Isolation and characterization of two growth factor-stimulated protein kinases that phosphorylate the epidermal growth factor receptor at threonine 669 (original) (raw)
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Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 1992
A synthetic peptide modeled after the major threonine (T669) phosphorylation site of the epidermal growth factor (EGF) receptor was an efficient substrate (apparent K m = 0.45 mM) for phosphorylation by purified p44 "p~. a MAP kinasc from sea star oocytcs. The peptide was also pbosphorylated by a related human M~P kinase, which was idea:ified by immunological criteria as IM2 m'pk. Within 5 rain of treatment of human cervical carcinoma A431 cells with EGF or phorbol myfistate acetate (PMA), a greater than 3-fold activation of p42 '~°pk was measured. However, Mona Q chromatography of A431 cell extracts afforded the resolution of at least three additional T669 peptide kinases, some of which may be new members of the MAP kinase family. One of these (peak I), which weakly adsorbed to Mann Q, phosphorylated myelin basic protein (MBP) and other .MAP Idnase substrates, immunoreacted as a 42 kDa protein on Western blots with four different MAP kinase antibodies, and behaved as a ~ 45 kDa protein upon Super~'.,e 6 gel filtration. Another T669 peptide kinase (peak IV), which bound more tightly to Mona Q than p42 m°pk (peak II), exhibited a nearly identical substrate specificity profile to that of p42 "apk. but it immunorcacted as a 40 kDa protein only with anti-p44 'm'k antibody on Western blots, and eluted from Superose 6 in a high molecular mass complex of greater than 400 kDa. By immunological criteria, the T669 peptide kinase in Mona O peak III was tentatively identdied as an active form of p34 cdc2 associated with cyclin .'L The Mona Q peaks llI and IV kinases were modestly stimulated following either EGF or PMA treatments of A431 cells, and they exhibited a greater T669 peptide/MBP ratio than p42 "~pk. These findings indicated that multiple praline-directed kinases may mediate phosphorylation of the EGF receptor.
European Journal of Biochemistry, 1992
The human epidermal-growth-factor receptor (EGF-R) is a 170-kDa transmembrane glycoprotein that mediates the mitogenic response of cells to EGF and transforming growth factor c(. Culture conditions have been developed for the large-scale expression of the cytoplasmic domain of the EGF-R in insect cells using a recombinant baculovirus. From 6 1 Sf9 cells, grown to high density using a bioreactor, 20 mg of the EGF-R kinase was purified to greater than 95% purity. Purification, which was carried out in the absence of detergents using classical purification methods, yielded an EGF-R protein that was not phosphorylated on tyrosine. This procedure has enabled us to produce high quality enzyme for both structural and biochemical studies on the EGF-R kinase. The in vitro activity of the cytoplasmic domain of the EGF-R kinase was modulated by multiple assay factors which include substrates, divalent cations and conformational modulators. Kinetic analysis in the presence of Mn2+ gave an apparent V,,, value of 20 nmol min-' mg-' and K , values of 4.5 pM for ATP and 1.43 mM for angiotensin 11. This corresponds to a turnover number of 1.4 mol min-' mol-' .
Journal of Biological Chemistry, 1985
This is an Open Access article under the CC BY license. Epidermal Growth Factor Receptor Phosphorylation 14539 ing affinity of the receptor in both purified and membrane systems has been studied to elucidate the role of these phosphorylation events in the control of cell proliferation. EXPERIMENTAL PROCEDURES Materials Peptides PI, P2, P3, and RS were synthesized by Cambridge Research Biochemicals, and peptide RS was also obtained from Peninsula Laboratories. Human epidermoid carcinoma A431 cells were grown as described in Ref. 21 and plasma membranes from these cells were isolated by the method of Thom et al. (22). The EGF receptor was purified from these cells using monoclonal antibody 9A as described previously (23). [y-''P]ATP (>5000 Ci/mmol) was from Amersham. EGF was prepared from adult male mouse submaxillary glands (24) and iodinated using the glucose oxidase method (25). Protein kinase C was purified from bovine brains (26). Methods Tyrosine Kinase Assay-For measurement of tyrosine kinase activity in solubilized A431 cell plasma membranes, 10 pg of membrane protein was incubated with 50 mM Hepes, pH 7.4, 5% glycerol, 0.2% Triton X-100, 150 mM NaCI, 2 mM MnC12, 12 mM MgC4, 100 p M Na3V04, and 20 p~ [y-32P]ATP (5 Ci/mmol) in a total volume of 50 11 with varying concentrations of substrate peptide. Reactions were carried out for 2 min at 30 or 0 "C in the presence or absence of 200 nM EGF as stated. The reaction was stopped by the addition of 45 pl of 5% trichloroacetic acid followed by 5 pl of 30 mg/ml bovine serum albumin. The mixture was kept at 0 "C for 15 min then centrifuged at 10,000 X g for 15 min at 4 "C. 50 pl of each supernatant was spotted onto phosphocellulose paper squares (1.5 cm, Whatman P l l) which were then washed in 30% acetic acid (3 X 10 min), and adsorbed radioactivity was measured. When purified receptor was used, incubations were carried out as above, except membranes were replaced by 2.5 ng of receptor protein. To determine the amount of ["PI phosphate incorporated into purified receptor in the course of a kinase assay, the trichloroacetic acid-precipitated protein pellet was redissolved in 50 pl of 1 M NaOH, and the suspension was heated at 60 "C for 3 min, vortexed vigorously, and spotted onto Whatman 3 " paper squares (1.5 cm). These were washed in 10% trichloroacetic acid (3 X 30 min), and adsorbed radioactivity was measured. Time Course of the Incorporation of Phosphate into Each Autophosphorylation Site of the EGF Receptor-100 pg of A431 Thom membranes (0.7 pmol of EGF receptor) were incubated in the phosphorylation buffer described above, without ATP but including 1 pg/ ml EGF, for 30 min at 18 "C (total volume 100 pl). [y-3'P]ATP (100 Ci/mmol) was added (final concentration 5 p~) and the mixture was incubated at 0 "C between 0.5 and 5 mi n. The reaction was stopped
The Journal of biological chemistry, 1987
We have examined the effect of tyrosine phosphorylation of microtubule-associated protein 2 (MAP2) by the epidermal growth factor (EGF) receptor kinase on its functions. Incubation of MAP2 with the EGF receptor in the presence of ATP resulted in a great decrease in the ability of MAP2 to promote tubulin polymerization. Under a variety of conditions, the decrease in the ability correlated with the extent of phosphorylation of MAP2. Furthermore, another function of MAP2, the actin filament cross-linking activity, was also inactivated by the incubation of MAP2 with the EGF receptor and ATP. The loss of this activity also correlated well with the extent of phosphorylation. These data indicate that tyrosine phosphorylation of MAP2 by the EGF receptor kinase inactivates both the tubulin polymerizing activity and actin filament cross-linking activity of MAP2. Thus, this study has clearly shown that tyrosine phosphorylation could modify the function of a cytoskeletal protein.
Role of epidermal growth factor-stimulated protein kinase in control of proliferation of A431 cells
Journal of Cellular Biochemistry, 1982
Epidermal growth factor (EGF), which stimulates tyrosine-specific protein kinase activity both in vivo and in vitro, inhibits proliferation of A43 1 human epidermoid carcinoma cells. After mutagenesis clonal cell lines that were resistant to the growth inhibitory effects of EGF were selected. All six variants examined contained decreased EGF-stimulated protein kinase. 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 variant cell lines with up to tenfold reductions in both activities. This result suggests that both EGF binding and kinase activities reside in the same or closely coupled molecules. The effect of EGF on growth of two resistant variants was examined in detail. Clone 29 contains -50% and clone 4 contains -20 % of the EGF-stimulated protein kinase activity of the parental A43 1 cell line. In serum-supplemented medium, EGF stimulated proliferation of clone 29 but did not affect growth of clone 4. In a 1:l mixture of DME and F-12 medium without serum, EGF caused both clone 29 and clone 4 to grow as well as in 10% serum. These variants, which were selected for resistance to the growth inhibitory effects of EGF, thus exhibit a strong mitogenic response to EGF. This result suggests that resistance to the growth inhibitory effect of EGF may involve both a decrease in EGF-stimulated protein kinase and an alteration in the response pathway.
Molecular and cellular biology, 1992
We have compared the protein tyrosine kinase activities of the chicken epidermal growth factor receptor (chEGFR) and three ErbB proteins to learn whether cancer-activating mutations affect the kinetics of kinase activity. In immune complex assays performed in the presence of 15 mM Mn2+, ErbB proteins and the chEGFR exhibited highly reproducible tyrosine kinase activity. Under these conditions, the ErbB and chEGFR proteins had similar apparent Km [Km(app)] values for ATP. The ErbB proteins appeared to be activated, as they had at least 3-fold-higher relative Vmax(app) for autophosphorylation and approximately 2-fold higher relative Vmax(app) for the phosphorylation of the exogenous substrate TK6 (a bacterially expressed fusion protein containing the C-terminal domain of the human EGFR). The ErbB kinases had both higher Km(app) and higher Vmax(app) for the phosphorylation of the exogenous substrate TK6 than did the chEGFR. The ratios of the Vmax(app) to the Km(app) for TK6 phosphoryla...
Journal of Biological Chemistry, 1993
A putative mitogen-activated protein kinase (MAPK) has recently been identified, which potentially phosphorylates the human epidermal growth factor (EGF) receptor at a physiological site (Thr-669) and is distinguished from other MAPKdextracellular signal-regulated protein kinases (ERKs) on the basis of chromatographic, immunological, and kinetic data. Here we report that this newly discovered MAPK is physically associated with the EGF receptor in A431 cells and with the related receptor/tyrosine kinase HEW (encoded by c-neu) in enzyme preparations obtained from Wilm's tumors. This human EGF receptor-associated kinase is characterized as a 40-kDa Thr-669 kinase that exists in a high molecular mass complex with the respective growth factor receptor. EGF treatment of A431 cells stimulates the tyrosine phosphorylation of p40 and increases Thr-669 kinase activity in p4O-containing fractions. The 40-kDa kinase is recognized by affinity-purified polyclonal antibodies directed against the sea star p44"pk and a Pan-ERK antibody directed against the conserved subdomain VI11 of MAPKdERKs, but is not recognized by antibodies selective for the rat p44erk1 and/or the p42ma*k/eru isoforms, thus identifying the EGF receptorassociated kinase as a novel MAPK that may regulate receptor function in vivo. Mitogen-activated protein kinases (MAPKs),' also known as extracellular signal-regulated protein kinases (ERKs), constit u t e a rapidly growing family of protein-serindthreonine ki-Science Foundation (to F. L. H.), the National Institutes of Health (to D.
Cancer Research, 1989
Two cell lines established from tumors of the head and neck area at different clinical stages were found to differ in the expression and in the tyrosine kinase activity of the epidermal growth factor (EGF) receptor. Cell line 183A was derived from an early-stage tumor and cell line 1483 was derived from a tumor that had metastasized to lymph nodes. The 1483 cells displayed a higher plating efficiency and clonogenicity in soft agar, suggesting a more tumorigenic phenotype over the 183A cells. Analyses of EGF receptor levels by using Rl anti-EGF receptor serum indicated that the 1483 cells expressed 5-fold more receptor than did the 183A cells. EGF receptors isolated from each cell line were active for kinase activity in an immune complex kinase assay, using monoclonal Rl anti-EGF receptor antibody. The autophosphorylation activity of both receptors was stimulated by addition of EGF to isolated membrane preparations and intact cells, although the EGF receptor of the 1483 cells was much less responsive to EGF than the receptor from 183A cells. In addition, the 1483 receptor consistently incorporated about twice as much phosphate as did the 183A receptor in an immune complex kinase assay. These data suggest that the basal tyrosine kinase activity of the EGF receptor from 1483 cells may be more active than the EGF receptor kinase from 183 cells.