Factor Receptor Protein Tyrosine Kinase Research Articles

Semicochliodinol A and B: inhibitors of HIV-1 protease and EGF-R protein tyrosine kinase related to asterriquinones produced by the fungus Chrysosporium merdarium.

The known bisalkylated 2,5-dihydroxybenzoquinones didemethylasterriquinone D and isocochliodinol as well as the new metabolites semicochliodinol A and B have been isolated as inhibitors of HIV-1 protease from the culture broth of the fungus Chrysosporium merdarium P-5656. The structures were elucidated by spectroscopic methods. The NMR spectra of two compounds were completely assigned. The metabolites inhibit HIV-1 protease with an IC50 value as low as 0.17 microM and epidermal growth factor receptor protein tyrosine kinase at 15 to 60 microM and are therefore valuable lead compounds for these targets. Molecular modelling of the HIV-1-protease-inhibitor complexes showed hydrogen bonding between the dihydroxybenzoquinone moiety of didemethylasterriquinone D and isocochliodinol to both active-site aspartic acids (Asp25/Asp25') of the protease and the indole parts of the inhibitors filling the P2 and P2' pockets of the protease.

Requirement for c-Src Catalytic Activity and the SH3 Domain in Platelet-derived Growth Factor BB and Epidermal Growth Factor Mitogenic Signaling

The Src family protein-tyrosine kinases are required for mitogenic signaling from the platelet-derived growth factor (PDGF), colony stimulating factor-1, and epidermal growth factor (EGF) receptor protein-tyrosine kinases (RPTK) (Twamley-Stein, G. M., Pepperkok, R., Ansorge, W., and Courtneidge, S. A. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 7696-7700; Roche, S., Koegl, M., Barone, M. V., Roussel, M. F., and Courtneidge, S. A.(1995) Mol. Cell. Biol. 15, 1102-1109). In NIH3T3 fibroblasts, c-Src, Fyn, and c-Yes associate with the activated PDGF receptor, are substrates for receptor phosphorylation, and are themselves activated. Src family catalytic function is required for RPTK mitogenic signaling as evidenced by the SH2-dependent dominant negative phenotype exhibited by kinase-inactive Src and Fyn mutants (Twamley-Stein, G. M., Pepperkok, R., Ansorge, W., and Courtneidge, S. A.(1993) Proc. Natl. Acad. Sci. U. S. A. 90, 7696-7700). Here, we have generated clonal Src- murine fibroblast cell lines overexpressing various murine c-Src mutants and studied the effect of these mutant Src proteins on PDGF- and EGF-induced mitogenesis. Two c-Src SH3 domain mutants, Y133F and Y138F, each inhibited PDGF BB- and EGF-induced DNA synthesis in quiescent cells. This demonstrates an involvement of the Src SH3 domain in PDGFbeta and EGF receptor mitogenic signaling. Since both Tyr-133 and Tyr-138 are located on the ligand binding surface of the SH3 domain, these results suggest that the c-Src SH3 domain is required for PDGF and EGF mitogenic signaling. The dominant negative effect of either single mutant on PDGF receptor signaling was reversed by a second SH2-inactivating mutation. We conclude that the c-Src SH3 domain function requires the SH2 domain in the case of the PDGF receptor, presumably because binding of c-Src to the receptor via its SH2 domain is a prerequisite for the SH3 domain function. In contrast, SH2 function is apparently not essential for the SH3 function in EGF receptor signaling.

The requirements for G1 checkpoint progression of Trypanosoma brucei S 427 clone 1

Trypanosoma brucei S 427 clone 1 accumulated in G1 when incubated under growth-limiting conditions. Further incubation of the G1-restricted organisms in medium containing 10% fetal bovine serum (FBS) and 2 mM hydroxyurea resulted in their reversible arrest after a G1 checkpoint beyond which serum was not required for progress into and through S. Progress of the G1-restricted T. brucei through the G1 checkpoint was linear and required continuous incubation with exogenous serum growth factors. These were principally low and high density lipoproteins; both lipoproteins triggered G1 progression in a dose- and time-dependent manner whilst their removal by immunoaffinity chromatography severely reduced the capacity of FBS to stimulate G1 progression. Serum-induced progress of T. brucei through G1 was Ca2+-independent, but required gene transcription, protein synthesis, and continuous kinase activity that was inhibited by tyrphostin 51 and DAPH 1 which typically inhibit epidermal growth factor receptor protein tyrosine kinase activity. The tyrphostin 51-sensitive catalytic activity was not required for T. brucei protein synthesis, glycolysis, or S phase progression but was required for tyrosine phosphorylation of several polypeptides, none of which was specifically associated with serum-induced G1 progression.

Radiation-Induced Autophosphorylation of Epidermal Growth Factor Receptor in Human Malignant Mammary and Squamous Epithelial Cells

In an effort to identify events initiating up-regulation of epidermal growth factor receptor after single and repeated radiation exposures, we investigated the role of epidermal growth factor receptor, a receptor protein tyrosine kinase, in radiation-induced signal transduction. Human malignant mammary, MCF-7, and squamous, A431, cells showed low baseline phospho-tyrosine levels of epidermal growth factor receptor, permitting reproducible dose-dependent stimulation of epidermal growth factor receptor autophosphorylation after exposure to epidermal growth factor. MCF-7 cells exhibited a mean 2.3-fold increase (95% confidence interval: 1.91, 2.65; P < 0.0001) in levels of epidermal growth factor phosphorylation in response to exposures of 2 Gy, which was substantially less than the epidermal growth factor receptor Y phosphorylation induced by epidermal growth factor. A quantitatively similar radiation response was seen in A431 cells. In the dose range of 1 to 4 Gy, no clear dose response was seen. There was a rapid induction of radiation-induced epidermal growth factor receptor Y phosphorylation, starting within 2 min, with maximum values between 0.5 and 5 min after radiation exposure followed by a slower decline to baseline levels after 20 min. The data presented identify the epidermal growth factor receptor protein tyrosine kinase associated with the plasma membrane as one target for ionizing radiation in the dose range used in radiotherapy.

Nucleotide Binding by the Epidermal Growth Factor Receptor Protein-tyrosine Kinase: TRINITROPHENYL-ATP AS A SPECTROSCOPIC PROBE

The nucleotide binding properties of the epidermal growth factor (EGF) receptor protein-tyrosine kinase were investigated with the fluorescent nucleotide analog 2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate (TNP-ATP). TNP-ATP was found to be an active substrate for the autophosphorylation reaction of the recombinant EGF receptor protein-tyrosine kinase domain (TKD). Whereas the Vmax for the TNP-ATP-dependent autophosphorylation reaction was approximately 200-fold lower than that of ATP, the Km for this reaction was similar to that observed with ATP. The nucleotide analog was also shown to be an inhibitor of the ATP-dependent autophosphorylation and substrate phosphorylation reactions of the TKD. Spectroscopic studies demonstrated both a high affinity binding of TNP-ATP to the recombinant TKD and a markedly enhanced fluorescence of the bound nucleotide analog. The fluorescence of enzyme-bound TNP-ATP was attenuated in the presence of ATP, which enabled determination of the dissociation constants for both ATP and the Mn2+ complex of ATP. A truncated form of the EGF receptor TKD lacking the C-terminal autophosphorylation domain exhibited an enhanced affinity for TNP-ATP, which indicated that the autophosphorylation domain occupied the peptide substrate binding site of the TKD and modulated the binding of the nucleotide substrates.

Molecular design and biological activity of potent and selective protein kinase inhibitors related to balanol

Background: The protein kinase C (PKC) family of serine/threonine-specific protein kinases is involved in many cellular processes, and the -unregulated activation of PKC has been implicated in carcinogenesis. PKC inhibitors thus have significant potential as chemotherapeutic agents. Recently, the fungal metabolite balanol was shown to be an exceptionally potent inhibitor of PKC. We previously developed a practical and efficient total synthesis of balanol. We set out to use this synthetic molecule, and several synthetic analogs, to probe the mechanism of PKC inhibition and to determine the effect of balanol on the activity of other protein kinases. Results: As well as inhibiting PKC, balanol is a potent inhibitor of cyclic AMP-dependent protein kinase (PKA), another protein serine/threonine kinase. Balanol does not, however, inhibit the Src or epidermal growth factor receptor protein tyrosine kinases. The inhibition of both PKC and PKA by balanol can be overcome by high concentrations of ATP, and molecular modeling studies suggest that balanol may function as an ATP structural analog. Although balanol discriminates rather poorly between PKC and PKA, only minor modifications to its molecular structure are required to furnish compounds that are highly specific inhibitors of PKA. Conclusions: A number of balanol analogs have been designed and synthesized that, unlike balanol itself, exhibit dramatic selectivity between PKA and PKC. Thus, despite the substantial homology between the catalytic domains of PKA and PKC, there is enough difference to allow for the development of potent and selective inhibitors acting in this region. These inhibitors should be useful tools for analyzing signal transduction pathways and may also aid in the development of drugs with significant therapeutic potential.

Localization of two mouse genes encoding the protein tyrosine kinase receptor-related protein RYK

We have mapped the gene encoding the murine RYK growth factor receptor protein tyrosine kinase by genetic linkage analysis with recombinant inbred strains of mouse. Two distinct Ryk loci (Ryk-1 and Ryk-2) were identified. Ryk-1 mapped to Chromosome (Chr) 9, whereas Ryk-2 mapped to Chr 12. A similar arrangement of RYK-related loci was previously determined in the human. Synteny has already been established between murine Chr 9 in the region of Ryk-1, and human chromosome 3q11-12, the location of the human RYK-1 gene. However, the Ryk-2/RYK-2 loci on murine Chr 12 and human Chr 17p13.3 define a new region of synteny.

Sequence Requirements for Binding of Src Family Tyrosine Kinases to Activated Growth Factor Receptors

Activation of growth factor receptor protein tyrosine kinases frequently results in the binding of numerous proteins to their tyrosine-phosphorylated cytoplasmic domains. These interactions involve the SH2 domains of the binding proteins and phosphorylated tyrosines on the receptor molecules, with the specificity of interaction dictated by the amino acid composition surrounding the phosphorylated tyrosine. In the case of the platelet-derived growth factor (PDGF) receptor, the major binding site for Src family tyrosine kinases is in the juxtamembrane domain and includes tyrosine 579 (Mori, S., Rönnstrand, L., Yokote, K., Engström, A., Courtneidge, S. A., Claesson-Welsh, L., and Heldin, C-H. (1993) EMBO J. 12, 2257-2264). To analyze in more detail which amino acids surrounding the phosphorylated tyrosine at position 579 were important for high affinity interaction with Src family kinases, we synthesized a series of phosphopeptides corresponding to this binding site in which single amino acids were individually changed and tested their ability to compete with the PDGF receptor for binding of Fyn. We found that not only the three residues carboxyl-terminal to the phosphorylated tyrosine were important but that also residues at positions -1 and +4 relative to the tyrosine were required. Phosphorylation of both tyrosines 579 and 581 significantly increased competition efficiency. The activated colony stimulating factor-1 (CSF-1) receptor, which is known to associate with Src family kinases, has a sequence in its juxtamembrane region similar to that surrounding Tyr-579 of the PDGF receptor, and a phosphopeptide modeled on this sequence competed the association of Fyn with the receptor in vitro. Furthermore, mutational analysis demonstrated that these sequences were required for the efficient association of Src family kinases with the activated CSF-1 receptor in vivo. Phosphopeptides corresponding to the Src family binding sites of both PDGF and CSF-1 receptors activated Src kinase activity in vitro. These observations support a model in which the enzymatic activity of Src family tyrosine kinases is controlled by intra- and intermolecular interactions of tyrosine-phosphorylated peptides with the SH2 domain of the kinases.

A conserved amino-terminal Shc domain binds to phosphotyrosine motifs in activated receptors and phosphopeptides.

Signal transduction by growth factor receptor protein-tyrosine kinases is generally initiated by autophosphorylation on tyrosine residues following ligand binding. Phosphotyrosines within activated receptors form binding sites for the Src homology 2 (SH2) domains of cytoplasmic signalling proteins. One such protein, Shc, is tyrosine phosphorylated in response to a large number of growth factors and cytokines. Phosphorylation of Shc on tyrosine residue Y317 allows binding to the SH2 domain of Grb2, and hence stimulation of the Ras pathway. Shc is therefore implicated as an adaptor protein able to couple normal and oncogenic protein-tyrosine kinases to Ras activation. Shc itself contains an SH2 domain at its carboxyl terminus, but the function of the amino-terminal half of the protein is unknown. We have found that the Shc amino-terminal region binds to a number of tyrosine-phosphorylated proteins in v-src-transformed cells. This domain also bound directly to the activated epidermal growth factor (EGF) receptor. A phosphotyrosine (pY)-containing peptide modeled after the Shc-binding site in polyoma middle T antigen (LLSNPTpYSVMRSK) was able to compete efficiently with the activated EGF receptor for binding to the Shc amino terminus. This competition was dependent on phosphorylation of the tyrosine residue within the peptide, and was abrogated by deletion of the leucine residue at position -5. The Shc amino-terminal domain also bound to the autophosphorylated nerve growth factor receptor (Trk), but bound significantly less well to a mutant receptor in which tyrosine Y490 in the receptor's Shc-binding site had been substituted by phenylalanine. These data implicate the amino-terminal region of Shc in binding to activated receptors and other tyrosine-phosphorylated proteins. Binding appears to be specific for phosphorylated tyrosine residues within the sequence NPXpY, which is conserved in many Shc-binding sites. The Shc amino-terminal region bears only very limited sequence identify to known SH2 domains, suggesting that it represents a new class of phosphotyrosine-binding modules. Consistent with this view, the amino-terminal Shc domain is highly conserved in a Drosophila Shc homologue. Binding of Shc to activated receptors through its amino terminus could leave the carboxy-terminal SH2 domain free for other interactions. In this way, Shc may function as an adaptor protein to bring two tyrosine-phosphorylated proteins together.

Paeciloquinones A, B, C, D, E and F: new potent inhibitors of protein tyrosine kinases produced by Paecilomyces carneus. I. Taxonomy, fermentation, isolation and biological activity.

Paeciloquinones A to F as well as versiconol have been isolated as inhibitors of protein tyrosine kinase from the culture broth of the fungus Paecilomyces carneus P-177. The novel anthraquinones inhibit epidermal growth factor receptor protein tyrosine kinase in the micromolar range. Two compounds, paeciloquinones A and C, are potent and selective inhibitors of the v-abl protein tyrosine kinase with an IC50 of 0.4 microM. Dependent on the fermentation conditions, partially different sets of paeciloquinones may be produced. An HPLC method allows separation of all major active components.

Epidermal growth factor-dependent association of phosphatidylinositol 3-kinase with the erbB3 gene product.

The ErbB3 protein is a member of the ErbB subfamily of receptor protein tyrosine kinases. In the present study, the mechanism by which the ErbB3 protein is phosphorylated and the signal-transducing functions of this phosphorylated protein were investigated. When phosphorylated by the epidermal growth factor receptor in vitro, the ErbB3 protein strongly associated with the regulatory p85 subunit and the catalytic activity of phosphatidylinositol (PI) 3-kinase. The association of PI 3-kinase with ErbB3 in human breast cancer cells was found to be correlated with the constitutive phosphorylation of ErbB3 on tyrosine residues. In MDA-MB-468 breast cancer cells in which the ErbB3 protein is not constitutively phosphorylated, stimulation with epidermal growth factor led to the phosphorylation of ErbB3 on tyrosine residues and the formation of a functional signal transduction complex involving the ErbB3 protein and PI 3-kinase. These results suggest that the ErbB3 protein can be phosphorylated on tyrosine residues by a cross-phosphorylation mechanism and that the phosphorylated ErbB3 protein can couple other growth factor receptor protein tyrosine kinases to the PI 3-kinase pathway in a manner similar to the insulin receptor substrate 1 protein.

Overexpression of the tyrosine phosphatase PTP1B is associated with human ovarian carcinomas

OBJECTIVE: Our purpose was to determine whether protein tyrosine phosphatase 1B is overexpressed in ovarian cancers, possibly altering the balance of intracellular tyrosine phosphorylation. STUDY DESIGN: The expression of protein tyrosine phosphatase 1B was assayed in frozen sections from 54 human ovarian carcinomas and seven normal ovaries by immunochemical staining with monoclonal antibody AE4-2J, which is specific for protein tyrosine phosphatase 1B. The expression of protein tyrosine phosphatase 1B - specific messenger ribonucleic acid in tumors was determined by Northern analysis. The results were analyzed statistically by means of Fisher's exact test. RESULTS: Minimal staining was observed in normal ovarian epithelium. In contrast, 43 of 54 (79.6%) tumors displayed increased protein tyrosine phosphatase 1B expression, which is statistically associated with malignancy. Overexpression was associated with the expression of the p185c-erbB-2, p170EGFR, and p165mCSFR growth factor receptor protein tyrosine kinases. Protein tyrosine phosphatase 1B messenger ribonucleic acid expression was inconsistently increased in tumor cells. CONCLUSION: Increased expression of protein tyrosine phosphatase 1B in ovarian cancers that also express protein tyrosine kinases suggests that protein tyrosine phosphatase 1B may play a role in the growth regulation of ovarian cancers. (AM J OBSTET GYNECOL 1994;170:1177-83.)

Nitrostyrene Derivatives of Adenosine 5′‐Glutarates Modified with an Alkyl Spacer and their inhibitory activity on epidermal growth factor receptor protein tyrosine kinase

Abstractβ‐Nitrostyrene derivatives of adenosine 5′‐glutarates are potent and selective bisubstrate‐type inhibitors of the epidermal growth factor receptor protein tyrosine kinase (EGF‐R PTK). In an attempt to improve the inhibitory activity, this type of compounds was modified with alkyl spacers of varying length between the nitrostyrene and the glutaryl units. The spacers consisted of 1, 3, 4, and 5 atoms to give compounds of the benzyl, oxyethyl, oxypropyl, and oxybutyl series, respectively (Schemes 1 and 2). Adenosine 5′‐esters were prepared in the benzyl and oxypropyl series only. Compared to the compounds in the parent series without spacer (IC50 = 0.7–12 μM), most of the modified compounds inhibited the EGF‐R PTK only marginally or were inactive (IC50 ≥ 100 μM). The only exceptions were the free acids 19 and 20 with IC50 values of ca. 5 μM. It is noteworthy that esterification of these two hydrogen glutarates with either MeOH or adenosine yielded inactive compounds, which is in contrast to the corresponding substances without spacers.

Aggregation-induced activation of the epidermal growth factor receptor protein tyrosine kinase.

Various agents are able to stimulate the EGF receptor protein tyrosine kinase in the absence of ligand binding. To characterize their mechanism of action, we investigated their effects on the kinase activity of the intracellular domain of the EGF receptor (EGFR-IC). EGFR-IC (67 kDa) lacking the extracellular domain and transmembrane segment of the EGF receptor, but retaining kinase and autophosphorylation domains, was produced and purified as a soluble, cytoplasmic protein from Sf9 insect cells infected with a recombinant baculovirus. EGFR-IC was able to undergo autophosphorylation in a manner similar to full-length EGFR. Synthetic substrate peptides showed similar affinity to EGFR-IC as to the full-length receptor. The activity of the EGFR-IC was found to be dependent on divalent cations, Mn2+ being a more potent activator than Mg2+. Agents capable of aggregating the kinase by direct interaction (cross-linking antibodies, polycations) or through altering the surrounding solvent structure and thereby decreasing protein solubility [ammonium sulfate, poly(ethylene glycol), 2-methyl-2,4-pentanediol] activated the kinase in a manner which correlated with their ability to precipitate the EGFR intracellular domain. The widely different chemical nature of these agents suggests that they do not act by direct interaction with specific allosteric regulatory sites, but rather by facilitating the interactions between kinase molecules. These results support the hypothesis that full-length receptor aggregation itself, induced by ligand binding to the extracellular domain, results in intracellular domain interactions and the activation of kinase activity.

Arylamides of hydroxylated isoquinolines as protein-tyrosine kinase inhibitors

Aryl-substituted amides of isomeric 6,7- and 7,8-dihydroxyisoquinoline-3-carboxamides ( 2 and 3)_ were prepared. Divergent structural requirements were observed for inhibiting p56 lck and epidermal growth factor receptor (EGFR) protein-tyrosine kinases (PTKs), with the 7,8-dihydroxy substitution pattern being essential for p56 lck activity, and the arylamide being required for EGFR potency. The work presents a useful approach toward the design of inhibitors which can discriminate between different PTKs.

PC12 cell neuronal differentiation is associated with prolonged p21 ras activity and consequent prolonged ERK activity

Expression of oncogenic ras in PC12 cells causes neuronal differentiation and sustained protein tyrosine phosphorylation and activity of extracellular signal-regulated kinases (ERKs), p42 erk2 and p44 erk1 . Oncogenic N- ras-induced neuronal differentiation is inhibited by compounds that block ERK protein tyrosine phosphorylation or ERK activity, indicating that ERKs are not only activated by p21 ras but serve as the primary downstream effectors of p21 ras . Treatment of PC12 cells with nerve growth factor or fibroblast growth factor results in neuronal differentiation and in a sustained elevation of p21 ras activity, of ERK activity, and of ERK tyrosine phosphorylation. Epidermal growth factor, which does not cause neuronal differentiation, stimulates only transient (<1 hr) activation of p21 ras ERKs. These data indicate that transient activation of p21 ras and, consequently, ERKs is not sufficient for induction of neuronal differentiation. Prolonged ERK activity is required: a consequence of sustained activation of p21 ras by the growth factor receptor protein tyrosine kinase.

Nitrostyrene Derivatives of Adenosine 5′‐Glutarates as Selective Inhibitors of the Epidermal Growth Factor Receptor Protein Tyrosine Kinase

AbstractThe syntheses and biological activities of some nitrostyrene derivatives of adenosine 5′‐glutarates, a novel class of selective, bi‐substrate‐type inhibitors of the EGF receptor protein tyrosine kinase with IC50 values around 1 μM. Only marginal inhibition of the tyrosine kinases v‐able and c‐src and of the serine/threonine kinase PKC was observed. Compounds 8, 9, 11, and 12 – lacking the adenosine moiety – were ten times less active than the most potent derivatives, whereas 17 – lacking the nitrostyryl part – showed no inhibitory activity at all. Most of the compounds showed potent antiproliferative activity against an EGF‐dependent mouse keratinocyte cell line.

Potentiation of epidermal growth factor receptor protein-tyrosine kinase activity by sulfate

The protein-tyrosine kinase activity of the epidermal growth factor (EGF) receptor is critical for EGF-stimulated cell growth, although little is known about the molecular details of its enzymatic activity. Previous studies have found that EGF receptor kinase activity can be stimulated by factors such as ammonium sulfate ((NH 4) 2SO 4), but the manner in which (NH 4) 2SO 4 induces this effect is unclear. Therefore, we have explored the processes by which (NH 4) 2SO 4 potentiated tyrosine kinase activity to better understand not only the molecular events involved in (NH 4) 2SO 4 activation, but also the kinetic properties and mechanism of the EGF receptor. In this study, the addition of an optimum concentration of (NH 4) 2SO 4 (250 mM) resulted in a 5-fold stimulation of kinase activity toward the peptide substrate, angiotensin II. The sulfate group is primarily involved in this action, since other salts containing SO 4 2− increased kinase activity similarly, whereas salts containing Cl − and F − had less of an effect, and divalent salts such as HPO 4 2− were inhibitory at doses of 1 mM or more. In addition, EGF receptor kinase activation by (NH 4) 2SO 4 did not strictly correlate with changes in the ionic strength or conductivity of the solution. However, several lines of evidence suggest that SO 4 2− directly alters the kinetic properties of the EGF receptor kinase: (1) the maximum velocity ( V max) and K m (ATP) for EGF receptor phosphorylation of angiotensin II were substantially higher in the presence of (NH 4) 2SO 4. (2) EGF receptor kinase activity in the absence of (NH 4) 2SO 4 required either Mn 2+ or Mg 2+, yet in the presence of (NH 4) 2SO 4, only Mn 2+ supported the increase in kinase activity. (3) Ammonium sulfate addition altered the product inhibition pattern of ADP versus angiotensin II, suggesting that an enzyme-angiotensin II-ADP complex can form in the presence of (NH 4) 2SO 4 but not in its absence. (4) The near-maximal rate of self-phosphorylation was not affected by (NH 4) 2SO 4, but the apparent K m(ATP) was greatly increased. From these results, we propose a model for (NH 4) 2SO 4 stimulation of EGF receptor kinase activity in which SO 4 2− interacts directly with the receptor or receptor-Mn 2+-ATP complex and alters reactant binding and the catalytic efficiency of the tyrosine kinase.

Alteration of the kinetic properties of the epidermal growth factor receptor tyrosine kinase by basic proteins.

Previous studies have shown that lysine- and arginine-rich proteins can enhance the activity of tyrosine and serine/threonine protein kinases. However, the kinetics and mechanism of this activation are not fully understood. Therefore we investigated the ability of poly(amino acids) and the arginine-rich protein, protamine, to alter the kinetic properties of epidermal growth factor (EGF) receptor protein-tyrosine kinase activity using immunoaffinity-purified receptor isolated from human epidermoid carcinoma (A431) cells. Poly(L-lysine), poly(L-arginine) and protamine stimulated EGF receptor kinase activity by 3-5-fold at non-saturating doses of ATP and peptide substrate, while poly(L-glutamate) had no effect. Initial kinetic studies demonstrated an increase in the maximum velocity and a decrease in the apparent Km for the peptide substrate angiotensin II in the presence of the basic effectors. Further analysis of the kinetic mechanism by product inhibition revealed that protamine altered the pattern of ADP inhibition towards the peptide substrate but not towards ATP. The change was indicative of the receptor's ability to form an enzyme-angiotensin II-ADP ternary complex in the presence of protamine but not in its absence. In addition, the basic effectors had a substantially decreased influence on the kinase activity of a C-terminally truncated form of the EGF receptor. Thus the changes in kinase activity may be partially mediated by the C-terminal region of the receptor, which contains the sites of receptor self-phosphorylation. These results suggest that the basic domains of proteins can interact with the EGF receptor to induce changes in its kinetic properties, especially with regard to reactant recognition and binding.

Mechanism of desensitization of the epidermal growth factor receptor protein-tyrosine kinase.

The intrinsic protein-tyrosine kinase activity of the epidermal growth factor (EGF) receptor is required for signal transduction. Increased protein-tyrosine kinase activity is observed following the binding of EGF to the receptor. However, signaling is rapidly desensitized during EGF treatment. We report that EGF receptors isolated from desensitized cells exhibit a lower protein-tyrosine kinase activity than EGF receptors isolated from control cells. The mechanism of desensitization of kinase activity can be accounted for, in part, by the EGF-stimulated phosphorylation of the receptor at Ser1046/7, a substrate for the multifunctional calmodulin-dependent protein kinase II in vitro. Mutation of Ser1046/7 by replacement with Ala residues blocks desensitization of the EGF receptor protein-tyrosine kinase activity. Furthermore, this mutation causes a marked inhibition of the EGF-stimulated endocytosis and down-regulation of cell surface receptors. Thus, the phosphorylation site Ser1046/7 is required for EGF receptor desensitization in EGF-treated cells. This regulatory phosphorylation site is located at the carboxyl terminus of the EGF receptor within the subdomain that binds src homology 2 regions of signaling molecules.