EGF-dependent Phosphorylation Research Articles

Functional heterogeneity of proto-oncogene tyrosine kinases: the C terminus of the human epidermal growth factor receptor facilitates cell proliferation.

Previous reports have indicated that the C termini of the membrane-associated tyrosine kinases encoded by c-src and c-fms proto-oncogenes have a negative effect on their biological activity and that this effect is mediated by their C-terminal tyrosine residue. To determine whether this was true for the human epidermal growth factor (EGF) receptor, which is also a membrane-associated tyrosine kinase proto-oncogene, we have constructed two premature termination mutants, dc19 and dc63, that delete the C-terminal 19 and 63 amino acids, respectively, from the human full-length receptor (hEGFR). The smaller deletion removes the C-terminal tyrosine residue, while the larger deletion removes the two most C-terminal tyrosines; similar deletions are found in v-erbB. As previously shown for the gene encoding the full-length EGF receptor, the two C-terminal mutants induced EGF-dependent focal transformation and anchorage-independent growth of NIH 3T3 cells. However, both dc19 and dc63 were quantitatively less efficient than the gene encoding the full-length receptor, with dc63 being less active than dc19. Although the C-terminal mutants displayed lower biological activity than the gene encoding the full-length receptor, the mutant receptors were found to be similar in several respects to the full-length receptor. These parameters included receptor localization, stability in the absence of EGF, receptor half-life in the presence of EGF, EGF binding, extent of EGF-dependent autophosphorylation in vitro, and EGF-dependent phosphorylation of an exogenous substrate in vitro. Therefore, the C-terminal 63 amino acids of the human receptor have no detectable influence on EGF-dependent early events. We conclude that in contrast

Rapid isolation of plasmalemma from cultured A431 cells: Characterization of epidermal growth factor receptors

A rapid method for the purification of plasma membrane from a relatively small number of A431 cells is described. The method is a simple, two-step differential centrifugation in the presence of Ca 2+ that requires a total centrifugation time of 7 min. The membrane preparations contained a high level of epidermal growth factor (EGF) receptor activity demonstrated by both the quantity of specific ligand binding and the amount of EGF-dependent phosphorylation of the receptor and an exogenous substrate. EGF-dependent autophosphorylation identified the EGF receptor in the purified membranes as an undegraded 170-kDa protein.

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

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

EGF-dependent phosphorylation of the EGF receptor in plasma membranes isolated from young and senescent WI-38 cells.

Tyrosine-specific phosphorylation of the receptor for epidermal growth factor (EGF) in plasma membranes isolated from WI-38 cells is EGF-dependent and occurs to an equivalent extent and on identical tryptic peptides in preparations from cells of various in vitro ages. There is a marked reduction, however, in phosphorylation of receptor molecules from senescent as compared with young WI-38 cells, if enzyme activity is assayed in an immune complex following solubilization of plasma membranes with Nonidet P-40 (NP-40). Differences in the level of receptor phosphorylation in young vs. senescent NP-40 extracts are not resolved by changing the temperature at which the assay is performed, or the length of incubation. Moreover, addition of NP-40 or chloroform-methanol extracts of young cells to assays measuring receptor phosphorylation in senescent cell NP-40 preparations does not augment the senescent enzyme activity. The immunopurified senescent receptor is, however, capable of catalyzing phosphorylation of exogenous substrates. These results indicate that the loss of receptor autophosphorylation in solubilized preparations may result from a differential sensitivity of the senescent cell receptor to the detergent. This finding provides a marker for senescence and suggests subtle changes in protein structure, conformation, or regulation of the EGF receptor in senescent cells.

Modified ganglioside as a possible modulator of transmembrane signaling mechanism through growth factor receptors: A preliminary note

Two types of modified GM3 strongly alter EGF-dependent phosphorylation of the EGF receptor in opposite directions, i.e., de-N-acetyl-GM3 (amino-GM3; NeuNH2 alpha 2----3Gal beta 1----4Glc beta 1----1 Ceramide) strongly promotes tyrosine phosphorylation of the EGF receptor of A431 cells, while lyso-GM3 (NeuNAc alpha 2----3Gal beta 1----4 Glc beta----1 Sphingosine) as well as GM3 inhibit tyrosine phosphorylation of the EGF receptor in the same cells under the same conditions. A hypothesis is proposed that de-N-acylation of gangliosides, in either the sialic acid or ceramide moiety, is a crucial event in triggering a positive or negative transmembrane signal.

Distinct effects of Na+ and Li+ on the induction of morphological changes in A431 cells mediated by epidermal growth factor.

Epidermal growth factor (EGF) induced the formation of thin sheetlike extensions (lamellipodia) and filamentous extensions at the edges of colonies of A431 cells. To determine the necessary processes for the induction of the morphological changes mediated by EGF, the effects of a variety of ions on these changes were examined. In a NaCl solution supplemented with CaCl2, MgCl2 and glucose, no EGF-induced morphological changes were observed. However, when the NaCl was replaced with LiCl, fingerlike extensions were formed, but sheetlike extensions were not. Addition of vanadate to the NaCl solution also induced fingerlike extensions in cells treated with EGF. In contrast, sheetlike lamellipodia were formed in EGF-treated cells by the addition of K+ or PO4(3-) to the NaCl solution or by the addition of PO4(3-) to the LiCl solution. These findings indicate that Li+, K+, PO4(3-) and vanadate are involved in the processes of EGF-induced morphological changes. Since vanadate and Li+ have been shown to inhibit phosphatases, an EGF-dependent phosphorylation step may play an important role in the induction of the morphological changes observed.

Epidermal growth factor stimulates the phosphorylation of the calcium-dependent 35,000-dalton substrate in intact A-431 cells.

We have previously reported the isolation of a 35-kDa protein from A-431 cells that, in the presence of Ca2+, is an excellent in vitro substrate for the epidermal growth factor (EGF) receptor/kinase present in membrane preparations (Fava, R. A., and Cohen, S. (1984) J. Biol. Chem. 259, 2636-2645). In this communication we demonstrate that the phosphorylation of the 35-kDa protein is markedly enhanced in intact, 32P-labeled, A-431 cells following exposure of the cells to EGF. The 35-kDa protein immunoprecipitated from cells treated with EGF is phosphorylated to a 20-120-fold greater extent than comparable preparations from control cells. Both phosphotyrosine and phosphoserine residues are detected in the protein after treatment of the cells with EGF. EGF-dependent phosphorylation of the 35-kDa protein is barely detected unless the intact cells are exposed to EGF for periods greater than 5 min. We suggest that endosomes containing internalized EGF X receptor/kinase complexes are primarily responsible for the observed phosphorylation of the 35-kDa protein in intact cells.

Nonfunctional epidermal growth factor receptor in cells transformed by Kirsten sarcoma virus

The cell membrane receptor for epidermal growth factor (EGF) appears to be a glycoprotein of M r 170,000 and mediates the mitogenic and metabolic responses of cells with EGF receptors (EGF-R). Normal rat kidney (NRK) have about 3 × 10 5 EGF-R per cell. Upon transformation of NRK cells by Kirsten sarcoma virus, the transformed derivative (KNRK) loses the ability to bind 125I-EGF. Membranes from NRK and KNRK cells were included in EGF-dependent phosphorylation reactions to search for evidence of the EGF-R. A phosphorylated protein of M r 170,000 was detected in both NRK and KNRK membranes. The M r 170,000 protein was identified to be EGF-R by immunoprecipitation with monoclonal antibody to the receptor. Furthermore, two-dimensional peptide mapping using trypsin and chymotrypsin digestions of the iodinated receptors from both NRK and KNRK cells showed essentially identical patterns. These data indicate that the EGF-R is present in KNRK cells with apparently the same protein structure as the NRK counterpart.

Characterization of the epidermal-growth-factor-dependent phosphorylation system from normal mouse-liver sinusoidal plasma membranes.

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.

Reduced binding of epidermal growth factor by avian sarcoma virus-transformed rat cells

Rat cells transformed by Rous sarcoma virus and Fujinami sarcoma virus bound 5–10% of the amount of epidermal growth factor (EGF) bound by normal cells. Scatchard plot analysis indicated that the reduction in binding by transformed cells was due to a decreased number of receptors rather than to altered binding affinity. In experiments with temperature sensitive mutants of Rous sarcoma virus and Fujinami sarcoma virus significant loss of EGF binding occurred within one hour of shift from non-permissive to permissive temperature. Conditioned media from various normal and transformed cell lines were examined for the ability to inhibit EGF binding to normal cells or to cause “down regulation” of EGF receptors. No activity of either type was found. EGF-dependent phosphorylation in isolated membrane preparations was also examined. Membranes from normal cells displayed EGF-dependent phosphorylation of a M r 180,000 protein presumed to be the EGF receptor. This activity was absent in membranes from transformed cells. The data suggest a close correlation between activation of avian sarcoma virus transforming gene products and modulation of the EGF growth regulatory system.

DMSO increases tyrosine residue phosphorylation in membranes from murine erythroleukemia cells

Phosphorylation of membranes from murine erythroleukemia cells was performed in the presence and absence of the polar solvent dimethyl sulfoxide. Quantitation of the phosphoamino acid content revealed that DMSO stimulated phosphotyrosine accumulation by three-fold; serine and threonine phosphorylation decreased significantly. We had previously shown that DMSO stimulated tyrosine residue phosphorylation of the hepatic epidermal growth factor receptor. EGF had little effect in MEL membranes; therefore, DMSO results in accumulation of phosphotyrosine in cell membranes that do not exhibit significant EGF-dependent phosphorylation.

Inhibition of tyrosine protein kinases by halomethyl ketones.

A chloromethyl ketone derivative of lactic acid was shown to inhibit protein phosphorylation in plasma membranes of Ehrlich ascites tumor cells [Johnson, H. J., Zimniak, A., & Racker, E. (1982) Biochemistry 21, 2984-2989]. We now show that this inhibitor as well as three halomethyl ketone derivatives of amino acids and peptides specifically inhibits tyrosine protein kinase activity in intact plasma membranes and Triton extracts of plasma membrane of A-431 tumor cells. The most effective inhibitor is a bromomethyl ketone derivative of leucine that inhibits the phosphorylation of a protein that migrates to the same position as the EGF receptor in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Inhibition of phosphorylation took place in the presence or absence of added EGF, and the inhibitor did not interfere with the binding of EGF to the receptor nor with the dephosphorylation of the EGF-stimulated phosphoprotein. EGF-dependent phosphorylation in a Triton extract of plasma membranes from normal placenta was considerably less sensitive to the bromomethyl ketone derivative of leucine. The tyrosine protein kinase activity of the transformation gene product of Fujinami virus was particularly sensitive to the bromomethyl ketone derivative of leucine, while the src gene product of Rous sarcoma virus was comparatively less sensitive. The bromomethyl ketone inhibitor interfered with the phosphorylation of the EGF receptor by [gamma-32P]-8-azido-ATP but much less with the light-sensitive binding. This observation and the lack of interference with EGF binding suggest that the inhibitor interacts with the protein kinase portion of the receptor complex.

Proteolytic cleavage of epidermal growth factor receptor. A Ca2+-dependent, sulfhydryl-sensitive proteolytic system in A431 cells.

The Mr = 160,000 epidermal growth factor (EGF) receptor in A431 cells is partially cleaved during membrane isolation to a Mr = 145,000 polypeptide containing both EGF binding and phosphate acceptor sites. We show that the proteolytic degradation of the EGF receptor depends upon the presence of Ca2+ in the medium used to scrape the cells from the substratum. Only the high molecular weight form of the receptor is detected in membranes prepared in the absence of Ca2+. Ca2+-dependent proteolysis occurs rapidly (t1/2 approximately 5 min) following cell scraping. Proteolysis results in a decrease in EGF-dependent phosphorylation of the receptor while retaining EGF binding capacity. In addition, membranes containing the uncleaved form of the receptor reveal a substantial increase in EGF-dependent phosphorylation of proteins with Mr approximately 80, 89, and 185 X 10(3). In the presence of Ca2+, addition of iodoacetic acid to the scraping medium strongly inhibits receptor fragmentation, whereas other inhibitors (phenylmethylsulfonyl fluoride, leupeptin, and pepstatin) have no effect. The results implicate a role for a Ca2+-dependent, SH-sensitive protease in EGF receptor degradation. Prevention of proteolysis yields membrane preparations with highly active EGF-dependent kinase system.

Alteration of epidermal growth factor-dependent phosphorylation during rat liver regeneration.

Epidermal growth factor (EGF) stimulates membrane protein phosphorylation in a human cell line, A-431. The known hepatic mitogenic action of EGF and the reduction in EGF receptor number that occurs during liver regeneration led us to study whether EGF-dependent protein kinase activity was present in rat liver and whether its activity was altered after partial hepatectomy. Liver membranes, preincubated with or without EGF, were phosphorylated (0 degrees C, 15 sec) and subjected to NaDodSO4/polyacrylamide gel electrophoresis and autoradiography. In microsomal fractions, EGF at 5-2000 ng/ml produced a dose-related stimulation of 32P incorporation into a single 170,000-dalton protein (p170). In plasma membranes, a similar EGF-dependent phosphorylation was present and was substantially enriched relative to the microsomal fraction. Acid hydrolysis of labeled microsomal fraction followed by phosphoamino acid determination revealed that EGF stimulated 32P incorporation into phosphotyrosine residues. The EGF-dependent phosphorylation of p170 was compared in microsomal fractions isolated from rats 36 hr after partial hepatectomy or sham operation. In the absence of EGF, in vitro labeling of p170 was similar. EGF stimulated the labeling of p170 in both groups, but the response was clearly diminished after partial hepatectomy. In the presence of EGF, the labeling of p170 in microsomal fraction from regenerating livers was only 47 +/- 6% of that observed in membranes from sham-operated rats (p less than 0.005). Reduction of EGF-dependent phosphorylation during liver regeneration paralleled the loss of binding of 125I-labeled EGF. An increase in the EGF-independent phosphorylation of a 130,000-dalton protein was also observed after partial hepatectomy. The increase in the amount of this phosphoprotein was roughly equal to the loss of EGF-stimulated p170 phosphorylation. Several additional proteins showed increased phosphorylation in membranes from partially hepatectomized rats. These findings indicate that alterations in membrane tyrosine residue phosphorylation occur during regulated growth in vivo.