Activation of the epidermal growth factor receptor tyrosine protein kinase in the absence of receptor oligomerization

The epidermal growth factor (EGF) receptor exists in a monomeric (170 kDa) form and in several aggregated states (360 kDa, greater than 500 kDa). The hypothesis that the oligomerization of the receptor is required for the stimulation of the kinase was tested by correlating the oligomeric state of the receptor with the protein kinase activity. EGF and sphingosine stimulate the phosphorylation of an exogenous peptide substrate by the receptor to an equal extent. Chemical cross-linking using disuccinimidyl suberate and the analysis of EGF receptor complexes by Western blotting demonstrated that EGF caused the aggregation of receptors. Similar results were obtained when [32P]phosphate-labeled receptors were cross-linked using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride. These results were confirmed by sucrose density gradient sedimentation analysis. In contrast to the effects of EGF, incubation of EGF receptors with sphingosine did not cause the oligomerization of the receptors. These data demonstrate that the EGF receptor kinase can be stimulated independently of the aggregation of the receptors.     

Activation mechanism of solubilized epidermal growth factor receptor tyrosine kinase.

Dimerization of epidermal growth factor receptor (EGFR) leads to the activation of its tyrosine kinase. To elucidate whether dimerization is responsible for activation of the intracellular tyrosine kinase domain or just plays a role in the stabilization of the active form, the activated status of wild-type EGFR moiety in the heterodimer with kinase activity-deficient mutant receptors was investigated. The kinase activity of the wild-type EGFR was partially activated by EGF in the heterodimer with intracellular domain deletion (sEGFR) or ATP binding-deficient mutant (K721A) EGFRs, while the wild-type EGFR in the heterodimer of wild-type and phosphate transfer activity-deficient mutant receptor D813N could be fully activated. After treatment with EGF, the ATP binding affinity and the V(max) of the wild-type EGFR increased. In the presence of sEGFR, a similar increase in the affinity for ATP was observed, but V(max) did not change. A two-step activation mechanism for EGFR was proposed: upon binding of EGF, the affinity for ATP increased and then, as a result of interaction between the neighboring tyrosine kinase domain, V(max) increased.     

Suppression of protein tyrosine kinase activity of the epidermal growth factor receptor by epidermal growth factor

Epidermal growth factor (EGF) receptor protein kinase activity, estimated by the use of peptide substrates, was reduced by as much as 70% after the treatment of intact A431 human carcinoma cells with EGF. The apparent decrease in protein kinase activity was observed after immunoprecipitation of the receptor or after purification of the receptor by lectin chromatography. By the use of [35S]methionine, it was determined that the total amount of receptor obtained was the same whether or not cells were treated with EGF. EGF stimulated the purified receptor protein kinase activity in vitro; however, the EGF-stimulated activity of receptor from EGF-treated cells continued to be reduced by as much at 70% compared to the EGF-stimulated activity from untreated cells. The reduction in receptor protein kinase activity induced by EGF may represent a feedback mechanism by which responsiveness to the growth factor is regulated.     

Generation of recombinant cytoplasmic domain of epidermal growth factor receptor with intrinsic protein tyrosine kinase activity

We have generated a recombinant baculovirus using the high expression vector pVL941 containing the complementary DNA encoding the intracellular domain of the human epidermal growth factor receptor (EGFR-IC). Upon infection of Spodoptera frugiperda insect cells, protein tyrosine kinase-active EGFR-IC was produced. The expressed protein has a molecular weight of 61,000 and is specifically recognized by antibodies directed against peptides representing different regions of human EGFR-IC. Upon sonication of infected cells, EGFR-IC was detected in both the soluble and insoluble fractions of the cell lysate. About 20-50% of the expressed EGFR-IC was soluble. Metabolic labeling and protein analyses showed that EGFR-IC comprised 7% of newly synthesized proteins in the cytoplasmic lysate and 0.1-0.2% of the total soluble protein. We have used a three-step purification procedure (fast-Q-Sepharose and phenyl-Sepharose column chromatographies and 30% ammonium sulfate precipitation) to purify EGFR-IC to 85% purity with 15-20% recovery from the initial soluble lysate. A yield of 3-4 mg of purified EGFR-IC has been consistently produced from 20 roller bottles with 2-4 x 10(8) infected cells/bottle. The tyrosine kinase activity was retained through purification. The enzyme demonstrated much higher autophosphorylation activity in the presence of Mn2+ than Mg2+. Phosphopeptide mapping revealed the same autophosphorylation sites utilized by EGFR-IC as those identified in wild-type EGFR. EGFR-IC-catalyzed phosphorylation of either a synthetic peptide representing the major autophosphorylation site or angiotensin II showed that the baculovirus-expressed EGFR-IC exhibits similar enzymatic kinetic characteristics to the intact activated EGFR kinase.     

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.     

Antibody-induced dimerization activates the epidermal growth factor receptor tyrosine kinase

The relationship between epidermal growth factor receptor (EGF-R) protein tyrosine kinase activation and ligand-induced receptor dimerization was investigated using several bivalent anti-EGF-R antibodies directed against various receptor epitopes. In A431 membrane preparations and permeabilized cells, all antibodies were able to activate the EGF-R tyrosine kinase, as measured by EGF-R autophosphorylation and phosphorylation of other substrates on tyrosine residues. EGF-R tyrosine kinase activation correlated strongly with the induction of EGF-R dimerization. (i) Both processes specifically occurred in a narrow antibody concentration range; (ii) both processes required the presence of detergent; and (iii) both processes depended on antibody bivalence since monovalent Fab fragments were inactive yet regained full activity after cross-linking by a second bivalent antibody. These data demonstrate that antibody bivalence is essential and sufficient for EGF-R activation and that activation occurs regardless of the EGF-R epitope recognized. Finally, EGF-R dimerization was shown not to depend on receptor autophosphorylation since it still occurred in the absence of ATP. Also, partial inhibition of the tyrosine kinase activity by the specific EGF-R tyrosine kinase inhibitor tyrphostin AG 213 did not affect formation of EGF-R dimers. Taken together these results demonstrate that induction of EGF-R dimerization is sufficient and in case of antibody action, essential, for activation of the EGF-R tyrosine kinase and thus provide strong support for an intermolecular mechanism of EGF-R tyrosine kinase activation.     

Calmodulin inhibits the epidermal growth factor receptor tyrosine kinase.

We demonstrate in this report that the epidermal growth factor (EGF) receptor from rat liver can be isolated by calmodulin affinity chromatography by binding in the presence of Ca2+ and elution with a Ca(2+)-chelating agent. The bulk of the EGF receptor is not eluted by a NaCl gradient in the presence of Ca2+. We ascertained the identity of the isolated receptor by immunoblot and immunoprecipitation using a polyclonal antibody against an EGF receptor from human origin. The purified receptor is autophosphorylated in tyrosine residues in an EGF-stimulated manner, and EGF-dependent phosphorylation of serine residues was also detected. Both the EGF and the transforming growth factor-alpha stimulate the tyrosine-directed protein kinase activity of the isolated receptor with similar affinities. Furthermore, we demonstrate that calmodulin inhibits the EGF-dependent tyrosine-directed protein kinase activity associated to the receptor in a concentration-dependent manner. This inhibition is partially Ca2+ dependent and is not displaced by increasing the concentration of EGF up to an EGF/calmodulin ratio of 10 (mol/mol). In addition, calmodulin was phosphorylated in an EGF-stimulated manner in the presence of a basic protein (histone) as cofactor and in the absence, but not in the presence, of Ca2+.     

Activation of p38 mitogen-activated protein kinase promotes epidermal growth factor receptor internalization

Endocytic trafficking plays an important role in the regulation of the epidermal growth factor receptor (EGFR). To address if cellular kinases regulate EGFR internalization, we used anisomycin, a potent activator of kinase cascades in mammalian cells, especially the stress-activated mitogen-activated protein (MAP) kinase subtypes. Here, we report that activation of p38 MAP kinase by anisomycin is sufficient to induce internalization of EGFR. Anisomycin and EGF employ different mechanisms to promote EGFR endocytosis as anisomycin-induced internalization does not require tyrosine kinase activity or ubiquitination of the receptor. In addition, anisomycin treatment did not result in delivery and degradation of EGFR at lysosomes. Incubation with a specific inhibitor of p38, or depletion of endogenous p38 by small interfering RNAs, abolished anisomycin-induced internalization of EGFR while having no effect on transferrin endocytosis, indicating that the effect of p38 activation on EGFR endocytosis is specific. Interestingly, inhibition of p38 activation also abolished endocytosis of EGFR induced by UV radiation. Our results reveal a novel role for p38 in the regulation of EGFR endocytosis and suggest that stimulation of EGFR internalization by p38 might represent a general mechanism to prevent generation of proliferative or anti-apoptotic signals under stress conditions.     

Ratiometric assay of epidermal growth factor receptor tyrosine kinase activation

Activation of cells is frequently followed by tyrosine phosphorylation of proteins. To quantify this process, we developed a ratiometric enzyme-linked immunosorbent assay (ELISA) using epidermal growth factor receptors (EGFR) as a model. Microtiter dishes were coated with anti-EGFR monoclonal antibodies to capture the receptor followed by parallel detection of receptor and phosphotyrosine content with secondary antibodies. The ratio of these two parameters was found to directly reflect EGFR activation and was insensitive to the effect of receptor downregulation. Our assay could resolve differences in EGFR activation due to small changes (less than 1 ng/ml) in ligand. We found that phosphotyrosine detection by ELISA was 8- to 32-fold more sensitive than Western blot detection and could be reliably detected using as little as 4 ng of cellular lysate. Detection of EGFR levels by ELISA was 30 times more sensitive than Western blot analysis and was reliable for as low as 8 ng of cellular lysate per well. Because of the wide linear range of the ELISA, we could directly compare receptor activation in cell types with different EGFR expression levels. Our assay provides a rapid and sensitive method of determining EGFR activation status and could be easily modified to evaluate any tyrosine-phosphorylated protein.     

Abl tyrosine kinase regulates endocytosis of the epidermal growth factor receptor

Signal attenuation from ligand-activated epidermal growth factor receptor (EGFR) is mediated in part by receptor endocytosis and trafficking to the lysosomal degradative compartment. Uncoupling the activated EGFR from endocytosis and degradation has emerged as a mechanism for oncogenic activation of the EGFR. The Abl nonreceptor tyrosine kinase is activated by ligand-stimulated EGFR, but the role of Abl in EGFR signaling has not been defined. Here we uncovered a novel role for the activated Abl kinase in the regulation of EGFR endocytosis. We show that activated Abl impairs EGFR internalization. Moreover, we show that activated Abl phosphorylates the EGFR primarily on tyrosine 1173, and that mutation of this site to phenylalanine restores ligand-dependent endocytosis of the EGFR in the presence of activated Abl. Furthermore, we show that activated Abl allows the ligand-activated EGFR to escape Cbl-dependent down-regulation by inhibiting the accumulation of Cbl at the plasma membrane in response to epidermal growth factor stimulation and disrupting the formation of the EGFR.Cbl complex without affecting Cbl protein stability. These findings reveal a novel role for Abl in promoting increased cell-surface expression of the EGFR and suggest that Abl/EGFR signaling may cooperate in human tumors.     

The epidermal growth factor receptor tyrosine kinase phosphorylates connexin32

Oncoprotein 18 or stathmin was isolated from bovine brain, characterized and novel features of its function as a microtubule depolymerizing factor were tested.The effect of phosphorylation of stathmin on its function as a microtubule depolymerizing factor has been tested in vitro. Five different protein kinases, protein kinase A, MAP kinase, cdc2 kinase, glycogen synthase kinase 3 and casein kinase 2, were used to modify stathmin, since it is known that these kinases could phosphorylate several residues that are modified in vivo and could have important roles in stathmin function. The residues phosphorylated in vitro by the different protein kinases were identified and in some cases they correspond to those modified in vivo.Recombinant unphosphorylated stathmin and native stathmin, which was previously dephosphorylated with alkaline phosphatase, showed similar microtubule depolymerizing activity. This activity is higher than that of stathmin phosphorylated by protein kinase A, MAP kinase or cdc 2 kinase, whereas phosphorylation of the protein with casein kinase 2 or glycogen synthase kinase 3 resulted in a slight increase of the depolymerizing activity.     

Acidic fibroblast growth factor receptor purified from bovine liver is a novel protein tyrosine kinase

Acidic fibroblast growth factor (aFGF) receptor was purified from plasma membranes of bovine liver using Triton X-100 extraction, wheat germ lectin-Sepharose 4B gel affinity chromatography, and DEAE-cellulose anion-exchange chromatography. As previously reported for the aFGF receptor in murine fibroblasts (Huang, S. S., and Huang, J. S. (1986) J. Biol. Chem. 261, 9568-9571), the purified aFGF receptor was also found to be a 135-kDa glycoprotein which showed an intrinsic and ligand-stimulated autophosphorylation activity. The 32P-labeled aFGF receptor was specifically immunoprecipitated by anti-FGF receptor (anti-flg/bek/cek gene product) antiserum. In contrast to other growth factor receptors/protein tyrosine kinases, the protein tyrosine kinase activity (autophosphorylation) of the aFGF receptor was stimulated (approximately 1.5-fold) by low concentrations of Mn2+, Mg2+, and Ca2+ (optimal concentrations of approximately 0.1, approximately 0.1, and 1 microM, respectively) but inhibited by higher concentrations of Mn2+, Mg2+, Ca2+, and pyrophosphate (greater than or equal to 20, greater than or equal to 50, greater than or equal to 10, and greater than or equal to 100 microM, respectively). However, addition of Mn2+ and pyrophosphate at a ratio of 1:1 not only reversed the inhibitory effect but also enhanced the kinase activity about 3-4-fold. The apparent Km of ATP for intrinsic and ligand-stimulated protein kinase activity of the aFGF receptor was estimated to be 25 microM. The preferred exogenous substrates for the protein tyrosine kinase activity of the aFGF receptor were found to be myelin basic protein and histone. Poly-L-arginine, an inhibitor for aFGF binding to the receptor, appeared to stimulate the mitogenesis or cell growth of responsive cells by mimicking aFGF activity.     

Inhibition of epidermal growth factor receptor tyrosine kinase ameliorates collagen-induced arthritis

Rheumatoid arthritis (RA) is an autoimmune synovitis characterized by the formation of pannus and the destruction of cartilage and bone in the synovial joints. Although immune cells, which infiltrate the pannus and promote inflammation, play a prominent role in the pathogenesis of RA, other cell types also contribute. Proliferation of synovial fibroblasts, for example, underlies the formation of the pannus, while proliferation of endothelial cells results in neovascularization, which supports the growth of the pannus by supplying it with nutrients and oxygen. The synovial fibroblasts also promote inflammation in the synovium by producing cytokines and chemokines. Finally, osteoclasts cause the destruction of bone. In this study, we show that erlotinib, an inhibitor of the tyrosine kinase epidermal growth factor receptor (EGFR), reduces the severity of established collagen-induced arthritis, a mouse model of RA, and that it does so by targeting synovial fibroblasts, endothelial cells, and osteoclasts. Erlotinib-induced attenuation of autoimmune arthritis was associated with a reduction in number of osteoclasts and blood vessels, and erlotinib inhibited the formation of murine osteoclasts and the proliferation of human endothelial cells in vitro. Erlotinib also inhibited the proliferation and cytokine production of human synovial fibroblasts in vitro. Moreover, EGFR was highly expressed and activated in the synovium of mice with collagen-induced arthritis and patients with RA. Taken together, these findings suggest that EGFR plays a central role in the pathogenesis of RA and that EGFR inhibition may provide benefits in the treatment of RA.     

Enhancement of tyrosine kinase activity of the Drosophila epidermal growth factor receptor homolog by alterations of the transmembrane domain

The Drosophila epidermal growth factor receptor homolog (DER) displays sequence similarity to both the epidermal growth factor (EGF) receptor and the neu vertebrate proteins. We have examined the possibility of deregulating the tyrosine kinase activity of DER by introducing structural changes which mimic the oncogenic alterations in the vertebrate counterparts. Substitution of valine by glutamic acid in the transmembrane domain, in a position analogous to the oncogenic mutation in the rat neu gene, elevated the in vivo kinase activity of DER in Drosophila Schneider cells sevenfold. A chimera containing the oncogenic neu extracellular and transmembrane domains and the DER kinase region, also showed a threefold elevated activity relative to a similar chimera with normal neu sequences. Double truncation of DER in the extracellular and cytoplasmic domains, mimicking the deletions in the v-erbB oncogene, did not however result in stimulation of in vivo kinase activity. The chimeric constructs were also expressed in monkey COS cells, and similar results were obtained. The ability to enhance the DER kinase activity by a specific structural modification of the transmembrane domain demonstrates the universality of this activation mechanism and strengthens the notion that this domain is intimately involved in signal transduction. These results also support the inclusion of DER within the tyrosine-kinase receptor family.     

Activation of a cytosolic serine protein kinase by epidermal growth factor

Exposure of A-431 cells to epidermal growth factor (EGF) results in a rapid enhancement (approximately 10-fold) of cytosolic serine protein kinase activity. The increase in serine kinase activity may be detected using a number of peptide and protein substrates. Enhancement of kinase activity occurs within 1 min of exposure of the cells to EGF and reaches a maximum in 5 min. Similar results were obtained with a variety of cell lines. We have partially purified the EGF-activated kinase from A-431 cells. It has an apparent molecular mass of approximately 100 kDa by gel filtration. One distinguishing property of the enzyme is its sensitivity to inhibition by micromolar quantities of polyarginine; polylysine has no effect. The EGF-activated kinase is unaffected by cyclic nucleotides, Ca2+/calmodulin, Ca2+/diolein/phosphatidylserine, or heparin. The enhancement of cytosolic serine kinase activity in A-431 cells appears to be an early event in cell "activation" by a number of biological response modifiers including EGF, bradykinin, 12-O-tetradecanoylphorbol-13-acetate, and histamine.     

Pharmacogenomics of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors

The EGFR is a validated anticancer target whose successful exploitation has added novel agents to our current treatment protocols. Subsets of patients have shown to benefit the most from these therapies, and though these differential responses have yet to be completely defined, they are mostly of genetic nature. Egfr amplifications have shown to increase sensitivity to both small molecule inhibitors and specific monoclonal antibodies targeting the EGFR. A somatic/germline egfr intron 1 CA repeat sequence polymorphism has shown to have an important role in the control of EGFR protein expression, and has been linked to an increased risk of familial breast cancer, a worse outcome in patients with colorectal cancer, and anti-EGFR treatment efficacy in preclinical models. Egfr activating mutations have been recently described in lung cancer linking a cluster of genotypes with sensitivity to EGFR tyrosine kinase pharmacological inhibition. Despite the initial excitement that this discovery elicited, follow-up reports have not unequivocally confirmed this finding, and these drugs have been solidly efficacious both in individual patients and in diseases generally lacking egfr mutations such as pancreas cancer. We are witnessing exciting developments in the field of the pharmacogenomics of cancer, and this has particularly evolved in the area pertaining EGFR tyrosine kinase inhibitors. This review will discuss the background and currently available preclinical and clinical data.     

Inhibition of epidermal growth factor receptor tyrosine kinase by chalcone derivatives.

In our previous study, butein, a chalcone derivative, was found to be an inhibitor of tyrosine kinases and the inhibition was ATP-competitive. In this work, chalcone and seven chalcone derivatives were used to analyse the relationship between the structure of these compounds and their inhibition of tyrosine kinase activity. Three of chalcone derivatives, including butein, marein and phloretin, were found to have an ability to inhibit the tyrosine kinase activity of epidermal growth factor receptor (EGFR) in vitro. IC(50) was 8 microM for butein, 19 microM for marein and 25 microM for phloretin. The structural characterisations of these inhibitors suggest that the hydroxylations at C4 and C4' of these molecules may be required for them to act as EGFR tyrosine kinase inhibitors. The inhibition of EGF-induced EGFR tyrosine phosphorylation by butein was also observed in human hepatocellular carcinoma HepG2 cells, while marein and phloretin were inactive at the doses tested. Molecular modelling suggests that butein, marein and phloretin can be docked into the ATP binding pocket of EGFR. Hydrogen bonds and hydrophobic interaction appear to be important in the binding of these inhibitors to EGFR.     

Protein kinase A mediates cAMP-induced tyrosine phosphorylation of the epidermal growth factor receptor

An increase in the intracellular cAMP concentration induces tyrosine phosphorylation of the epidermal growth factor receptor (EGFR) followed by activation of extracellular signal-regulated kinases 1/2 (ERK1/2). In this report we demonstrate that these effects of cAMP are mediated via activation of protein kinase A (PKA). Chemical inhibition of PKA suppressed forskolin-induced EGFR tyrosine phosphorylation and ERK1/2 activation in PC12 cells. Furthermore, forskolin failed to induce significant tyrosine phosphorylation of the EGFR and ERK1/2 activation in PKA-defective PC12 cells. Forskolin-induced EGFR tyrosine phosphorylation was also observed in A431 cells and in membranes isolated from these cells. Phosphoamino acid analysis indicated that the recombinant catalytic subunit of PKA elicited phosphorylation of the EGFR on both tyrosine and serine but not threonine residues in A431 membranes. Together, our data indicate that activation of PKA mediates the effects of cAMP on the EGFR and ERK1/2. While PKA may directly phosphorylate the EGFR on serine residues, PKA-induced tyrosine phosphorylation of the EGFR occurs by an indirect mechanism.     

Protein kinase C inhibition of the epidermal growth factor receptor tyrosine protein kinase activity is independent of the oligomeric state of the receptor

Treatment of A431 human epidermoid carcinoma cells with 4-phorbol 12-myristate 13-acetate (PMA) causes an inhibition of the high affinity binding of epidermal growth factor (EGF) to cell surface receptors and an inhibition of the EGF receptor tyrosine protein kinase activity. The hypothesis that PMA controls EGF receptor function by regulating the oligomeric state of the receptor was tested. Dimeric EGF receptors bound to 125I-EGF were identified by covalent cross-linking analysis using disuccinimidyl suberimidate. Treatment of cells with PMA in the presence of 20 nM 125I-EGF caused no significant change in the level of labeled cross-linked monomeric and dimeric receptor species. Investigation of the in vitro autophosphorylation of receptor monomers and dimers cross-linked with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide demonstrated that the treatment of cells with PMA caused an inhibition of the tyrosine phosphorylation of both monomeric and dimeric EGF receptors. We conclude that the inhibition of the EGF receptor tyrosine protein kinase activity caused by PMA is not associated with the regulation of the oligomeric state of the EGF receptor.     

Alkyl isothiocyanates suppress epidermal growth factor receptor kinase activity but augment tyrosine kinase activity

Aim: We have reported the in vitro and in vivo anticancer activities of 6-(methylsulfinyl)hexyl isothiocyanate (6-MITC) derived from a Japanese spice, wasabi. In order to obtain some clues about the mechanism of the anticancer activity, we have studied the effect of alkyl isothiocyanates (MITCs) on protein kinase activities. Methods: The anti-autophosphorylation activity of MITCs with respect to the epidermal growth factor (EGF)-stimulated receptor kinase of A431 epidermoid carcinoma cells was examined by incorporation of radioactive ATP into an acid-insoluble fraction. Their anti-phosphorylation activity with respect to the non-receptor protein kinase was analyzed by a standard SDS-PAGE method. Results: All the tested MITCs interfered with the EGF-stimulated receptor kinase activity in a dose-dependent manner, although their effects were less than 1/10 of that of erbstatin in μg/ml. On the other hand, the MITCs did not interfere with non-receptor kinases (kinase A, kinase C, tyrosine kinase and calmodulin dependent kinase III), but enhanced non-receptor tyrosine kinase. Discussion: A possible anticancer mechanism of MITCs may involve the suppression of EGF receptor kinase activity and augmentation of non-receptor PTK.