Alterations in tubulin immunoreactivity; relation to secondary structure

Blood sinusoidal plasma membrane subfractions were isolated from normal mouse liver in the presence of the proteinase inhibitors PhMeSO2F and iodoacetamide. They were purified from smooth microsomal and Golgi vesicle contaminants. The phosphorylation reaction was studied at 33 degrees C, in the presence of 2 mM MnCl2. Addition of epidermal growth factor (EGF) to the preparations stimulated 32P incorporation from [gamma-32P]ATP or [gamma-32P]GTP essentially into one 170 000 Mr protein. Some incorporation was observed in a minor 120 000-Mr component which appears to be a degradation product of the 170 000-Mr component. No EGF-dependent phosphorylation of other membrane proteins or various exogenous proteins could be detected in vitro. The dephosphorylation of the 170 000-Mr component was observed after 4 min of incubation at 33 degrees C. This dephosphorylation reaction was inhibited by addition of 5 mM p-nitrophenyl phosphate but not by addition of micromolar Zn2+, Be2+ or orthovanadate. The 170 000-Mr protein specifically bound 125I-labeled EGF and thus appeared to be the hepatic EGF receptor. The EGF stimulatable kinase activity considerably enhances incorporation of 32P into tyrosine residues of the 170 000-Mr EGF receptor at 33 degrees C. Tryptic peptide maps of the 32P-labeled 170 000-Mr protein revealed a multiplicity of phosphorylated sites. Seven 32P-labeled phosphopeptides were observed after EGF stimulation, three of them being largely prominent. Tryptic peptide maps of the 170 000-Mr protein after it was covalently linked to 125I-labeled EGF showed only one 125I-labeled peptide, the migration of which appeared different from that of 32P-labeled phosphopeptides. These findings were confirmed by V8 protease unidimensional peptide mapping of the 170 000-Mr protein, labeled with 32P or 125I-EGF.     

A direct radioimmunoassay for human epidermal growth factor receptor using 32P-autophosphorylated receptor

A simple and reproducible radioimmunoassay of the epidermal growth factor (EGF) receptor which uses 32P-labeled EGF receptor and anti-receptor monoclonal antibodies is reported. In vitro phosphorylation of A431 cell membranes with [gamma-32P]ATP in the presence of 20% dimethyl sulfoxide (which stimulates autophosphorylation of the EGF receptor) and 10 microM Na3VO4 (a potent inhibitor of phosphotyrosyl protein phosphatase) provides radiolabeled EGF receptor for radioimmunoassay without further purification. The most selective phosphorylation of the EGF receptor is achieved at ATP concentrations of 0.1-0.2 microM, which corresponds to the reported Km value for the autophosphorylation reaction of the EGF receptor (W. Weber, P.J. Bertics, and G.N. Gill, 1984, J. Biol. Chem. 259, 14631-14939). The incorporation of 32P into EGF receptors increases in proportion to the increase of ATP concentration up to 6 mol of labeled phosphate at 2.0 microM ATP. The label is entirely on tyrosine residues. The cell membranes can be stored at -70 degrees C for 3 months without loss of immunoreactivity and autophosphorylating activity. Standard curves for the radioimmunoassay were constructed employing either A431 cell membranes or whole cell homogenates containing a known amount of EGF receptor. The assay can detect 7 X 10(10) EGF receptor molecules or 20 ng of the receptor protein, and can quantitatively distinguish the difference in EGF receptor numbers between A431 cells and 29E2 and KB cells with 10-fold and 15-fold fewer receptors than A431 cells, respectively. 29E2 cells and KB cells express twofold more immunoreactive EGF receptors than EGF-binding sites. In contrast, A431 cells possess the same number of immunoreactive sites and receptor sites for EGF binding. To assess total EGF receptor expression, it is necessary to use a method which detects EGF receptors regardless of their intrinsic kinase activity, or capacity to bind EGF. This radioimmunoassay detects immunoreactive receptor molecules, even those which do not bind EGF.     

Bovine heart pyruvate dehydrogenase kinase stimulation by monovalent ions

The effects of monovalent ions on endogenous pyruvate dehydrogenase (PDH) kinase activity in purified bovine heart pyruvate dehydrogenase complex were investigated. Activity of PDH kinase was stimulated 1.9-, 1.95-, 1.65-, and 1.4-fold by 10 mM K+, Rb+, NH+4, and Cs+, respectively, whereas Na+ and Li+ had no effect on PDH kinase activity. The crystal radii of stimulatory ions were in the range of 1.33 to 1.69 A while the crystal radii of nonstimulatory ions were in the range of 0.6 to 0.94 A. Stimulation of PDH kinase by monovalent ions was not pH dependent. Protein dilution studies showed that monovalent ion stimulation was measurable within 10 s after protein addition to PDH kinase assays. Furthermore, stimulation occurred at all protein concentrations tested. At ATP concentrations from 12.5 to 25 microM, K+ and NH+4 stimulation was constant from 0 to 110 and 0 to 30 mM, respectively. At higher ATP concentrations, from 50 to 500 microM, K+ and NH+4 stimulation peaked at approximately 30 and 3 mM, respectively, and thereafter declined as the ion concentration increased. Maximal PDH kinase stimulation by K+ or NH+4 also declined as Na+ was increased from 0 to 120 mM, but at a fixed salt concentration of 120 mM, both K+ and NH+4 stimulated PDH kinase activity. Phosphopeptide analysis demonstrated that K+ and NH+4 stimulated phosphorylation at sites 1 and 2, but that site 3 phosphorylation was relatively constant under all conditions. Thiamin pyrophosphate and 5,5'-dithiobis-(2-nitrobenzoate) blocked monovalent ion stimulation half-maximally at 4 and 6 microM, respectively. However, neither thiamin pyrophosphate nor 5,5'-dithiobis-(2-nitrobenzoate) significantly inhibited PDH kinase activity in the absence of monovalent ions. The results indicate that heart PDH kinase stimulation by monovalent ions does not occur by changing the binding equilibrium between PDH and dihydrolipoyl transacetylase core. Instead, monovalent ions bind and exert their regulatory effects at or near the active site of PDH kinase.     

Regulation of the epidermal growth factor receptor by phosphorylation

The receptor for epidermal growth factor (EGF) is a glycosylated transmembrane phosphoprotein that exhibits EGF-stimulable protein tyrosine kinase activity. On EGF stimulation, the receptor undergoes a self-phosphorylation reaction at tyrosine residues located primarily in the extreme carboxyl-terminal region of the protein. Using enzymatically active EGF receptor purified by immunoaffinity chromatography from A431 human epidermoid carcinoma cells, the self-phosphorylation reaction has been characterized as a rapid, intramolecular process which is maximal at 30-37 degrees C and exhibits a very low Km for ATP (0.2 microM). When phosphorylation of exogenous peptide substrates was measured as a function of receptor self-phosphorylation, tyrosine kinase activity was found to be enhanced two to threefold at 1-2 mol of phosphate per mol of receptor. Analysis of the dependence of the tyrosine kinase activity on ATP concentration yielded hyperbolic kinetics when plotted in double-reciprocal fashion, indicating that ATP can serve as an activator of the enzyme. Higher concentrations of peptide substrates were found to inhibit both the self- and peptide phosphorylation, but this inhibition could be overcome by first self-phosphorylating the enzyme. These results suggest that self-phosphorylation can remove a competitive/inhibitory constraint so that certain exogenous substrates can have greater access to the enzyme active site. In addition to self-phosphorylation, the EGF receptor can be phosphorylated on threonine residues by the calcium- and phospholipid-dependent protein kinase C. The sites on the EGF receptor phosphorylated in vitro by protein kinase C are identical to the sites phosphorylated on the receptor isolated from A431 cells exposed to the tumor promoters 12-O-tetradecanoylphorbol 13-acetate or teleocidin. This phosphorylation of the EGF receptor results in a suppression of its tyrosine kinase and EGF binding activities both in vivo and in vitro. The EGF receptor can thus be variably regulated by phosphorylation: self-phosphorylation can enhance tyrosine kinase activity whereas protein kinase C-catalyzed phosphorylation can depress enzyme activity. Because these two phosphorylations account for only a fraction of the phosphate present in the EGF receptor in vivo, other protein kinases can apparently phosphorylate the receptor and these may exert additional controls on EGF receptor/kinase function.     

Immunoaffinity purification of the epidermal growth factor receptor. Stoichiometry of binding and kinetics of self-phosphorylation

Epidermal growth factor (EGF) receptor protein has been purified in a single high-yield step by immunoaffinity chromatography of extracts of A431 cells. A monoclonal antibody directed against the EGF binding site of the receptor was immobilized to Sepharose 4B as a specific immune absorbent and competitive elution with EGF was used to obtain purified EGF receptor protein with tyrosine kinase activity. The stoichiometry of EGF binding was determined by comparing 125I-EGF binding to A431 cells with the mass of EGF receptor protein in those cells as measured by immunoaffinity chromatography, radioimmunoassay, and immune precipitation. Each measurement indicated one EGF binding site/EGF receptor protein molecule. Study of the kinetics of autophosphorylation revealed rapid incorporation of 1 mol of phosphate/mol of enzyme followed by slower incorporation of additional phosphate groups. The autophosphorylation reaction has a Km for ATP (0.2 microM) which is about 10-fold lower than that for phosphorylation of exogenous substrates. The kinetically preferred autophosphorylation is an intramolecular reaction.     

Differential in vitro phosphorylation of clathrin light chains by the epidermal growth factor receptor-associated protein tyrosine kinase and a pp60c-src-related spleen tyrosine kinase.

The epidermal growth factor (EGF) receptor-associated protein tyrosine kinase activity has been suggested to play important roles in the EGF-enhanced, clathrin-coated pit-mediated receptor internalization (W. S. Chen, C. S. Lazar, M. Peonie, R. Y. Tsien, G. N. Gill, and M. G. Rosenfeld, 1987, Nature 328, 820-823) but the kinase substrate important for this process has not been identified. This study demonstrates that the EGF receptor, partially purified from A431 epidermoid carcinoma cells, catalyzes the phosphorylation of one of the two clathrin light chains, clathrin light chain a (LCa). The phosphorylation activity is stimulated by EGF and immunoprecipitated by an EGF receptor monoclonal antibody. The phosphorylation occurs exclusively on tyrosine residues. Amino acid composition of the major tryptic phosphopeptide of the EGF receptor-phosphorylated LCa corresponds closely to that of residues 1 to 97 of LCa. A stoichiometry of 0.2 mol phosphate/mol LCa was attained after 60 min at 30 degrees C and a Km value of 1.7 microM was determined for the reaction. LCa of either neuronal or non-neuronal origin could serve as a substrate. In addition to the EGF receptor tyrosine kinase, a particulate src-related protein tyrosine kinase purified from bovine spleen (C. M. E. Litwin, H.-C. Cheng, and J. H. Wang, 1991, J. Biol. Chem. 226, 2557-2566) was shown in this study to also phosphorylate the light chains. However, in contrast to the EGF receptor phosphorylation, both clathrin light chains a and b were phosphorylated by the spleen kinase, suggesting that the two tyrosine kinases have differential site specificities. Given the specificity of LCa phosphorylation by the EGF receptor, we propose that LCa phosphorylation on a tyrosine residue(s) may be important in EGF-induced receptor internalization.     

Catalytic properties of glucose-6-phosphate dehydrogenase from pea leaves.

A homogeneous preparation of glucose-6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49) with a specific activity of 3.88 U/mg protein was isolated from pea (Pisum sativum L.) leaves. The molecular mass of the G6PDH is 79 +/- 2 kD. According to SDS-PAGE, the molecular mass of the enzyme subunit is 40 +/- 3 kD. The Km values for glucose-6-phosphate and NADP are 2 and 0.5 mM, respectively. The enzyme has a pH optimum of 8.0. Mg2+, Mn2+, and Ca2+ activate the enzyme at concentrations above 1 mM. Galactose-6-phosphate and fructose-6-phosphate inhibit the G6PDH from pea leaves. Fructose-1, 6-bisphosphate and galactose-1-phosphate are enzyme activators. NADPH is a competitive inhibitor of the G6PDH with respect to glucose-6-phosphate (Ki = 0.027 mM). ATP, ADP, AMP, UTP, NAD, and NADH have no effect on the activity of the enzyme.     

cAMP-dependent protein kinase stimulates epidermal growth factor-dependent phosphorylation of epidermal growth factor receptors

Epidermal growth factor (EGF)-dependent transfer of radiolabeled phosphate from [gamma-32P]ATP to 160-kDa EGF receptor solubilized from human epidermoid carcinoma A431 cell surface membranes was stimulated up to 3-fold by addition of 3',5'-cAMP and purified cAMP-dependent protein kinase. Phosphorylation of EGF receptors was stimulated to the same extent when cAMP-dependent protein kinase catalytic subunit was substituted for 3',5'-cAMP and cAMP-dependent protein kinase. Phosphoamino acid analysis revealed that the extent of phosphorylation of EGF receptor at tyrosine residues was the same regardless of whether cAMP-dependent protein kinase catalytic subunit was present in or omitted from the system. Increased EGF receptor phosphorylation occurring in response to cAMP-dependent protein kinase catalytic subunit was accounted for by phosphorylation at serine or threonine residues. In samples phosphorylated in the presence of cAMP-dependent protein kinase catalytic subunit, phosphate was present in tyrosine, serine, and threonine in a ratio of 32:60:8. Two-dimensional mapping of radiolabeled phosphopeptides produced from EGF receptors by digestion with trypsin revealed the generation of one additional major phosphoserine-containing peptide when cAMP-dependent protein kinase was present with EGF in the EGF receptor kinase system. Degradation of 160-kDa EGF receptors to a 145-kDa form by purified Ca2+-activated neutral protease produced a 145-kDa fragment with phosphoserine content increased over that present initially in the 160-kDa precursor.     

Amiloride directly inhibits growth factor receptor tyrosine kinase activity

Addition of amiloride to A431 human epidermoid carcinoma cell membranes inhibited autophosphorylation of the epidermal growth factor (EGF) receptor. The tyrosine phosphorylation of histone H2B catalyzed by an affinity-purified preparation of EGF receptor was also inhibited by amiloride. The inhibition was noncompetitive with respect to histone but competitive with ATP, suggesting that amiloride may act as an ATP analogue which causes the formation of nonproductive enzyme-substrate complexes. The tyrosine phosphorylation of histone H2B catalyzed by the purified EGF receptor was inhibited by amiloride at concentrations identical to those previously reported to block EGF action on cell proliferation (Ki = 350 microM). Amiloride similarly inhibited the tyrosine phosphorylation of the human placental insulin receptor and the platelet-derived growth factor receptor of Swiss 3T3 cells. Immunoprecipitation of the EGF receptor from A431 cells labeled for 24 h with [32P]phosphate demonstrated that amiloride decreased the phosphorylation of the EGF receptor on serine and threonine residues and blocked the effect of EGF to cause phosphorylation of the receptor on tyrosine residues. Phosphoamino acid analysis of total cell proteins indicated that amiloride inhibited the increase in phosphotyrosine levels caused by EGF. We conclude that amiloride directly inhibits the tyrosine kinase activity of the receptors for EGF, insulin, and platelet-derived growth factor in in vitro and can mediate such actions in vivo. This effect of amiloride demonstrates that it is unsuitable as a drug to test the hypothesis that the stimulation of the Na+/H+ antiporter is essential for mitogenic signaling by growth factor receptors.     

The nuclear matrix from rat liver is capable of phosphorylating exogenous tyrosine-containing substrates

The nuclear matrix isolated from rat liver phosphorylated exogenous tyrosine-containing substrates angiotensin II and synthetic polymer (Glu, Tyr; 4:1). The phosphorylation reaction was dependent on Mn2+ or Mg2+, but the former was the preferred ion. Km values for poly(Glu,Tyr; 4:1) and ATP were 0.2 mM and 4 microM, respectively. Angiotensin II showed a lower affinity for the kinase than poly(Glu,Tyr; 4:1). The isoflavone genistein, a specific inhibitor for tyrosine phosphorylation, inhibited the tyrosine kinase activity in the nuclear matrix.     

Tannic acid, a potent inhibitor of epidermal growth factor receptor tyrosine kinase

Increasing evidence supports the hypothesis that tannic acid, a plant polyphenol, exerts anticarcinogenic activity in chemically induced cancers. In the present study, tannic acid was found to strongly inhibit tyrosine kinase activity of epidermal growth factor receptor (EGFr) in vitro (IC50 = 323 nM). In contrast, the inhibition by tannic acid of p60(c-src) tyrosine kinase (IC50 = 14 microM) and insulin receptor tyrosine kinase (IC50 = 5 microM) was much weaker. The inhibition of EGFr tyrosine kinase by tannic acid was competitive with respect to ATP and non-competitive with respect to peptide substrate. In cultured cells, growth factor-induced tyrosine phosphorylation of growth factor receptors, including EGFr, platelet-derived growth factor receptor, and basic fibroblast growth factor receptor, was inhibited by tannic acid. No inhibition of insulin-induced tyrosine phosphorylation of insulin receptor and insulin-receptor substrate-1 was observed. EGF-stimulated growth of HepG2 cells was inhibited in the presence of tannic acid. The inhibition of serine/threonine-specific protein kinases, including cAMP-dependent protein kinase, protein kinase C and mitogen-activated protein kinase, by tannic acid was only detected at relatively high concentration, IC50 being 3, 325 and 142 microM respectively. The molecular modeling study suggested that tannic acid could be docked into the ATP binding pockets of either EGFr or insulin receptor. These results demonstrate that tannic acid is an in vitro potent inhibitor of EGFr tyrosine kinase.     

Bovine heart pyruvate dehydrogenase kinase stimulation by alpha-ketoisovalerate

Purified bovine heart pyruvate dehydrogenase complex was used to investigate the effects of monovalent cations and alpha-ketoisovalerate on pyruvate dehydrogenase (PDH) kinase inhibition by thiamin pyrophosphate. Initial velocity patterns for thiamin pyrophosphate inhibition were consistent with hyperbolic non-competitive or hyperbolic uncompetitive inhibition at various K+ concentrations between 0 and 120 mM. The Kis, Kid, and Kin for thiamin pyrophosphate were in the range of 0.009 to 5.1 microM over the range of K+ concentrations tested. In the absence of K+, 1 mM alpha-ketoisovalerate had no effect on PDH kinase inhibition by thiamin pyrophosphate, whereas in the presence of 20 mM K+, alpha-ketoisovalerate stimulated PDH kinase activity almost 2-fold over the range of 0-80 microM thiamin pyrophosphate. Half-maximal stimulation by alpha-ketoisovalerate occurred at about 200 microM in the presence of 100 microM thiamin pyrophosphate and 20 mM K+. Similar but less extensive changes occurred in the presence of 100 microM thiamin pyrophosphate and 1 mM NH4+. Initial velocity patterns for PDH kinase inhibition by thiamin pyrophosphate in the presence of 2 mM alpha-ketoisovalerate were mixed noncompetitive, but alpha-ketoisovalerate increased the Vm and Km for adenosine 5'-triphosphate in the presence of inhibitor. In the presence of thiamin pyrophosphate, PDH kinase remained stimulated after chromatography on Sephadex G-25 to remove alpha-ketoisovalerate. The results indicate that acylation of pyruvate dehydrogenase complex by alpha-ketoisovalerate results in PDH kinase stimulation but only in the presence of monovalent cations and thiamin pyrophosphate.     

Angiotensin II stimulates rat astrocyte mitogen-activated protein kinase activity and growth through EGF and PDGF receptor transactivation

We showed that the intracellular tyrosine kinases src and pyk2 mediate angiotensin II (Ang II) stimulation of growth and ERK1/2 mitogen-activated protein (MAP) kinase phosphorylation in astrocytes. In this study, we investigated whether the membrane-bound receptor tyrosine kinases platelet-derived growth factor (PDGF) and epidermal growth factor (EGF) receptors mediate Ang II stimulation of ERK1/2 and astrocyte growth. Ang II significantly stimulated PDGF and EGF receptors in a dose- and time-dependent manner. The PDGF receptor and the EGF receptor were maximally stimulated with 100 nM Ang II (0.98+/-0.18- and 4.4+/-1.4-fold above basal, respectively). This stimulation occurred as early as 5 min, and was sustained for at least 15 min for both receptor tyrosine kinases. Moreover, 1 microM AG1478 and 0.25 microM PDGFRInhib attenuated Ang II stimulation of the EGF and PDGF receptors, respectively. Ang II-induced phosphorylation of ERK1/2 and astrocyte growth was mediated by both PDGF and EGF receptors. This report also provides novel findings that co-inhibiting EGF and PDGF receptors had a greater effect to decrease Ang II-induced ERK1/2 (90% versus 49% and 71% with PDGF receptor and EGF receptor inhibition, respectively), and astrocyte growth (60% versus 10% and 32% with PDGF receptor and EGF receptor inhibition, respectively). In conclusion we showed in astrocytes that the PDGF and the EGF receptors mediate Ang II-induced ERK1/2 phosphorylation and astrocyte growth and that these two receptors may exhibit synergism to regulate effects of the peptide in these cells.     

The insulin-like growth factor II receptor is phosphorylated by a tyrosine kinase in adipocyte plasma membranes

Incorporation of 32P from [gamma-32P]ATP into tyrosine residues of the insulin-like growth factor (IGF)-II receptor was observed in a Triton X-100-insoluble fraction of rat adipocyte plasma membranes. IGF-II receptor phosphorylation proceeded to a stoichiometry of approximately 0.5 mol of phosphate/IGF-II binding site after 10 min of incubation at 4 degrees C. A Km for ATP of 6 microM was calculated for this phosphorylation reaction. Addition of IGF-II caused an approximately 2-fold increase in tyrosine phosphorylation of the IGF-II receptor in this preparation. In contrast, phosphorylation of angiotensin II by the Triton X-100 washed membranes was not stimulated by IGF-II. Incubation of purified receptor immobilized on IGF-II agarose or of receptor-enriched low density microsomal membranes with [gamma-32P]ATP did not result in appreciable incorporation of [32P]phosphate into the IGF-II receptor nor into exogenous substrates. These data suggest that the IGF-II receptor is not a tyrosine protein kinase capable of autophosphorylation but that it is a substrate for a tyrosine protein kinase endogenous to the adipocyte plasma membrane. The stimulatory effect of IGF-II on the tyrosine phosphorylation of its receptor may be due to a conformational change which converts the receptor to a better substrate for this tyrosine kinase.     

Reversibility of the epidermal growth factor receptor self-phosphorylation reaction. Evidence for formation of a high energy phosphotyrosine bond

The reversibility of the epidermal growth factor (EGF) receptor self-phosphorylation reaction was studied using highly purified receptor from A431 human epidermoid carcinoma cells. Self-phosphorylation is inhibited by the reaction product ADP in a dose-dependent manner exhibiting an IC50 approximately 2 microM. In addition, phosphorylated EGF receptor can be rapidly dephosphorylated in the presence of ADP. The dephosphorylation reaction results in equimolar production of ATP and loss of phosphate from the receptor. The reverse reaction is dependent on time and ADP exhibiting a t1/2 of 15 s and a Km(ADP) = 0.40 +/- 0.14 microM. The dephosphorylation reaction can be effectively inhibited by an exogenous peptide substrate for the forward reaction, i.e., the src-peptide (a synthetic peptide corresponding to one of the self-phosphorylation sites in p60v-src). This suggests that the dephosphorylation reaction is intrinsic to the EGF receptor. The equilibrium constant, K, for the self-phosphorylation reaction was estimated to be 0.5-1.6 using kinetic and reactant/product concentration analyses. Assuming that the standard free energy change, delta G0, for ATP hydrolysis is -9.5 kcal/mol, an observed delta G0 for hydrolysis of the EGF receptor phosphotyrosine bond was calculated to be -9 to -10 kcal/mol. These results indicate that the EGF receptor self-phosphorylation reaction, which appears important in the regulation of EGF receptor function, is readily reversible and that the phosphotyrosine bond formed by this reaction is of relatively high energy.     

C-kinase phosphorylates the epidermal growth factor receptor and reduces its epidermal growth factor-stimulated tyrosine protein kinase activity

The Ca2+- and phospholipid-dependent protein kinase (C-kinase) binds tightly in the presence of Ca2+ to purified membranes of A431 human epidermoid carcinoma cells. The major membrane substrate for C-kinase is the epidermal growth factor (EGF) receptor. Phosphorylation of the EGF receptor is Ca2+-dependent and occurs at threonine and serine residues. After tryptic digestion of the receptor, three major phosphothreonine-containing peptides were identified. These are identical with three new phosphopeptides present in the EGF receptor isolated from A431 cells treated with either of the tumor promoters 12-O-tetradecanoylphorbol 13-acetate or teleocidin. C-kinase catalyzes phosphorylation at these same sites in purified EGF receptor protein. These results indicate that, in A431 cells exposed to tumor promoters, C-kinase catalyzes phosphorylation of a significant population of EGF receptor molecules. This phosphorylation of EGF receptors results in decreased self-phosphorylation of the EGF receptor at tyrosine residues both in vivo and in vitro and in decreased EGF-stimulated tyrosine kinase activity in vivo.     

Insulin-EGF receptor chimerae mediate tyrosine transphosphorylation and serine/threonine phosphorylation of kinase-deficient EGF receptors.

To study cross-talk between unoccupied epidermal growth factor (EGF) receptors and activated EGF receptor kinases, we have used double-transfected cells, IHE2 cells, expressing both an enzymatically active insulin-EGF chimeric receptor and an inactive kinase EGF receptor mutant. Using immunoaffinity-purified receptors, we show that insulin increased phosphorylation of the insulin-EGF chimeric beta subunit and of the kinase-deficient EGF receptor. Stimulation of intact IHE2 cells with insulin leads to a rapid tyrosine autophosphorylation of the insulin-EGF chimeric beta subunit and to tyrosine phosphorylation of the unoccupied kinase-deficient EGF receptor. Insulin-stimulated transphosphorylation of the kinase-deficient EGF receptor yields the same pattern of tryptic phosphopeptides as those in EGF-induced autophosphorylation of the wild-type human EGF receptor. We conclude that insulin, through activation of the insulin-EGF chimeric receptor, mediates transphosphorylation of the kinase-deficient EGF receptor, further confirming that EGF receptor autophosphorylation may proceed by an intermolecular mechanism. In addition to receptor tyrosine phosphorylation, we find that exposure of cells to insulin results in enhanced phosphorylation on serine and threonine residues of the unoccupied kinase-deficient EGF receptor. These results suggest that insulin-EGF chimeric receptor activation stimulates at least one serine/threonine kinase, which in turn phosphorylates the kinase-deficient EGF receptor. Finally, we show that transphosphorylation and coexpression of an active kinase cause a decrease in the number of cell surface kinase-deficient EGF receptors without increasing their degradation rate.     

Evidence that the tyrosine kinase domain of a small fraction of epidermal growth factor receptor molecules is exposed on the outer surface of A431 cells

Intact A431 cells were labeled with [gamma-32P]ATP. The major phosphorylation product of the ecto-kinase activity of A431 cells had the molecular mass of 170 kd and was identified as EGF receptor by specific immunoprecipitation. This phosphorylation was not stimulated by EGF added to the reaction buffer, but replacement of MgCl2 by MnCl2 in the buffer remarkably stimulated phosphorylation. An exogenous protein substrate, alpha-casein, was also phosphorylated by intact A431 cells. The analyses for phospho-amino acids of both EGF receptor and alpha-casein revealed that phosphorylation occurred mainly at phosphotyrosine residues. Tryptic phospho-peptides of the EGF receptor of intact A431 cells labeled with [gamma-32P]ATP were fractionated by HPLC. The elution patterns were essentially the same as that of the autophosphorylated EGF receptor, indicating that the phosphorylation sites of EGF receptor labeled in vivo with [gamma-32P]ATP are located in three tyrosine residues in the carboxyl terminus. These results indicate that the carboxyl-terminal tyrosine kinase domain of a small fraction of the EGF receptor molecules of an A431 cell is exposed on the outer surface of the cells.     

Differences in the sites of phosphorylation of the insulin receptor in vivo and in vitro

Phosphorylation of the insulin receptor was studied in intact well differentiated hepatoma cells (Fao) and in a solubilized and partially purified receptor preparation obtained from these cells by affinity chromatography on wheat germ agglutinin agarose. Tryptic peptides containing the phosphorylation sites of the beta-subunit of the insulin receptor were analyzed by reverse-phase high performance liquid chromatography. Phosphoamino acid content of these peptides was determined by acid hydrolysis and high voltage electrophoresis. Separation of the phosphopeptides from unstimulated Fao cells revealed one major and two minor phosphoserine-containing peptides and a single minor phosphothreonine-containing peptide. Insulin (10(-7) M) increased the phosphorylation of the beta-subunit of the insulin receptor 3- to 4-fold in the intact Fao cell. After insulin stimulation, two phosphotyrosine-containing peptides were identified. Tyrosine phosphorylation reached a steady state within 20 s after the addition of insulin and remained nearly constant for 1 h. Under our experimental conditions, no significant change in the amount of [32P]phosphoserine or [32P]phosphothreonine associated with the beta-subunit was found during the initial response of cells to insulin. When the insulin receptor was extracted from the Fao cells and incubated in vitro with [gamma-32P]ATP and Mn2+, very little phosphorylation occurred in the absence of insulin. In this preparation, insulin rapidly stimulated autophosphorylation of the receptor on tyrosine residues only and high performance liquid chromatography analysis of the beta-subunit digested with trypsin revealed one minor and two major phosphopeptides. The elution position of the minor peptide corresponded to that of the major phosphotyrosine-containing peptide obtained from the beta-subunit of the insulin-stimulated receptor labeled in vivo. In contrast, the elution position of one of the major phosphopeptides that occurred during in vitro phosphorylation corresponded to the minor phosphotyrosine-containing peptide phosphorylated in vivo. The other major in vitro phosphotyrosine-containing peptide was not detected in vivo. Our results indicate that: tyrosine phosphorylation of the insulin receptor occurs rapidly following insulin binding to intact cells; the level of tyrosine phosphorylation remains constant for up to 1 h; the specificity of the receptor kinase or accessibility of the phosphorylation sites are different in vivo and in vitro.(ABSTRACT TRUNCATED AT 400 WORDS)