Stimulation of phosphorylation of lipocortin at threonine residues by epidermal growth factor (EGF) and the EGF receptor: addition of protein kinase P with polylysine inhibits this effect.

In this paper we show that epidermal growth factor (EGF) stimulates the phosphorylation of lipocortin 1, at threonine as well as at tyrosine residues, by a highly purified preparation of the EGF receptor. The phosphorylation of threonine residues is catalyzed by an enzyme that contaminates the receptor preparations, since crude extracts of A431 plasma membranes contain larger amounts of the threonine kinase than does the receptor preparation. Protein kinase P (2.5 ng) inhibits both threonine and tyrosine phosphorylation of lipocortin 1 while greatly stimulating the autophosphorylation of the EGF receptor. Acetyllipocortin 1 is poorly phosphorylated at tyrosine residues by the EGF receptor kinase, but it becomes readily phosphorylated in the presence of polylysine. The most likely explanation for this observation is that there is an interaction between polylysine and acetyllipocortin that converts the latter into a suitable substrate for the EGF receptor. These and other experiments described in this paper point to a role of surface charges in the susceptibility of substrates to attach by protein kinases.     

Epidermal growth factor stimulates tyrosine phosphorylation of phospholipase C-II independently of receptor internalization and extracellular calcium.

Epidermal growth factor (EGF) rapidly stimulates the formation of inositol 1,4,5-trisphosphate in a variety of cell types. Previously we have found that in intact cells stimulation of phospholipase C (PLC) activity by EGF is correlated with the retention of increased amounts of PLC activity by a phosphotyrosine immunoaffinity matrix, suggesting that the EGF-receptor tyrosine kinase phosphorylates PLC. We now define parameters of the mechanism by which EGF addition to A-431 cells stimulates phosphotyrosine immunoisolation of PLC activity and demonstrate that EGF addition to A-431 cells increases tyrosine phosphorylation of PLC. EGF rapidly and reversibly stimulated the anti-phosphotyrosine recovery of increased PLC activity when cells were treated with growth factor at 3 degrees C, indicating that receptor internalization is not required and that the phosphorylation event occurs prior to formation of inositol 1,4,5-trisphosphate. Also, the EGF stimulation of anti-phosphotyrosine recovery of PLC activity occurred in the absence of extracellular Ca2+. Stimulation of PLC activity in intact cells by other agonists, such as bradykinin or ATP, did not result in increased anti-phosphotyrosine recovery of PLC activity, suggesting two separate mechanisms exist in A-431 cells for hormone-stimulated formation of inositol phosphates. Finally, using monoclonal antibodies that specifically recognize three distinct PLC isozymes, we show that an approximately 145-kDa PLC isozyme (PLC-II) is present in A-431 cells and that EGF treatment of A-431 cells stimulates phosphorylation of PLC-II on both tyrosine and serine residues.     

Phorbol esters potentiate tyrosine phosphorylation of epidermal growth factor receptors in A431 membranes by a calcium-independent mechanism.

Incubation of membranes prepared from A431 cells with either epidermal growth factor (EGF) or phorbol 12-myristate 13-acetate (PMA) stimulates the transfer of 32phosphate from [gamma-32P]ATP into 8-10 membrane proteins. The major phosphorylated protein migrates on NaDodSO4/polyacrylamide gels with an apparent Mr of 180,000, corresponding to the previously identified EGF receptor. Stimulation of EGF receptor phosphorylation by PMA does not require Ca2+, suggesting that prior activation of protein kinase C is not a prerequisite for phosphate transfer. PMA-enhanced phosphorylation proceeds at 4 degrees C and requires Mn2+, both properties of tyrosine-specific protein kinases. Phospho amino acid analysis of the Mr 180,000 receptor band shows that only tyrosine residues are phosphorylated when A431 membranes are treated with either EGF or PMA. Moreover, proteolysis reveals that these residues are located in the same peptides of the receptor. These results demonstrate that a potent tumor-promoting phorbol ester can mimic a critical early response usually elicited by EGF.     

Monoclonal antibody against epidermal growth factor receptor is internalized without stimulating receptor phosphorylation.

The down-regulation and internalization of the epidermal growth factor (EGF) receptors induced by two separate anti-EGF monoclonal antibodies (mAbs), IgG1 mAb-225 and -455, and by EGF was examined. mAb-225 competitively inhibits EGF binding and it is internalized to an extent comparable to EGF. The antibody down-regulates surface EGF receptors in a dose-dependent manner. In contrast, mAb-455 does not competitively inhibit the binding of EGF or mAb-225, but it specifically immunoprecipitates the EGF receptor. mAb-455 also down-regulates the EGF receptor. Unlike EGF, which elicits phosphorylation of the receptor at tyrosine, threonine, and serine residues, neither of these antibodies elicits phosphorylation of the EGF receptor in intact A431 cells or in KB cells. Our studies suggest that EGF-stimulated phosphorylation of the receptor is not required for the internalization of the ligand-receptor complex.     

Signal transduction by the epidermal growth factor receptor after functional desensitization of the receptor tyrosine protein kinase activity.

Previous work identified a protein kinase activity that phosphorylates the epidermal growth factor (EGF) receptor at Thr669. An assay for this protein kinase activity present in homogenates prepared from A431 human epidermoid carcinoma cells was developed using a synthetic peptide substrate corresponding to residues 663-681 of the EGF receptor (peptide T669). Here we report that a greater initial rate of T669 phosphorylation was observed in experiments using homogenates prepared from EGF- or phorbol ester-treated cells compared with control cells. EGF and 4 beta-phorbol 12-myristate 13-acetate (PMA) caused a 6-fold and a 2-fold increase in protein kinase activity, respectively. A kinetic analysis of T669 phosphorylation demonstrated that the increase in protein kinase activity observed was accounted for by an increase in Vmax. To examine the interaction between protein kinase C and signal transduction by the EGF receptor, the effect of pretreatment of cells with PMA on the subsequent response to EGF was investigated. Treatment of cells with PMA caused greater than 90% inhibition of the EGF-stimulated tyrosine phosphorylation of the EGF receptor and abolished the EGF-stimulated formation of soluble inositol phosphates. In contrast, PMA was not observed to inhibit the stimulation of T669 protein kinase activity caused by EGF. Thus, the apparent functional desensitization of the EGF receptor caused by PMA does not inhibit signal transduction mediated by the T669 protein kinase. Our results demonstrate that EGF receptor transmodulation alters the pattern of signal-transduction pathways that are utilized by the EGF receptor.     

Peroxynitrite Inhibits Epidermal Growth Factor Receptor Signaling in Caco-2 Cells

Intestinal mucosa serves as an important barrier that may be disrupted by inflammation. A complex system of cellular and humoral factors, including epidermal growth factor (EGF), maintains the integrity of this barrier. We hypothesized that peroxynitrite, generated in inflamed intestinal epithelium, can alter the EGF receptor function by nitrating tyrosine residues and blocking ligand-activated tyrosine autophosphorylation. Caco-2 cells or A431 cell membranes were treated with peroxynitrite or its decomposed form. Cell proliferation was measured by [3H]thymidine uptake. Immunoblot and immunoprecipitation were used to assess the tyrosine phosphorylation and nitration. Binding of epidermal growth factor to its receptor was detected by affinity labeling with 125I-EGF. Peroxynitrite inhibited EGF-induced Caco-2 cell proliferation and binding of EGF to its receptor in a concentration-dependent manner. Peroxynitrite abolished EGF-stimulated receptor autophosphorylation and nitrated EGF receptor tyrosine residues. Peroxynitrite generated during inflammation may disrupt the EGF-induced signaling in intestinal epithelial cells.     

Tumor-promoting phorbol diesters cause the phosphorylation of epidermal growth factor receptors in normal human fibroblasts at threonine-654.

The effect of tumor-promoting phorbol diesters to potentiate the action of epidermal growth factor (EGF) on cell proliferation is associated with phosphorylation of EGF receptors, acute depression of EGF binding, and inhibition of EGF receptor tyrosine kinase activity. In the present studies, normal human fibroblasts and A431 carcinoma cells were labeled with [32P]phosphate and treated with and without 10 nM 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA). The EGF receptors then were isolated by immunoprecipitation and digested with trypsin. Analysis of the labeled receptor phosphopeptides by reversed-phase HPLC revealed that PMA induces the phosphorylation of a unique phosphopeptide containing [32P]phosphothreonine. Comparison of several chemical and physical properties of the 32P-labeled phosphopeptide with the primary structure of the EGF receptor suggested the identify Lys-Arg-Thr(P)-Leu-Arg. This was confirmed by direct demonstration that a synthetic peptide of this structure comigrates during HPLC and electrophoresis with the 32P-labeled phosphopeptide isolated from the EGF receptors of normal human fibroblasts. The phosphorylated site on the peptide corresponds to threonine-654 of the EGF receptor, which is located on the cytoplasmic side of the plasma membrane nine residues distant from the transmembrane domain. These data indicate that phosphorylation of the EGF receptor in human fibroblasts and A431 cells at threonine-654 may regulate the EGF receptor tyrosine kinase activity and the binding of EGF.     

Different responses to EGF in two human carcinoma cell lines, A431 and UCVA-1, possessing high numbers of EGF receptors

EGF binding capacity was examined in 9 different human cell lines which were derived from colon, rectum and pancreas tumors. Among these cell lines, a pancreatic carcinoma cell line, UCVA-1, was found to possess a high number (0.9 X 10(6)/cell) of EGF receptors. This number is comparable to that of EGF receptors in human vulva epidermoid carcinoma A431 cells (2 X 10(6)/cell). However, it was found that, unlike A431 cells, the growth of UCVA-1 cells, in serum-containing and serum-free conditions, was not inhibited by EGF. The UCVA-1 cells have EGF receptor of Mr = 170 K and of two affinity types: Kd1 = 72 X 10(-9) M and Kd2 = 2 X 10(-8) M. The EGF receptors in UCVA-1 cells are less susceptible to proteolytic cleavage than those in A431 cells. In UCVA-1 cells, EGF is apparently processed via a receptor-mediated endocytosis. The UCVA-1 cell membrane contained EGF-stimulated protein kinase as was found in A431 cells. The stimulation of phosphorylation by EGF was only approximately 20% in UCVA-1 while it was over 100% in A431. When angiotensin II was used as a substrate, the relative activity of EGF-dependent tyrosine-specific protein phosphorylation was approximately 8 times less in UCVA-1 cell membrane. The EGF-stimulated phosphorylation was mostly on EGF receptors for both cell lines. However, several other components (Mr = 100 K, 80 K, 72 K and 65 K) were readily detected in A431 cells. These observations indicate that the EGF receptor/protein kinase relation differs in these two cell lines and suggests that it may be related to the growth-inhibitory effect of EGF seen in A431.     

Phosphorylation process induced by epidermal growth factor alters the oncogenic and cellular neu (NGL) gene products.

The rat neu oncogene encodes a cell surface glycoprotein, p185, that possesses tyrosine kinase activity. The p185 polypeptide exhibits structural similarity to the epidermal growth factor receptor (EGFR) at both the deduced amino acid and nucleic acid level. However, the neu oncogene and the gene encoding the EGFR have been shown to reside on distinct chromosomes. Comparative analysis of the sequences of the normal neu cDNA and of the neu cDNA from neuroblastomas has revealed a single point mutation leading to a valine-to-glutamic acid substitution in the transmembrane anchoring domain. This mutation converts the neu gene to a transforming gene in rodents. In humans, the gene is called ERBB2 (also NGL and HER2), and amplification and over-expression of its products have been detected in certain tumors. The rat embryonal fibroblast cell line (Rat-1) appears to express both EGFR and cellular p185 polypeptides. We have found that EGF stimulates the phosphorylation of p185 in these cells at tyrosine as well as serine and threonine residues in a specific and dose-dependent manner. This activity occurs even though radiolabeled EGF cannot bind to immunopurified p185. The EGF effect is apparently unique since platelet-derived growth factor, insulin, and transforming growth factor beta all fail to phosphorylate p185 at tyrosine. The EGF-induced effect requires interaction of the EGFR and its cognate ligand because cell lines that lack EGFR cannot be shown to phosphorylate p185, even when exposed to large amounts of EGF. Oncogenic rodent p185 and the human p185 homologue ERBB2 that is overexpressed in human breast tumor cells also can be shown to become phosphorylated on tyrosine residues by the action of EGF. Collectively, these data demonstrate that EGF mediates phosphorylation of p185 at tyrosine as well as serine/threonine through cellular kinases by a receptor-specific mechanism.     

Effect of protein kinase P on phosphorylations catalyzed by the epidermal growth factor.

Protein kinase P (PK-P) activated by histones or certain other basic compounds has been purified previously from yeast [Yanagita, Y., Abdel-Ghany, M., Raden, D., Nelson, N. & Racker, E. (1987) Proc. Natl. Acad. Sci. USA 84, 925-929]. It is shown here that PK-P is present in solubilized membranes of A-431 carcinoma cells where it changes the epidermal growth factor (EGF) receptor kinase activity. Polylysine, a weak PK-P activator, inhibited the autophosphorylation of the EGF receptor both in the absence and presence of EGF. Increased PK-P activity induced by histone 1, a potent activator, gave rise to increased autophosphorylation of the EGF receptor as well as phosphorylation at tyrosine residues of numerous other endogenous membrane components. The stimulation by histone was particularly striking in the presence of EGF. A similar stimulation was achieved with polylysine and EGF on addition of yeast PK-P. However, addition of yeast PK-P in the presence of histone 1 markedly inhibited the EGF-stimulated phosphorylation of endogenous membrane proteins. We conclude from these results that the effect of PK-P on the EGF receptor takes place in three phases: at low levels PK-P inhibits the autophosphorylation, at intermediate levels it stimulates the autophosphorylation as well as the EGF-dependent phosphorylation of numerous other membrane proteins, and at high levels it inhibits the phosphorylation of these proteins.     

Insect cell-expressed p180erbB3 possesses an impaired tyrosine kinase activity.

Protein kinases share a number of highly conserved or invariant amino acid residues in their catalytic domains, suggesting that these residues are necessary for kinase activity. In p180erbB3, a receptor tyrosine kinase belonging to the epidermal growth factor (EGF) receptor subfamily, three of these residues are altered, suggesting that this protein might have an impaired protein tyrosine kinase activity. To test this hypothesis, we have expressed human EGF receptor and bovine p180erbB3 in insect cells via baculovirus infection and have compared their autophosphorylation and substrate phosphorylation activities. We have found that, while the EGF receptor readily undergoes EGF-stimulated autophosphorylation and catalyzes the incorporation of phosphate into the model substrates (E4Y1)n (random 4:1 copolymer of glutamic acid and tyrosine) and GST-p85 (glutathione S-transferase fusion protein with the 85-kDa subunit of phosphatidylinositol 3-kinase), p180erbB3 autophosphorylation and substrate phosphorylation are at least 2 orders of magnitude less efficient. However, p180erbB3 is capable of binding the ATP analog 5'-p-fluorosulfonylbenzoyladenosine, indicating that the lack of observed kinase activity is probably not due to nonfunctional or denatured receptors expressed by the insect cells. On the basis of these results, we propose that p180erbB3 possesses an impaired intrinsic tyrosine kinase activity.     

Thyrotropin regulates autophosphorylation and kinase activity in both the insulin and the insulin-like growth factor-I receptors in FRTL5 cells.

TSH regulation of insulin and insulin-like growth factor-I (IGF-I) receptor kinases has been studied in FRTL5 cultured thyroid cells. Preincubation of intact cells with TSH increased by 2-fold insulin and IGF-I receptor autophosphorylation and phosphorylation of the p175 endogenous substrate for the receptors. Enhanced phosphorylations reached a maximum within 30 min, were maintained for 30 min more, and vanished after 120 min of TSH incubation. TSH dose-responses exhibited half-maximal and maximal effects at 1 and 10 pM, respectively. In vitro, insulin as well as IGF-I receptors purified from cells treated with 10 pM TSH also exhibited 2-fold enhanced receptor autophosphorylation and kinase activity toward the exogenous substrate poly(Glu,Tyr) (4:1). At variance with TSH, cell incubation with either 8-bromo-cAMP or the protein kinase-C activator 12-O-tetradecanoylphorbol-13-acetate inhibited insulin and IGF-I receptor kinases. In intact cells, TSH stimulation of insulin and IGF-I receptor kinases was accompanied by enhanced turnover of phosphate on autophosphorylated receptors, increased receptor tyrosine phosphorylation, and decreased receptor serine/threonine phosphorylation in response to insulin. Incubation of in vivo labeled insulin and IGF-I receptors with extracts from TSH-treated cells also decreased receptor phosphoserine and phosphothreonine content. Furthermore, preincubation of insulin and IGF-I receptors with extracts from TSH-treated cells enhanced in vitro autophosphorylation. The latter effect was inhibited by the serine/threonine phosphatase inhibitors fluoride and okadaic acid, but not by the tyrosine phosphatase inhibitor vanadate. The data suggest that in FRTL5 cells, TSH induces the activity of a Ser/Thr protein phosphatase, which dephosphorylates insulin and IGF-I receptors and enhances their endogenous kinases.     

Progesterone receptor subunits are high-affinity substrates for phosphorylation by epidermal growth factor receptor.

Purified preparations of epidermal growth factor (EGF) receptor were used to test hen oviduct progesterone receptor subunits as substrates for phosphorylation catalyzed by EGF receptor. Both the 80-kilodalton (kDa) (A) and the 105-kDa (B) progesterone receptor subunits were phosphorylated in a reaction that required EGF and EGF receptor. No phosphorylation of progesterone receptor subunits was observed in the absence of EGF receptor, even when Ca2+ was substituted for Mg2+ and Mn2+. Phospho amino acid analysis revealed phosphorylation at tyrosine residues, with no phosphorylation detectable at serine or threonine residues. Two-dimensional maps of phosphopeptides generated from phosphorylated 80- or 105-kDa subunits by tryptic digestion revealed similar patterns, with resolution of two major, several minor, and a number of very minor phosphopeptides. The Km of progesterone receptor for phosphorylation by EGF-activated EGF receptor was 100 nM and the Vmax was 2.5 nmol/min per mg of EGF receptor protein at 0 degrees C. The stoichiometry of phosphorylation/hormone binding for progesterone receptor subunits was 0.31 at ice-bath temperature and approximately 1.0 at 22 degrees C.     

Identification of insulin receptor substrate 1 serine/threonine phosphorylation sites using mass spectrometry analysis: regulatory role of serine 1223

Insulin receptor substrate 1 (IRS-1), an intracellular substrate of the insulin receptor tyrosine kinase, also is heavily phosphorylated on serine and threonine residues, and several serine phosphorylation sites alter the function of IRS-1. Because of the large number of serine/threonine residues, position-by-position analysis of these potential phosphorylation sites by mutagenesis is difficult. To circumvent this, we have employed matrix-assisted laser desorption/ionization time-of-flight and HPLC-electrospray ionization tandem mass spectrometry techniques to scan for serine and threonine residues that are phosphorylated in full-length human IRS-1 ectopically expressed in cells using an adenoviral vector. This approach revealed 12 phosphorylation sites on serine or threonine residues, 10 of which were novel sites. Seven of these sites were in proline-directed motifs, whereas five were in arginine-directed sites. Sequence inspection suggested that phosphorylation of Ser1223 might alter the interaction of IRS-1 with the protein tyrosine phosphatase Src homology domain 2 (SH2)-containing phosphatase-2 (SHP-2). Mutation of Ser1223 to alanine to prevent phosphorylation resulted in increased association of SHP-2 with IRS-1, decreased insulin-stimulated tyrosine phosphorylation of IRS-1 in CHO/IR cells, and decreased insulin-stimulated association of the p85 regulatory subunit of phosphatidylinositol-3-kinase with IRS-1. This mutation had no effect on association of IRS-1 with the insulin receptor. Sequence analysis showed the Ser1223 region to be widely conserved evolutionarily. These data suggest that phosphorylation of Ser1223 dampens association of IRS-1 with SHP-2, thereby increasing net insulin-stimulated tyrosine phosphorylation.     

The epidermal growth factor receptor and the product of the neu protooncogene are members of a receptor tyrosine phosphorylation cascade.

The protein product of the neu protooncogene, p185, is a tyrosine kinase with a high degree of sequence homology to the epidermal growth factor (EGF) receptor. Although p185 does not bind EGF, EGF stimulates tyrosine phosphorylation of p185. To determine the mechanism of this interaction we have used a vaccinia virus/bacteriophage T7-based transient gene expression system to induce production of normal and kinase-deficient forms of p185 in the absence and presence of EGF receptors. Tyrosine phosphorylation of kinase-deficient p185 was observed, but only in the presence of the EGF receptor. These findings strongly support the hypothesis that p185 is a substrate for the EGF receptor tyrosine kinase in a tyrosine kinase cascade.     

Tumor promoters block tyrosine-specific phosphorylation of the epidermal growth factor receptor.

Tyrosine-specific phosphorylation of the epidermal growth factor (EGF) receptor in hormonally stimulated A431 cells is blocked by three chemically distinct classes of tumor promoters. Tumor-promoting esters of the diterpene phorbol (phorbol 12-myristate 13-acetate, beta-phorbol 12,13-dibutyrate, and beta-phorbol 12,13-didecanoate), indole alkaloids (teleocidin and lyngbyatoxin A), and polyacetates ( aplysiatoxin and debromoaplysiatoxin ) all inhibited EGF-stimulated phosphorylation of the receptor. Non-tumor-promoting analogs (beta-phorbol, alpha-phorbol 12,13-didecanoate, and hydrolyzed teleocidin) had no effect on the levels of receptor phosphorylation. The ED50 values of the inhibitory effect (0.1-3 ng/ml) reflected the relative tumor-promoting abilities of these compounds in vivo. None of the tumor promoters tested significantly decreased the overall specific binding of 125I-labeled EGF to A431 cells. Scatchard analysis, however, revealed two apparent EGF receptors in this cell type. The dose-responses for tumor-promoter inhibition of EGF receptor tyrosine phosphorylation and high-affinity EGF binding were similar, suggesting that the same initial event is responsible for both effects. This demonstrates a correlation between modulation of EGF receptor binding and phosphorylation of tyrosine by tumor promoters. The data suggest a possible role for protein kinase C, the putative cellular receptor for these tumor promoters, in the mechanism of action.     

Purification and characterization of epidermal growth factor receptor/protein kinase from normal mouse liver.

We have purified the epidermal growth factor (EGF) receptor/protein kinase from the livers of normal mice by affinity chromatography. The biochemical properties of the liver receptor are very similar to those of the EGF receptor previously prepared from the human tumor cell line A-431 [Cohen, S., Ushiro, H., Stoscheck, C. & Chinkers, M. (1982) J. Biol. Chem. 257, 1523-1531]. The liver receptor for EGF is a glycoprotein of Mr 170,000. It binds 125I-labeled EGF and possesses an EGF-stimulable protein kinase activity specific for tyrosine residues. Both autophosphorylation and kinase activity toward exogenous substrates are demonstrable. The EGF receptor purified from normal mouse liver is antigenically related to the receptor purified from human A-431 cells.     

Müllerian inhibiting substance blocks autophosphorylation of the EGF receptor by inhibiting tyrosine kinase

The fetal regressor Müllerian inhibiting substance (MIS), in concentrations as low as picomolar, inhibited the growth of A-431 cells and the autophosphorylation of its epidermal growth factor (EGF) receptor. The inhibition of membrane phosphorylation was due neither to the reduction of the total number of EGF receptor binding sites, nor to stimulation of intrinsic phosphates, but rather to inhibition of tyrosine kinase activity. MIS control of EGF receptor autophosphorylation by tyrosine kinase may be one mechanism by which Müllerian duct regression in the embryo and the inhibition of A-431 proliferation is initiated. In addition, MIS as an inhibitor of phosphorylation may furnish a tool to probe the role of membrane phosphorylation in growth control.     

Altered epidermal growth factor (EGF)-stimulated protein kinase activity in variant A431 cells with altered growth responses to EGF

To determine the role of epidermal growth factor (EGF)-stimulated protein kinase in the biological effects caused by EGF, tyrosine-specific kinase activity has been quantitated in A431 human epidermoid carcinoma cells and six variant cell lines. Because EGF inhibited proliferation of A431 cells, variants resistant to this inhibition were selected by treatment with mutagen and maintenance for 1 month in 0.1 μM EGF. After cloning and growth for 6-20 generations without EGF, the resistance of the variants to the growth-inhibitory effect of EGF was confirmed. Whereas EGF increased cellular phosphotyrosine content ≈10-fold in parental A431 cells, EGF caused smaller or undetectable increases in the six variant cell lines. Solubilized membranes from the six variants displayed diminished EGF-stimulated phosphorylation of the EGF receptor and of antibodies to p60^src (the product of the Rous sarcoma virus transforming gene), which act as an exogenous substrate. The decrease in EGF-stimulated tyrosine-specific protein kinase activity varied from ≈40% (clone 16) to ≈8% (clone 18) of parental A431 activity. Phosphorylated EGF receptors from parental and variant cells migrated identically on sodium dodecyl sulfate/polyacrylamide gels. The number of EGF receptors in variant cells decreased in parallel with EGF-stimulated protein kinase activity, so that the specific activity of EGF-stimulated protein kinase per EGF receptor remained constant in the six variant cell lines with reductions in both activities to as low as 10%. These results suggest that this tyrosine-specific protein kinase activity mediates the growth-inhibitory effect of EGF on A431 cells and that both EGF binding and kinase activities reside in the same or tightly associated molecules.     

Catalysis of serine and tyrosine autophosphorylation by the human insulin receptor.

The protein kinase activity of human insulin receptors purified from Sf9 insect cells after infection with a recombinant baculovirus was evaluated. The following experimental observations led to the unexpected conclusion that this receptor protein catalyzes both serine and tyrosine autophosphorylation at significant stoichiometries. (i) Phosphorylation of lectin-purified insulin receptors with [gamma-32P]ATP resulted in rapid receptor tyrosine phosphorylation (7 mol of P per high-affinity binding site) and the delayed onset of insulin-stimulated receptor serine phosphorylation (about 7% of total phosphorylation). The tyrosine kinase inhibitor (hydroxy-2-naphthalenylmethyl)phosphonic acid (HNMPA), which has no effect on protein kinase C or cyclic AMP-dependent protein kinase activities, inhibited both the receptor serine and tyrosine phosphorylation. (ii) Phosphorylation of a synthetic peptide substrate composed of insulin receptor residues 1290-1319 on serines-1305/1306 by partially purified insulin receptors was also inhibited by HNMPA. (iii) Insulin receptors sequentially affinity-purified on immobilized wheat germ agglutinin and immobilized insulin showed no apparent contaminant proteins on silver-stained SDS/polyacrylamide gels yet catalyzed autophosphorylation on receptor serine and tyrosine residues when incubated with [gamma-32P]ATP. These results suggest that the catalytic site of the insulin receptor tyrosine kinase also recognizes receptor serine residues as substrates for the phosphotransfer reaction. Furthermore, insulin-stimulated receptor serine phosphorylation in intact cells may occur in part by an autophosphorylation mechanism subsequent to tyrosine phosphorylation of the insulin receptor.