THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1984 by The American Society of Biological Chemists, Inc.

Vol. 259, No. 15, Issue of August 10, pp. 9913-9921, 1984 Printed in U.S.A.

A Comparison of the Insulin- and Epidermal Growth Factor-stimulated Protein Kinases from Human Placenta*

(Received for publication, November 18, 1983)

Linda J. Pike, Elizabeth A. Kuenzel, John E. Casnellie, and Edwin G. Krebs From the Howard Hughes Medical Institute, University of Washington, Department of Pharmacology, Seattle, Washington 98195

The characteristics of the insulin- and epidermal growth factor (EGF)-stimulated tyrosine-specific pro- tein kinases in a wheat germ lectin-Sepharose-purified preparation of solubilized placenta membranes were compared. The specific activity of the insulin-stimu- lated kinase in this preparation was 72 nmol/min/mg whereas the specific activity of the EGF-stimulated kinase was 312 nmol/min/mg using a synthetic peptide as the phosphorylatable substrate. The two enzymes showed similar divalent metal ion requirements and nucleotide specificities. In addition, both kinases were inhibited by treatment with N-ethylmaleimide. How- ever, the EGF-stimulated enzyme was more sensitive to modification by this reagent than was the insulin- stimulated kinase. When examined for their ability to utilize a number of different proteins as substrates, the insulin- and EGF-stimulated kinases exhibited similar but not identical substrate specificities. These similar- ities at the molecular level may be the basis of the similarity between the actions of insulin and EGF at the cellular level.

A number of polypeptide growth factors, including insulin, EGF’, and platelet-derived growth factor, stimulate similar physiological responses including phosphatidylinositol turn- over, amino acid uptake, and hexose transport (Refs. 1-4; for review, see Refs. 5-7). Recently, it has been shown that each of these hormones stimulates the phosphorylation of proteins on tyrosine residues (8-16), suggesting that they work through a common molecular mechanism. Data from a number of different laboratories indicate that the growth factor receptors themselves are probably the tyrosine-specific protein kinases and that the activity of these kinases is increased as a result of hormone binding to the receptor (17-23).

It was of interest to know whether the similarity in the cellular response to the various growth factors was a reflection of similar characteristics of their receptor kinases. To examine this question, the substrate specificities of the insulin- and EGF-stimulated tyrosine kinases from a single source, human placenta, were compared. Avruch et al. (24) have previously compared the ability of insulin and EGF to stimulate endog- enous protein phosphorylation in detergent extracts of pla- centa membranes. In the present report, the phosphorylation of exogenous protein and peptide substrates by these two

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The abbreviations used are: EGF, epidermal growth factor; SDS, sodium dodecyl sulfate; HEPES, 4-(2-hydroxyethyl)-l-piperazine- ethanesulfonic acid; ATPrS, adenosine 5’-O-(thiotriphosphate).

growth factor-stimulated kinases are compared using wheat germ lectin-Sepharose-purified preparations of solubilized re- ceptors from placenta membranes. The characteristics of the two enzymes were found to be similar but not identical.

EXPERIMENTAL PROCEDURES

Materials-Casein, histone fi, histone fzb, insulin, phosphoserine, and phosphothreonine were from Sigma. Chicken gizzard smooth muscle light chains and tropomyosin were the gift of Dr. Arthur Edelman (University of Washington). Troponin I was provided by Dr. Edmond Fischer (University of Washington). The peptide with the sequence Arg-Arg-Leu-Ile-Glu-Asp-Ala-Glu-Tyr-Ala-Ala-Arg- Gly, which will be referred to as “synthetic peptide” throughout this study, was synthesized as described previously (25). EGF was pur- chased from Collaborative Research. Proinsulin was the gift of E. R. Chance (Eli Lilly). Wheat germ lectin-Sepharose was from Pharma- cia. ‘%I-Insulin was from the United States Public Health Service Diabetes Center, Seattle, WA and ‘%I-EGF was from New England Nuclear. Anti-EGF receptor antibodies were the generous gift of Dr. Stanley Cohen (Vanderbilt University). Anti-insulin receptor anti- bodies were the gift of Dr. c. Ron Kahn (Joslin Diabetes Center, Harvard Medical School).

tially as described by Siege1 et al. (26). Placentas were obtained within Membranes-Human placental membranes were prepared essen-

1 h of delivery. All procedures were carried out at 4 “C. The supporting membranes were removed and the tissue was washed extensively with 10 mM Tris, pH 7.4, 150 mM NaCl. The washed tissue was then homogenized three times for 15 s in a Waring blender in 2 volumes of 0.25 M sucrose, 25 mM benzamidine, 5 mM EDTA, 0.2 mM phen- ylmethanesulfonyl fluoride. The homogenate was filtered through one layer of cheesecloth and then centrifuged for 30 min at 10,000 X g. The supernatants were collected and made up to 100 mM NaCl and 0.2 mM MgClP by addition of concentrated solutions of these reagents. The homogenate was then centrifuged for 60 min at 30,000 X g. The pellets were collected and washed by homogenization in 50 ml of 40 mM imidazole, pH 7.2,0.2 M NaCl. The homogenate was then centri- fuged at 40,000 X g for 30 min. The pellets were resuspended in 30 ml of 40 mM imidazole, pH 7.2, 0.2 M NaCl, separated into 5-ml aliquots, and stored at -70 “C until use.

Solubilization and Purification of the Insulin- and EGF-stimulnted Protein Kimes-Placental membranes were solubilized by addition of Triton X-100 to a final concentration of 2% followed by stirring on ice for 45 min. The material was then centrifuged for 30 min at 100,000 X g. The supernatant was mixed with 30 ml of wheat germ lectin-Sepharose and rotated end-over-end for 2 h at 4 “C. The resin was then poured into a column and washed with 100 ml of 40 mM imidazole, 0.5 M NaCl, 10% glycerol, 0.05% Triton X-100, pH 7.2. The column was eluted with the same buffer containing 0.3 M N - acetylglucosamine. Four-ml fractions were collected and assayed for insulin- and EGF-stimulated kinase activity using the synthetic pep- tide as substrate. The two activities co-eluted from the lectin column, Fractions containing activity were pooled and stored frozen at -70 “C.

Assays-Phosphorylation assays were carried out in a total volume of 18 pl. A 6 - ~ 1 aliquot of the diluted wheat germ lectin-purified preparation containing 150 ng of protein was added as the source of enzyme. Other assay constituents were (in final concentrations): 20 mM imidazole, pH 7.2,85 mM NaCl, 50 mM N-acetylglucosamine, 5% glycerol, 0.05% Triton X-100, 200 p M [Y-~’P]ATP, 12 mM MgCl,, 2 mM MnC12, 20 mM p-nitrophenyl phosphate, 100 FM sodium vana-

9913

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9914 Insulin- and EGF-stimulated Protein Phosphorylation date, 2 mM dithiothreitol. The standard synthetic peptide phospho- rylation assay contained 2 mM peptide. The protein substrates were added at varying concentrations. When present, insulin and EGF were used at 30 and 500 nM, respectively. The growth factors and the kinase preparation were preincubated for 15 min at room temperature prior to addition of the substrates. The reactions were initiated by the addition of [y3'P]ATP and continued for 5 min at 30 'C during which time they were essentially linear. For assay of the peptide substrate, the reaction was stopped by addition of 30 pl of 5% trichloroacetic acid. An aliquot of the reaction mixture was spotted on a square of phosphocellulose paper and the papers were washed three times for 2 min in 75 mM H3POa (27). The papers were then dried and counted for 32P. Phosphorylation of chicken gizzard smooth muscle light chains and histones was measured in the same manner with the exception that the reactions were stopped by addition of 30% acetic acid. For assay of all other exogenous protein substrates, the reactions were stopped by addition of an SDS-containing buffer followed by boiling for 3 min (28). The samples were run on 12% polyacrylamide gels and stained with Coomassie blue. The wet gels were autoradiographed to locate the phosphorylated proteins and the appropriate bands were excised from the gel and counted for 32P. For all the data shown, with the exception of Table 11, the insulin- or EGF-stimulated phosphorylation was calculated by subtracting the amount of phosphorylation of the substrate occurring in the absence of growth factor from the phosphorylation observed in the presence of insulin or EGF.

Phosphorylation of proteins in the wheat germ lectin-purified preparation was carried out using the same assay conditions described above with the exception that no exogenous substrates were added. The reactions were terminated by addition of an SDS-containing buffer followed by boiling. The samples were run on a 9% polyacryl- amide gel, stained with Coomassie blue to locate the molecular weight standards, and then dried and autoradiographed.

Other Methods-SDS-polyacrylamide gels were run as described by Laemmli (28). Proteolysis gels were performed according to the method of Cleveland et al. (29). For this, the phosphorylated protein bands from a 12% polyacrylamide gel were cut out and placed in the wells of a 4% stacking gel over an 18% running gel. Fifty microliters of a 20 pg/ml solution of Staphylococcus aureus V8 protease in SDS- containing sample buffer was added and the gel was run at 5 mA for -16 h. The gel was then autoradiographed following a 1-h incubation in 10% trichloroacetic acid.

The binding of lZ5I-insulin and lZ51-EGF to placenta membranes was measured essentially as described by Carpenter et al. (30).

Phosphoamino acid analyses were performed as described previ- ously (16) using authentic samples of phosphoserine, phosphothreo- nine, and phosphotyrosine as standards.

Treatment of the wheat germ lectin-purified insulin- and EGF- stimulated kinases with N-ethylmaleimide was carried out in buffer containing 40 mM imidazole, 10% glycerol, 0.5 M NaC1,0.05% Triton X-100, pH 7.0. Concentrated solutions of N-ethylmaleimide were diluted 10-fold into wheat germ lectin-purified preparations in the above buffer to obtain the desired final concentration. Reactions were allowed to proceed for 15 min at 0 "C and were quenched by the addition of dithiothreitol to a final concentration of 12 mM. The treated wheat germ-purified preparations were then assayed using the standard peptide phosphorylation assay.

Immunoprecipitations-Following an incubation for 5 min at 30 "C, standard phosphorylation reaction mixtures were diluted 2-fold with RIPA buffer (50 mM HEPES, 50 mM NaCl, 0.5% deoxycholate, 0.5% Nonidet P-40, 0.1% SDS). Three microliters of either anti-insulin receptor antibody or anti-EGF receptor antibody were added to the diluted reaction mixtures which were then incubated overnight at 4 "C. One hundred microliters of a 10% suspension of washed Pan- sorbin were then added and the mixtures were allowed to stand for 1 h at 4 "C. The tubes were then centrifuged and the pellets were washed three times in RIPA buffer. The immunoprecipitates were solubilized by boiling in an SDS-containing buffer and analyzed by SDS-polyacrylamide gel electrophoresis.

RESULTS

In soluble extracts of placenta membranes, insulin and EGF stimulated the phosphorylation of the synthetic, tyrosine- containing peptide, Arg-Arg-Leu-Ile-Glu-Asp-Ala-Glu-Tyr- Ala-Ala-Arg-Gly, found to be a substrate for several tyrosine- specific protein kinases (16, 25, 31,32). In the soluble extract

of placental membranes, phosphorylation of the peptide was linear for 7 to 10 min and was stimulated by both insulin and EGF. Based on their ability to phosphorylate the synthetic peptide, the insulin- and EGF-stimulated tyrosine protein kinases were purified by chromatography on wheat germ lectin-Sepharose (Table I). The EGF-stimulated kinase was purified 37-fold with a 50% recovery while the insulin-stim- ulated kinase was purified 51-fold with an overall yield of 68%. The activities shown in Table I are initial reaction rates obtained using a concentration of peptide substrate equal to the K,,, value (see Fig. 1) and are thus approximately half of the maximal activities obtainable with this preparation.

Using the wheat germ lectin-purified material as the source of kinase, the initial rates of the phosphorylation reactions were linear for 10 min and were proportional to the amount of enzyme added. Fig. 1 shows the Lineweaver-Burk plots for reactions in which insulin and EGF stimulated the phospho- rylation of the synthetic, tyrosine-containing peptide. The insulin- and EGF-stimulated kinases exhibited similar K, values of 1-2 mM for the peptide. The EGF-stimulated kinase showed a 5-fold greater Vmax than the insulin-stimulated ki- nase, presumably due, at least in part, to the greater number of EGF receptors present in the starting membranes. Radi- oligand-binding assays indicated that the placenta mem- branes contained 1.5-2.5 pmol of insulin receptors/mg of protein and 6-8 pmol of EGF receptors/mg of protein. Be- cause the binding assay for soluble EGF receptors from tissues other than A431 cells is very inefficient (33, 34), a direct measurement of the number of EGF receptors in the solubi- lized preparations could not be obtained. However, the recov- eries of the insulin- and EGF-stimulated kinase activities in the wheat germ lectin-purified preparation were similar so that the ratio of the number of EGF receptors to insulin receptors was probably maintained in the wheat germ lectin- purified preparation.

The insulin- and EGF-stimulated kinases present in the wheat germ lectin-purified preparation were further charac- terized using the synthetic peptide substrate. Both insulin and EGF stimulated the phosphorylation of the peptide in a dose-dependent fashion (Fig. 2, A and B) . Half-maximal stim- ulation occurred at 2 and 17 nM for insulin and EGF, respec- tively. Proinsulin was also capable of stimulating peptide phosphorylation (Fig. 2 A ) . While the maximal activity seen in the presence of proinsulin was the same as that observed with insulin, proinsulin was 30-fold less potent than insulin, exhibiting half-maximal activity at 68 nM. This relative po- tency of insulin and proinsulin is characteristic of a response mediated via the insulin receptor (35).

The data in Table I1 demonstrate that the response to insulin and EGF was additive. Insulin stimulated a %fold increase in the incorporation of 32P into the synthetic peptide while EGF stimulated a 5-fold increase. When added together, insulin and EGF induced a 6-fold increase in the phosphoryl- ation of the peptide. The relative levels of insulin- and EGF- stimulated peptide phosphorylation varied from one prepa- ration to another but seemed to parallel the ratio of the number of insulin to EGF receptors present in the starting membranes as measured by radioligand binding.

Fig. 3 shows a comparison of the metal ion dependency for the insulin- and EGF-stimulated kinases. Both insulin and EGF stimulated more activity in the presence of Mn2+ than in the presence of M e . With either metal ion, the EGF- stimulated reaction exhibited a biphasic response, increasing up to 2 mM Mn2+ or 15 mM M P , and then falling off as the concentration of metal ion increased. The insulin-stimulated enzyme did not show a similar decrease in activity at high

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Insulin- and EGF-stimulated Protein Phosphorylation TABLE I

Purification of the insulin- and EGF-stimulated tyrosine protein kinases from human placenta Fresh human placenta was processed as described under "Experimental Procedures." The insulin- and EGF-

stimulated kinase activities were assayed for 5 min at 30 "C usine 2 mM svnthetic Deptide as the acceDtor substrate.

9915

EGF-stimulated kinase Insulin-stimulated kinase Total

protein Purification Yield Specific Purification Yield activitv nrt.ivitv

w nmollminlmg -fold % nmol/min/mg -fold % Triton X-100 extracts 56 2.47 0.335 Wheat eerm lectin-SeDharose 0.75 91.9 37 50 17.2 51 68

0.06 1 + Insulm J t / 0 0 4

V K m Vmox

imM1 inmol/m~n/mgi

+ 1"S"lI" 2 0 1 2

+ E G F I 4 312

-~

(&I 0.02

-0.8 -0.4 0 0.4 , 0.8 1.2 1.6 2.0 2 4

& (mM")

FIG. 1. Lineweaver-Burk plot of the phosphorylation of the synthetic peptide by wheat germ lectin-purified placenta preparations. Phosphorylation assays containing varying concen- trations of synthetic peptide were carried out as described under "Experimental Procedures." The data were calculated from the activ- ity that was stimulated by either insulin or EGF over basal levels of peptide phosphorylation. In general, it was found that insulin caused a 2-%fold increase in the phosphorylation rate and EGF caused a 4- 5-fold increase.

concentrations of metal ions. Both the insulin- and EGF- stimulated kinases showed greater activity in the presence of a combination of Mn2+ and M$+ than when either metal ion alone was used to fulfill the divalent cation requirement.

The insulin- and EGF-stimulated kinases exhibited similar K,,, values of approximately 70 NM for ATP (data not shown). To examine the ability of other nucleotides to interact with these enzymes, peptide phosphorylation assays were run in the presence of 50 p~ [y-32P]ATP and varying concentrations of several unlabeled nucleotides, which were examined for their ability to inhibit the phosphorylation reaction. As shown in Table 111, neither of the two growth factor-stimulated kinases appeared to interact with GTP but both kinases were inhibited significantly by adenine nucleotides. The ATP- binding site apparently has affinity for only di- or triphos- phonucleotides as ATPyS and ADP were both inhibitory whereas AMP was not. ATP+ was slightly more effective at inhibiting the EGF-stimulated kinase than the insulin-stim- ulated kinase.

Inhibition by N-Ethylmaleimide-Certain actions of insulin such as the stimulation of hexose transport are known to be inhibited by treatment of cells with sulfhydryl reagents (36- 38). It was therefore of interest to determine the effect of N- ethylmaleimide on the insulin-stimulated kinase and compare this with the effect of N-ethylmaleimide on the EGF-stimu- lated kinase. As shown in Fig. 4, both the EGF- and the insulin-stimulated kinases were inhibited by treatment with

ioglo (Growth Foctor Concentrohon 1 ( M I

B

100-

c

n"

40- 2

0 -11 -10 -9 -8 -7 -6 - 5

Log10 [EGF] (MI

FIG. 2. Dose-response curves for growth factor-stimulated peptide phosphorylation. Synthetic peptide phosphorylation by wheat germ lectin-purified placenta extracts was measured at a syn- thetic peptide substrate concentration of 2 mM in the presence of varying concentrations of (A) insulin, proinsulin, or ( B ) EGF. The data shown represent the activity stimulated by each growth factor over basal levels of peptide phosphorylation. Maximal response was 45.6 nmol/min/mg in the presence of insulin and proinsulin and 131.7 nmol/min/mg in the presence of EGF. The curves shown are from a representative experiment with each point performed in duplicate.

TABLE I1 Additivity of the response to insulin and EGF

A wheat germ lectin-purified placenta preparation was assayed for synthetic peptide phosphorylation as described under "Experimental Procedures" in the absence of growth factor or in the presence of 30 nM insulin, 500 nM EGF, or a combination of insulin plus EGF at these same concentrations.

Additions 32p Incornorated . ~

cpm No growth factor 18,226

+EGF +Insulin 36,810

116,424 +Insulin + EGF 136,340

N-ethylmaleimide. The EGF-stimulated kinase was more sen- sitive to N-ethylmaleimide treatment than was the insulin- stimulated kinase. Half-maximal inactivation of the EGF- stimulated enzyme occurred at approximately 0.1 mM N- ethylmaleimide compared to 4 mM for half-maximal inacti- vation of the response to insulin.

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9916 Insulin- and EGF-stimulated Protein Phosphorylation

A 1

0 4 8 12 16 20 24 28 50 8 -

110 A

1001

70 -

- 60- 0 50- r

2 40-

x v

L1 0 30-

20 - 10-

0- r 0

2j d

I I I I 1 L I

4 8 12 16 20 24 28 50 ”

[Me”] (mM)

FIG. 3. Effect of metal ions on insulin- and EGF-stimulated peptide phosphorylation. Insulin- ( A ) and EGF (B)-stimulated peptide phosphorylation was measured as described under “Experimental Procedures” with the exception that the type and concentration of divalent metal ion were varied. Squares represent the activity observed at varying concentrations of Me. Circles represent the activity in the presence of varying concentrations of Mn2+. The single point denoted by the triangles represents the activity under the standard assay conditions of 2 mM Mn2+ and 12 mM M$+.

TABLE I11 Effect of the addition of unlnbeled nucleotides on insulin- and EGF-

stimulated kinuse activity A wheat germ lectin-purified placenta preparation was assayed for

peptide phosphorylation essentially as described under “Experimental Procedures” except the concentration of [Y-~’P]ATP was 50 FM. Unlabeled nucleotides at the indicated concentrations were added with the labeled ATP to start the reactions. Data are presented as the percentage of the control activity observed in the absence of any addition.

Addition stimulated Insulin-

% control % control

EGF-stimulated

50 PM GTP 110 102 500 FM GTP 106 111 50 FLLM ATPyS 97 71

500 WM ATPyS 61 21 50 ~ L M ADP 76 66

500 p~ ADP 4 17 500 p~ AMP 108 88

Substrate Specificity-With the availability of a preparation which contained the insulin- and EGF-stimulated kinases at a similar stage of purification, it was of interest to compare the two enzymes to determine whether there were any differ- ences in their substrate specificities. These two growth factor- stimulated kinases were first tested for their ability to phos- phorylate endogenous substrates in the wheat germ lectin- purified preparation.

Fig. 5 demonstrates that insulin stimulated the phospho- rylation of an M , = 95,000 protein which presumably repre- sents the p subunit of the insulin receptor (8-11). Similarly, EGF stimulated the phosphorylation of an M, = 170,000 protein which is most likely the EGF receptor (39). In addition to increasing the phosphorylation of the M, = 170,000 protein, EGF also stimulated the phosphorylation of an M, = 95,000 protein which co-migrates with the protein phosphorylated in response to insulin. Likewise, insulin appears to increase the phosphorylation of an M , = 170,000 protein with a mobility identical to that of the protein phosphorylated in response to EGF.

100 -

80 - - 2 t

60- 0

O\“ 40-

FIG. 4. Effect of N-ethylmaleimide on insulin- and EGF- stimulated peptide phosphorylation. Wheat germ lectin-purified preparations of solubilized placenta membranes were treated with varying concentrations of N-ethylmaleimide ( N E M ) as described under “Experimental Procedures.” Following quenching of the reac- tion with dithiothreitol, the treated preparations were assayed for insulin- (0) or EGF (0)-stimulated peptide phosphorylation.

To determine whether insulin and EGF were capable of inducing phosphorylation of the EGF and insulin receptors, respectively, a wheat germ lectin-purified preparation of these receptors was phosphorylated in the absence of growth factors and in the presence of optimal concentration of insulin or EGF. Duplicate reaction mixtures were immunoprecipitated with anti-insulin receptor antibody or anti-EGF receptor an- tibody. The immunoprecipitations were then subjected to SDS-polyacrylamide gel electrophoresis and the gels were dried and autoradiographed. As shown in Fig. 6, both insulin and EGF increased the incorporation of 32P into an M, = 95,000 protein which was immunoprecipitated by the anti- insulin receptor antibody. Scans of the autoradiogram indi- cated that insulin caused a 3-fold increase in the phosphoryl- ation of the insulin receptor while EGF stimulated a 2.5-fold increase. Similarly, insulin and EGF each stimulated the phosphorylation of an M , = 170,000 protein which was pre-

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Insulin- and EGF-stimulated Protein Phosphorylation

"m -200,000

-1 16,000 - 92,000

200,000-

rwlrcs 1 16,000-

92,000- -66,000

66,000-

anti-EGF R

-43,000

FIG. 5. Endogenous protein phosphorylation stimulated by insulin and EGF. A wheat germ lectin-purified preparation of solubilized placenta membranes was phosphorylated as described under "Experimental Procedures" except that no exogenous protein substrates were added. Phosphorylation was for 5 min at 30 "C. The phosphorylated proteins were separated on a 9% polyacrylamide gel. The gel was dried and autoradiographed. Phosphorylations were performed in the presence of (a) no growth factor, (b) insulin, and (c) EGF. Molecular weight standards used were myosin, &galactosidase, phosphorylase b, bovine serum albumin, and ovalbumin.

cipitated by the anti-EGF receptor antibody. Scans of this autoradiogram demonstrated a 40% increase in EGF receptor phosphorylation in response to insulin and a 100% increase in response to EGF.

The dose-response curves for insulin-stimulated phospho- rylation of the insulin receptor and the EGF receptor were similar and showed half-maximal activation at 8 X lo-' M insulin. Half-maximal stimulation of the phosphorylation of both the EGF receptor and the insulin receptor occurred at 9 x lo-* M EGF (data not shown).

A number of exogenous proteins were also examined for their ability to serve as substrates for the insulin and EGF- stimulated kinases. The K, and Vmax for each substrate were calculated from Lineweaver-Burk plots in which only that fraction of the activity stimulated either by insulin or EGF was taken into account. This was done because the basal activity represents activity due to both kinases and there was no way to determine the contribution of each kinase to this activity. To facilitate comparisons, a single wheat germ lectin- purified preparation was used throughout these experiments. The starting membranes for this preparation possessed 2.2 pmol of insulin receptors/mg of protein and 8 pmol of EGF receptors/mg of protein.

Table IV summarizes the results from a series of experi- ments in which the kinetic parameters for smooth muscle light chains (40), casein, troponin I, histone f,, and histone

9917

~200,000

.116,000

w"92,000

-66,000 ant i - IR

FIG. 6. Immunoprecipitation of endogenous proteins phos- phorylated in response to insulin and EGF. Phosphorylation of a wheat germ lectin-purified preparation of solubilized placenta mem- branes was carried out as described in Fig. 5. Reaction mixtures contained no growth factor (lanes I and 41, 30 nM insulin (lanes 2 and 5), or 500 nM EGF (lanes 3 and 6 ) and were immunoprecipitated with either anti-EGF receptor (anti-EGF R ) antibody (lanes 1-3) or anti-insulin receptor (anti-IR) antibody (lanes 4-6). The immunopre- cipitates were analyzed by electrophoresis on a 6% SDS-polyacryl- amide gel and the labeled proteins were located by autoradiography of the dried gel.

TABLE IV Kinetic parameters for protein substrates phosphorylated by the

insulin- and EGF-stimulated kinases Varying concentrations of the listed proteins were phosphorylated

by a wheat germ lectin-purified placenta preparation in the absence of growth factor or in the presence of insulin or EGF. The K , and V,. for each protein was determined from a Lineweaver-Burk plot constructed from the data for that portion of the total activity that was specifically stimulated by either insulin or EGF.

Substrate Insulin-stimulated EGF-stimulated

K, V-. K, V"". nmol/min/mg nmollminlmg

Synthetic peptide 2.0 mM 72 1.4 mM 312 Smooth muscle 15 gM 11.9 27 pM 57.2

Casein 0.5 mg/ml 10.1 1.1 mg/ml 56.0 Troponin I 10 pM 1.2 18 WM 3.6 Histone f, 1.0 pM 11.2 0.5 pM 9.7 Histone f2b 1.1 WM 4.6 1.1 YM 4.4

light chain

fib were determined. The data from Fig. 1 for the synthetic peptide have been included in the table for comparison. While the K, values varied among the different substrates, the K, for a given substrate was nearly the same in the insulin- and EGF-stimulated reactions. The K , values for the utilization of the five protein substrates by the insulin- and EGF-stim- ulated kinases were all in the micromolar range, with histones exhibiting the lowest K,. This contrasts markedly with the millimolar K , noted for the kinases with the synthetic peptide as substrate. A number of other proteins including tropomy- osin, actin, ribonuclease, glutamate dehydrogenase, and lac-

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9918 Insulin- and EGF-stimulated Protein Phosphorylation

tate dehydrogenase were found not to be phosphorylated by these growth factor-stimulated kinases.

For the synthetic peptide, smooth muscle light chains, casein, and troponin I, the V,,, for the phosphorylation stim- ulated by EGF was 3- to &fold higher than that seen in the presence of insulin. For histone fl and histone f2b, the Vm,, for the EGF-stimulated reaction was the same as that for the insulin-stimulated reaction. The apparently decreased ability of the EGF-stimulated kinase to phosphorylate the histone substrates is not related to an effect of histones on the binding of EGF to its receptor since 1251-EGF binding to placenta membranes wag not decreased by the inclusion of histones in the binding assay (data not shown).

Phosphoamino acid analyses indicated that insulin and EGF stimulated the incorporation of phosphate only into tyrosine residues in these proteins (Fig. 7). Thus, the kinetic parameters determined above represent values for an insulin- or EGF-stimulated tyrosine-specific protein kinase.

To determine whether the insulin and EGF-stimulated enzymes phosphorylated the same site or sites on the protein substrates, the phosphorylated proteins were analyzed by partial proteolysis mapping according to the method of Cleve- land et al. (29). As shown in Fig. 8, the proteins phosphoryl- ated in the presence of EGF or insulin produced similar patterns of "P-labeled peptides suggesting that the same site or sites were phosphorylated in the presence of either growth

SM LC Casein Histone f l a b c a b c a b c

Ser(P)-

Thr (PI-

1 2 3 4 5 6 7 8 910

+ '

FIG. 8. Proteolysis gels of the proteins phosphorylated by wheat germ lectin-purified placenta preparations. Smooth muscle light chains (lanes I and 2). casein (lanes 3 and 4, histone fl, (lanes 5 and 6) , histone f2b (lanes 7 and 8), and troponin I (lanes 9 and 10) at the same concentrations used for Fig. 6 were phosphoryl- ated for 5 min at 30 "C. The reactions were stopped by the addition of Laemmli SDS sample buffer following by boiling for 3 min. The phosphorylated proteins were then analyzed as described under "Ex- perimental Procedures." Since 8-casein was the predominant protein phosphorylated in the mixture of caseins, this band was used for this analysis. The resulting gel was autoradiographed for 1 to 4 days to optimize the exposure of each lane. Odd-numbered lanes are from proteins phosphorylated in the presence of insulin. Even-numbered lanes are from proteins phosphorylated in the presence of EGF.

factor. Because these maps represent peptides from only partially proteolyzed substrates it is not possible to determine

data. A Tyr (PI- Q ' the number of sites phosphorylated in each protein from this

Histone f2b T N I Peptide a b c a b c a b c

v - Ser(P1-

Thr(P)-

Tyr (P)-

0

FIG. 7. Phosphoamino acid analyses of substrates phospho- rylated by wheat germ lectin-purified placenta preparations. Substrates were phosphorylated for 5 min at 30 "C as detailed under "Experimental Procedures." The reactions were stopped by addition of 30 pl of 2 N HCI and the entire mixture was subjected to hydrolysis for 2 h at 110 "C in 6 N HCI. The concentrations of substrate used in the phosphorylation assays were: synthetic peptide, 2 mM; smooth muscle light chains (SMLC), 51 pM; casein, 3.0 mg/ml; troponin I (TNI), 45 p ~ ; histone f,, 10 p ~ ; and histone f2br 10 pM. The arrows indicate the positions of the authentic phosphoamino acids. Lune a, no growth factor; lane b, plus insulin; lane c, plus EGF.

DISCUSSION

In Triton X-100 extracts of human placenta membranes, insulin and EGF stimulate the phosphorylation of a synthetic tyrosine-containing peptide. The insulin- and EGF-stimu- lated phosphorylating activities are both substantially en- riched by chromatography on wheat germ lectin-Sepharose. In the wheat germ lectin-purified preparation, growth factor- stimulated peptide phosphorylation is linear with time and proportional to enzyme concentration. The effect of insulin and EGF on peptide phosphorylation is additive, suggesting that these two growth factors stimulate this response through distinct receptors.

From receptor purification studies (411, it can be estimated that the insulin receptor constitutes roughly 1-5% of the protein in the wheat germ lectin-purified extracts of placenta membranes used in our studies. Assuming that the insulin receptor is the tyrosine-specific protein kinase, a calculated specific activity for the homogeneous kinase of 1 to 7 pmol/ min/mg is obtained from our data. This value is similar to the specific activity exhibited by the CAMP-dependent protein kinase using synthetic peptide substrates (42). Thus, the tyrosine-specific protein kinases stimulated by insulin, and by inference EGF, are highly active and are likely to be of physiological importance.

It is noteworthy that the specific activities for the purified kinases reported here are significantly higher than previously published values. The specific activity of the insulin-stimu-

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Insulin- and EGF-stimulated Protein Phosphorylation 9919

lated kinase (72 nmol/min/mg) is approximately 300-fold higher than that reported by Stadtmauer and Rosen (32) using the same peptide substrate on wheat germ lectin-puri- fied extracts of L1-3T3 cells. This specific activity is also 12- fold greater than that reported for an affinity-purified prep- aration of human placenta insulin receptors (43). Similarly, the specific activity of the EGF-stimulated kinase (360 nmol/ min/mg) is about 300-fold higher than that reported by Er- neux et al. (44) using a related peptide substrate with affinity- purified EGF receptors from A431 cells.

Part of the difference may be due to variations in assay conditions and tissues sources, and part may be due to differ- ences in the method of preparation used. The assay conditions employed in the present study are similar to those used by Petruzzelli et al. (11) with the exception that 100 FM Vo4 was added to inhibit ATPase activity and 2 mM dithiothreitol was added to stabilize the kinase. Together these two additions produce at most a 3- to 4-fold increase in the activity of the insulin- and EGF-stimulated kinases in the wheat germ lec- tin-purified preparation.' The other major difference in the present assay conditions is the use of relatively high (200 p ~ ) concentrations of ATP. Other investigators have reported using from 0.5 to 50 PM ATP (32, 43, 44). The K, for ATP for the insulin- and EGF-stimulated kinases was estimated to be 70 PM in the present study; thus, use of concentrations of ATP well below the K , could account for a severalfold de- crease in apparent specific activity of these enzymes relative to that presented here.

Because of incomplete descriptions of the procedures used by others, to prepare wheat germ lectin-purified material, direct comparison with the method reported here is difficult. There are several points at which variations in the method apparently do occur. Homogenization of the washed placenta in our procedure was done for a total of 45 s in a large volume Waring blender rather than for 5 min as reported by others (41, 43). In addition, the crude homogenate was filtered through cheesecloth, a technique which replaces the first low speed spin, helping to reduce the overall time of preparation of the membranes. With these changes, a fresh placenta can be processed into membranes in 2.5 to 3 h. The membranes can be stored indefinitely at -70 "C; however, upon thawing, they were used immediately and never allowed to reach tem- peratures above 4 "C. All buffers from this point on contained 10% glycerol to stabilize enzyme activity. Solubilization of the receptors was carried out on ice, rather than at room temperature (41, 43). The soluble preparation was immedi- ately applied batchwise to wheat germ lectin-Sepharose and rotated end-over-end for 2 h at 4 "C, not overnight (41,43) as reported previously. The resin was poured into a column and rapidly washed, eluted, and assayed for kinase activity. Using this procedure, placental membranes could be solubilized and purified on wheat germ lectin-Sepharose in less than 8 h. The material, which was aliquoted into 1-ml fractions and stored at -70 "c, was stable for several months. The key differences in our procedure thus appear to be rapid preparation of membranes and wheat germ lectin-purified material combined with a strict maintenance of temperature a t 4 "C.

Previous studies of the insulin- (11, 18, 24, 32, 43, 45) or EGF-stimulated kinases (12, 24, 30, 46) have suggested that the properties of the two enzymes are quite similar. However, few (24) of the previous reports examined these enzymes from the same tissue source at the same stage of purification using the same assay conditions. None has directly compared the utilization of exogenous substrates by the insulin- and EGF-

L. J. Pike and E. G. Krebs, unpublished data.

stimulated kinases. Thus, the question of how similar the specificities of these two kinases are had not been adequately addressed.

In the present studies, both enzymes showed greater activity when Mn'+ rather than Mg2+ was used to fulfill the divalent cation requirement. In addition, both kinases exhibited the highest activity when a combination of Mn2+ and M%+ was used. This has been reported previously for the insulin-stim- ulated kinase (11) but differs from the reported preference for Mg2+ by the EGF-stimulated kinase in A431 cells (30) or placenta membranes (46). This dual metal ion requirement may reflect a need for metal ions to inhibit competing activ- ities in the preparation. It is also possible that in addition to binding to ATP, divalent cations regulate the kinase activity directly (47).

The insulin- and EGF-stimulated kinases exhibited similar K,,, values for ATP and showed a strong selectivity for adenine as opposed to guanine nucleotides. Similar findings have been reported previously for both the insulin- and EGF-stimulated kinases (11,30,45). The utilization of [3'P]ATP by the EGF- stimulated kinase is more sensitive to inhibition by ATP+ than is the corresponding activity of the insulin-stimulated kinase. This suggests that this nucleotide analog binds with greater affinity to the EGF-stimulated enzyme and this points out a minor difference in the nucleotide specificities of the two kinases.

Both the insulin- and EGF-stimulated kinases were inhib- ited by treatment with N-ethylmaleimide, although the EGF- stimulated enzyme was more sensitive to modification by this reagent. These data suggst that both kinases possess sulfhy- dryl groups that are important for enzymatic activity. How- ever, since the growth factor-stimulated kinase activity of these two enzymes is dependent upon an intact hormone- binding site, as well as a functional kinase active site, the inhibition by N-ethylmaleimide could be related to modifica- tions in either functional domain. Alternatively, modlfication of receptor-kinase "coupling" without actual inactivation of the kinase could be responsible for the observed loss of hor- mone-sensitive kinase activity. It seems likely that the effect of N-ethylmaleimide on the insulin-stimulated kinase is not at the level of hormone binding since insulin binding to isolated membranes has been reported to be unaltered by treatment with N-ethylmaleimide (48, 49). If insulin-stimu- lated tyrosine phosphorylation is involved in mediating the effects of this hormone, inhibition of kinase activity either directly or indirectly by N-ethylmaleimide could explain the inhibitory effects of this reagent on processes such as insulin- stimulated hexose transport (36-38).

The substrate specificities of the insulin- and EGF-stimu- lated kinases appear to be quite similar. As has been reported previously by others (8-12, 39), we found that these two kinases undergo autophosphorylation. Our finding that insu- lin appears to stimulate the phosphorylation of the EGF receptor and that EGF appears to stimulate the phosphoryl- ation of the insulin receptor is novel. The most likely expla- nation for this observation is that the two kinases phospho- rylate one another since insulin does not interact with the EGF receptor (50) and EGF does not bind to the insulin receptor (26). Jacobs et al. (51) have recently reported similar cross-phosphorylation between the insulin receptor and the somatomedin-C receptor. Thus, such phosphorylations may be a general phenomenon and may possibly be of regulatory significance in uiuo.

The insulin- and EGF-stimulated kinases exhibited nearly identical K , values for each protein or peptide substrate examined. Thus, these two growth factor-stimulated enzymes

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9920 Insulin- and EGF-stimulated Protein Phosphorylation

could not be distinguished based on this kinetic parameter. However, some differences between the insulin- and EGF- stimulated kinases could be discerned when the Vmax values for the different substrates were compared. Of the six sub- strates examined, four showed a 5-fold greater Vmax in the presence of EGF than in the presence of insulin. This may be due to the higher number of EGF than insulin receptors present in the wheat germ lectin-purified preparation used in these experiments. Two of the substrates, histone fl and histone f2b, showed equivalent V,,, values in the presence of insulin and EGF. Since the same preparation was used to phosphorylate all six substrates, this implies that the turnover number of the insulin-stimulated kinase using histones as substrate improved approximately 5-fold relative to the turn- over number of the EGF-stimulated enzyme using the same substrate. Thus, the insulin- and EGF-stimulated kinases can be distinguished based on the relative turnover number of the two enzymes using different protein substrates.

Two comments can be made concerning the general utili- zation of the substrates examined in this study. First, the insulin- and EGF-stimulated kinases exhibited K , values for the protein substrates 3 orders of magnitude lower than those seen with the peptide as substrate. The micromolar K , values seen with the protein substrates are more in keeping with physiologically attainable concentrations than is the milli- molar K , value seen using the peptide. This difference be- tween the protein and peptide substrates indicates that some structural feature other than primary sequence is important in binding of the acceptor substrate to the kinase (52). Second, because the sites of phosphorylation of these proteins are unknown, no primary sequence specificity can be discerned from these data. However, it should be noted that these two growth factor-stimulated kinases do exhibit specificity since numerous proteins were not phosphorylated by these en- zymes.

The similarities noted between the in uitro characteristics of the insulin- and EGF-stimulated kinases suggests that the in vivo specificity of these two enzymes may overlap. A number of similarities in the effects of insulin and EGF have been noted (1, 4-6). These include stimulation of phospho- fructokinase activity (53) and glycolysis (54), as well as in- creased phosphatidylinositol metabolism (3, 55), ion fluxes (56, 57), and hexose transport (58). Insulin is known to alter the expression of certain genes (59) and this hormone has also been reported to stimulate DNA synthesis and cell growth in a few cell types (5). Similarly, EGF elicits changes in gene expression but does not affect cell growth in pituitary cells (60), yet is a potent mitogen for many other cells. The differ- ences between the insulin- and EGF-stimulated kinases noted with the histone substrates indicate that the substrate speci- ficities of the two enzymes are not identical. However, if the phosphorylation of proteins on tyrosine residues is important in mediating the effects of these growth factors, the similarity of the two kinases observed at the molecular level might be related to the similarity of the responses elicited by insulin and EGF at the cellular level.

1.

2.

3. 4.

5.

REFERENCES Hollenberg, M. D., and Cuatrecasas, P. (1975) J. Bwl. Chem.

Habenicht, A. J. R., Glomset, J. A., King, W. C., Nist, C., Mitchell, C. D., and Ross, R. (1981) J. Biol. Chem. 256 , 12329-12335

Sawyer, S. T., and Cohen, S. (1981) Biochemistry 20,6280-6286 Koontz, J. W., and Iwahashi, M. (1981) Science (Wash. D. C.)

Goldhe, I. D. (1981) Biochemical Actions ofHormones, Vol. VIII,

250,3845-3853

2 1 1,947-949

pp. 273-305, Academic Press, Inc., New York

6.

7. 8.

9.

10.

11.

12. 13.

14.

15.

16.

17.

18.

19. 20.

21.

22.

23.

24.

25.

26.

27. 28. 29.

30.

31.

32.

33.

34.

35.

36.

37. 38. 39.

40.

41.

42.

43.

44.

45.

46.

Carpenter, G., and Cohen, S. (1979) Annu. Reu. Biochem. 4 8 ,

Ross, R., and Vogel, A. (1978) Cell 14 , 203-210 Kasuga, M., Karlsson, F. A., and Kahn, C. R. (1982) Science

Van Obberghen, E., and Kowalski, A. (1982) FEBS Lett. 143,

Kasuga, M., Zick, Y., Blithe, D. L., Crettaz, M., and Kahn, C. R. (1982) Nature (Lord.) 298,667-669

Petruzzelli, L. M., Ganguly, S., Smith, C. J., Cobb, M. H., Rubin, C. S., and Rose, 0. M. (1982) Proc. Natl. Acad. Sci. U. S. A.

Ushiro, H., and Cohen, S. (1980) J. Biol. Chem. 255,8363-8365 Ek, B., Westermark, B., Wasteson, A., and Heldin, C.-H. (1982)

Ek, B., and Heldin, C.-H. (1982) J. Bwl. Chem. 257 , 10486-

Nishimura, J., Huang, J. S., and Deuel, T. F. (1982) Proc. Natl.

Pike, L. J., Bowen-Pope, D. F., Ross, R., and Krebs, E. G. (1983)

Roth, R. A., and Cassell, D. J. (1983) Science (Wash. D. C.) 2 1 9 ,

Kasuga, M., Fujita-Yamaguchi, Y., Blithe, D. L., and Kahn, C.

Shia, M. A., and Pilch, P. F. (1983) Biochemistry 22, 717-721 Van Obberghen, E., Rossi, B.., Kowalski, A., Gazzano, H., and

Ponzio, G. (1983) Proc. Natl. Acad. Sci. U. S. A. 80,945-949 Buhrow, S. A., Cohen, S., Garbers, D. L., and Staros, J. V. (1983) J. Biol. Chem. 258 , 7824-7827

Buhrow, S. A., Cohen, S., and Staros, J. V. (1982) J. Bwl. Chem.

Heldin, C.-H., Ek, B., and Ronnstrand, L. (1983) J. Biol. Chem.

Avruch, J., Nemenoff, R. A., Blackshear, P. J., Pierce, M. W., and Osathanondh, R. (1982) J. Biol. Chem. 257, 15162-15166

Casnellie, J . E., Harrison, M. L., Pike, L. J., Hellstrom, K. E., and Krebs, E. G. (1982) Proc. Natl. Acad. Sci. U. S. A. 79,282- 286

Siegel, T. W., Ganguly, S., Jacobs, S., Rosen, 0. M., and Rubin,

Roskoski, R. (1984) Methods Enzymol. 99,3-6 Laemmli, U. K. (1970) Nature (Lord.) 227,680-685 Cleveland, D. W., Fischer, S. G., Kirschner, M. W., and Laemmli,

Carpenter, G., King, L., Jr., and Cohen, S. (1979) J. Bwl. Chem.

Pike, L. J., Gallis, B., Casnellie, J. E., Bornstein, P., and Krebs,

Stadtmauer, L. A,, and Rosen, 0. M. (1983) J. Biol. Chem. 2 5 8 ,

Nexo, E., Hock, R. A., and Hollenberg, M. D. (1979) J. Biol.

Cohen, S., Fava, R. A., and Sawyer, S. T. (1982) Proc. Natl. Acad.

Freychet, P., Roth, J., and Neville, D. M. (1971) Proc. Natl. Acad.

Cadenas, E., Kaji, H., Park, C. R., and Rasmussen, H. (1961) J.

Czech, M. P. (1976) J. Cell. Physiol. 89,661-668 Czech, M. P. (1976) J. Biol. Chem. 251, 1164-1170 Cohen, S., Carpenter, G., and King, L., Jr. (1980) J. Biol. Chem.

Gallis, B., Edelman, A. M., Casnellie, J. E., and Krebs, E. G.

Fujita-Yamaguchi, Y., Choi, S., Sakamoto, Y., and Itakura, K.

Kemp, B. E., Graves, D. J., Benjamini, E., and Krebs, E. G. (1977)

Kasuga, M., Fujita-Yamaguchi, Y., Blithe, D. L., White, M. F.,

Erneux, C., Cohen, S., and Garbers, D. L. (1983) J. Biol. Chem.

Zick. Y.. Kasuea. M.. Kahn, C. R., and Roth, J. (1983) J. Bioi.

193-216

(Wash. D. C.) 215,185-187

179-182

79,6792-6796

Nature (Lond.) 295,419-420

10492

Acad. Sci. U. S. A. 79,4303-4307

J. BWl. Chem. 258,9383-9390

299-301

R. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 2137-2141

257,4019-4022

258,10054-10061

C. S. (1981) J. BWl. Chem. 256,9266-9273

U. K. (1977) J. Biol. Chem. 252,1102-1106

254,4884-4891

E. G. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 1443-1447

6682-6685

Chem. 254,8740-8743

Sci. U. S. A. 79,6237-6241

SC~. U. S. A. 68,1833-1837

Bwl. Chem. 2 3 6 , PC63-PC64

255,4834-4842

(1983) J. Biol. Chem. 258 , 13089-13093

(1983) J. Bid. Chem. 2 5 8 , 5045-5049

J. Biol. Chem. 252,4888-4894

and Kahn, C. R. (1983) J. Biol. Chem. 2 5 8 , 10973-10980

258,4137-4142

Chem.’ 258,175-80 ’

crinol. 18, 189-199 Carpenter, G., Poliner, L., and King. L. (1980) Mol. Cell. Endo-

by guest on May 19, 2018

http://ww

w.jbc.org/

Dow

nloaded from

Insulin- and EGF-stimulated Protein Phosphorylation 9921

47. White, M. F., Haring, H.-V., Kasuga, M., and Kahn, c. R. (1984) 54. Diamond, I., Legg, A., Schneider, J. A,, and Rozengurt, E. (1978)

48. Cuatrecasas, P. (1971) J. Bwl. Chem. 246, 7265-7274 49. Pilkis, s. J., Johnson, R. A., and Park, c. R. (1972) Diabetes 21 , 56. Rozengurt, E., and Heppel, L. A. (1975) proc, Natl. ad. sei, u. 50. O'Keefe, E., Hollenberg, M. D., and Cuatrecasas, P. (1974) Arch. 57. Czech, M. p. (1977) A ~ ~ ~ . R ~ ~ . Biockm. 46, 359-384

J . Biol. Chem. 259,255-264 J. Bwl. Chem. 253,866-871 55. Michell, R. H. (1975) Biochim. Bwphys. Acta 415,81-147

Suppl. 1,335 S. A. 72,4492-4495

Bio~hem. Biophys. 164,518-526 51. Jacobs, S., Kull, F. C., EaT, H. S., Svoboda, M. E., Van Wyk, J. 58* w. R., Nilsen-Hamilton, M.7 and Hamilton, R. T. (1981)

J., and Cuatrecasas, P. (1983) J. Bwl. Chem. 2 5 8 , 9581-9584 J. Cell. Physiol. 108 , 15-24 52. Casnellie, J. E., and Krebs, E. G. (1984) Adu. Enzyme Regul. 2 2 , 59. Granner, D.y T.t Sasaki, K.* and E. (1983)

53. Schneider, J. A., Diamond, J., and Rozengurt, E. (1978) J. Biol. 60. Johnson, L. K., Baxter, J. D., Vledavsky, I., and Gospodarowicz, 501-515 Nature (Lord.) 305 , 549-551

Chem. 253,872-877 D. (1980) Proc. Natl. Acad. Sci. U. S. A. 77,394-398

by guest on May 19, 2018

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w.jbc.org/

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nloaded from

L J Pike, E A Kuenzel, J E Casnellie and E G Krebskinases from human placenta.

A comparison of the insulin- and epidermal growth factor-stimulated protein

1984, 259:9913-9921.J. Biol. Chem. 

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