine. High concentrations of ammonium sulfate, previously shown to increase the V,,, for EGF receptor substrate phosphorylation without affecting the K,,, (33), also affected the activity of kd ~‘1022 EGFR (Table III). At an optimal concentration of 1.5 M, ammonium sulfate activated AI1 phosphorylation 12.5-fold for kd ~'1022 EGFR. Ammonium sulfate activated kd EGFR to a similar extent. Interestingly, the phosphorylation of src peptide was greatly inhibited by ammonium sulfate in both cases. Phosphorylation of 1173 peptide by kd ~‘1022 EGFR or kd EGFR was stimulated approximately 2-fold. TABLE

TABLE

II

Sphingosine Activation of kd ~‘1022 EGFR and kd EGFR

III

Ammonium Sulfate Activation of kd ~‘1022 EGFR and kd EGFR”

-Fold activation of substrate phosphorylation*

-Fold Activation of substrate phosphorylationb

Substrate

kd EGFR

kd ~‘1022 EGFR

Substrate

kd EGFR

kd ~‘1022 EGFR

Angiotensin II src peptide 1173 peptide

4.9 + 0.1 5.4 f 0.1 0.88 + 0.07

5.1 +_0.1 5.6 zk 0.2 1.8 + 0.3

Angiotensin II src peptide 1173 peptide

10.0 + 0.4 0.27 31 0.03 1.8 + 0.2

12.5 + 1.2 0.13 + 0.02 2.2 + 0.3

a Purified kd EGFR (1.6 pmol) or kd ~‘1022 EGFR (1.6 pmol) was assayed under standard conditions with 20 @M [y-s*P]ATP (l-2 X lo* cpm/pmol), without or with sphingosine, using 2 mM AII, 0.5 mM arc peptide, or 1 mM 1173 peptide as the substrate. * The -fold activation is the reaction velocity in the presence of sphingosine divided by the reaction velocity in the absence of sphingosine. Each value is the mean of duplicate measurements + range. Similar results were obtained in three separate experiments.

’ Purified kd EGFR (1.6 pmol) or kd ~‘1022 EGFR (1.6 pmol) was assayed under standard conditions with 20 ELM [y-32P]ATP (l-2 X lo4 cpm/pmol), without or with ammonium sulfate, using 2 mM AII, 0.5 mM src peptide, or 1 mM 1173 peptide as the substrate. * The -fold activation is the reaction velocity in the presence of ammonium sulfate divided by the reaction velocity in the absence of ammonium sulfate. Each value is the mean of duplicate measurements + range. Similar results were obtained in two separate experiments.

CARBOXYL

TRUNCATION

OF THE

EPIDERMAL

DISCUSSION This report demonstrates that (Y-terminal truncation of 164 amino acids from the EGF receptor or its cytoplasmic tyrosine kinase domain does not result in enhanced in vitro tyrosine kinase activity of the purified proteins, as measured by phosphorylation of peptide substrates. Instead, a significant reduction in activity with (?-terminal deletion is observed, especially comparing the cytoplasmic kinase domain, kd EGFR, to the (Y-terminal deletion, kd ~‘1022 EGFR. Although the decreased in vitro activity does not correlate with the observed in viva tyrosine kinase activity (5,8) (Fig. 3A), these results support the proposition that the C’-terminus is a positive regulator of EGF receptor activity (9, 11). Phosphorylation of the (Y-terminus may be necessary for the EGFR to assume a fully active conformation. This is especially evident when the extracellular domain is deleted, no longer allowing EGF to fully activate the tyrosine kinase. Thus, loss of the ligand binding domain combined with loss of most of the autophosphorylation domain creates a protein unable to assume a completely a.ctive conformation. This suggestion does not exclude a role for the C’-terminus as a negative inhibitory region. It is possible that a deletion larger than the 164 amino acid truncation studied is necessary to completely remove an inhibitory domain, as previously suggested (5). The ~‘1022 EGFR and kd ~‘1022 EGFR contain a site of autophosphorylation. Compared to autophosphorylation of five sites in the complete C’terminus, phosphorylation of only a single site may be insufficient to induce complete removal of inhibitory sequences and full kinase activation. The finding of Helin et al. (11) that mutation of three tyrosine sites of autophosphorylation reduced a.ctivity is compatible with this hypothesis. Analysis of greater deletions to a tyrosine kinase conserved core will provide a further understanding of the C’-terminal domain contribution to EGFR activity. Additionally, the EGFFl C’-terminal domain may be important in maintaining the conformational integrity of the protein. Removal of portions of the (Y-terminus may lead to protein instability, reflected in the lower relative activity of ~‘1022 EGFR and kd ~‘1022 EGFR. This instability may be overcome in uivo through interactions between the C-terminal deleted EGF receptors and other proteins/factors. Support for this hypothesis is provided by the recently solved crystal structure of the catalytic subunit of the CAMP-dependent protein kinase (34). The region C/-terminal to the conserved kinase core of the CAMP-dependent protein kinase apparently not only functions as an important structural element, but also plays a role in substrate recognition. Thus, experimentally observed effects of EGF receptor C’-terminal truncation on activity may be due to C’-terminal truncation effects on EGF receptor conformational stability and substrate recognition.

GROWTH

FACTOR

RECEPTOR

279

~‘1022 EGFR, kd EGFR, and kd ~‘1022 EGFR showed a striking preference for Mn2+ compared to the holo EGFR which utilized either Mn2+ or Mg2+. This difference between ~‘1022 EGFR and holo EGFR may reflect altered in uiuo responses to intracellular metal ion concentrations leading to differences in observed in vivo tyrosine kinase activity. The preference for Mn2+ became more pronounced coincident with more extensive truncation of the EGFR. Removal of the extracellular ligand binding domain and 164 amino acids at the (Y-terminus, as with kd ~‘1022 EGFR, virtually abolished any Mg2+-dependent tyrosine kinase activity. A 42-kDa tyrosine kinase domain produced by proteolytic cleavage of the EGFR showed a similar lack of activity in the presence of Mg2+ (32). Studies with the 42-kDa kinase domain suggested a distinct Mn2+ binding site based on the requirement for millimolar concentration of Mn2+ and enhanced modification of reactive cysteines in the presence of Mn2+. Koland and Cerione (33) report that Mn2+ can fully activate the holo EGFR in the absence of EGF and propose that Mn2+ binding to the tyrosine kinase domain causes an activating conformational change. The results presented here are consistent with this hypothesis. The insulin receptor and the insulin receptor kinase domain also show a strong preference for Mn2+ over Mg2+ (31, 35), with Mn2+ inducing a conformational change in the cytoplasmic domain as measured by circular dichroism (36). Since Mn2+ is only present in cells in the micromolar range, Wente et al. (36) suggested that Mn2+ activation of the insulin receptor tyrosine kinase may be less important in uiuo, but may mimic the effect of a physiological activator. A similar proposal for the EGF receptor is inferred, although much tighter Mn2+ binding in viva is not excluded. Ammonium sulfate and sphingosine, previously identified as EGFR tyrosine kinase activators (33, 37), were both able to activate kd ~‘1022 EGFR, indicating that the 164 amino acids at the C’-terminus are not necessary to mediate the effect of these activators. However, sphingosine- or ammonium sulfate-activated kd ~‘1022 EGFR remained significantly less active than activated kd EGFR. The substrate that was phosphorylated with the highest reaction velocity, the 1173 peptide, was the least affected by sphingosine or ammonium sulfate addition. The effects of ammonium sulfate depended on the peptide substrate used. Ammonium sulfate greatly inhibited the phosphorylation of src peptide by kd EGFR or kd ~‘1022 EGFR, in agreement with studies showing ammonium sulfate inhibition of src peptide phosphorylation by EGFR (38). Ammonium sulfate also inhibited the phosphorylation of a random copolymer of glutamate, alanine, and tyrosine by the proteolytically cleaved 42-kDa tyrosine kinase domain (32). Ammonium sulfate addition to the EGFR changes the nature of ADP inhibition of tyrosine kinase activity from competitive to noncompetitive with respect to the peptide substrate AII, indicating ammonium

280

WEDEGAERTNER

sulfate induces conformation changes in the active site of the EGFR (38). Sphingosine-mediated activation of the EGFR tyrosine kinase, and its effects on other signal transducing kinases, such as protein kinase C and src kinase inhibition (39, 40), may represent a physiological role for sphingosine and/or related sphingolipids as second messengers (41). In summary, we present an analysis of the effect of removal of 164 amino acids at the (Y-terminus of the EGF receptor on in vitro tyrosine kinase activity. Comparison of holo EGFR to ~‘1022 EGFR revealed similar in vitro tyrosine kinase activity. However, when the ligand binding extracellular domain was removed, kd ~‘1022 EGFR displayed significantly reduced in vitro tyrosine kinase activity compared to kd EGFR, implicating the C’-terminus as an important regulatory domain essential for optimal catalytic function. ACKNOWLEDGMENTS The authors thank Mr. Gordon M. Walton for purification and initial characterization of ~‘1022 EGFR and for helpful advice and discussion, and Dr. John Glenney for the generous gift of the PY-20 antibody affinity column.

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