scholarly journals Nucleotide-binding properties of kinase-deficient epidermal-growth-factor- receptor mutants

1998 ◽  
Vol 330 (1) ◽  
pp. 353-359 ◽  
Author(s):  
Kunrong CHENG ◽  
G. John KOLAND
Keyword(s):  
Amino Acid ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Egf Receptor ◽  
Nucleotide Binding ◽  
Protein Kinase Activity ◽  
Amino Acid Substitutions ◽  
Wild Type ◽  
Binding Properties ◽  
Epidermal Growth

The nucleotide-binding properties of wild-type epidermal- growth-factor (EGF)-receptor protein tyrosine kinase (PTK) and EGF-receptor mutants with site-specific amino acid substitutions known to attenuate protein kinase activity were analysed by a fluorescence competition assay employing the nucleotide analogue 2ʹ(3ʹ)-O-(2,4,6-trinitrophenyl)adenosine 5ʹ-triphosphate.Binding affinities for ATP and Mn·ATP complex were determined for the PTK domains of the wild-type and two mutant proteins. Surprisingly, mutation of the highly conserved Lys-721 residue in the nucleotide-binding site of the EGF- receptor PTK domain did not abolish ATP and Mn·ATP binding, although the binding affinity for the Mn·ATP complex was significantly reduced. A second kinase-inactivating mutation that targeted the highly conserved Asp-813 residue had little effect on the nucleotide-binding properties of the EGF-receptor PTK domain. These results indicated that the principle effect of these two kinase-inactivating amino acid substitutions is not to block nucleotide binding, but is instead an inhibition of the phospho-transfer reaction.

scholarly journals Localization of Phospholipase D1 to Caveolin-enriched Membrane via Palmitoylation: Implications for Epidermal Growth Factor Signaling

2002 ◽  
Vol 13 (11) ◽  
pp. 3976-3988 ◽  
Author(s):  
Jung Min Han ◽  
Yong Kim ◽  
Jun Sung Lee ◽  
Chang Sup Lee ◽  
Byoung Dae Lee ◽  
...  
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Egf Receptor ◽  
Point Mutations ◽  
Dominant Negative ◽  
Growth Factor Signaling ◽  
Wild Type ◽  
Caveolin 1 ◽  
Epidermal Growth ◽  
Egf Signaling

Phospholipase D (PLD) has been suggested to mediate epidermal growth factor (EGF) signaling. However, the molecular mechanism of EGF-induced PLD activation has not yet been elucidated. We investigated the importance of the phosphorylation and compartmentalization of PLD1 in EGF signaling. EGF treatment of COS-7 cells transiently expressing PLD1 stimulated PLD1 activity and induced PLD1 phosphorylation. The EGF-induced phosphorylation of threonine147 was completely blocked and the activity of PLD1 attenuated by point mutations (S2A/T147A/S561A) of PLD1 phosphorylation sites. The expression of a dominant negative PKCα mutant by adenovirus-mediated gene transfer greatly inhibited the phosphorylation and activation of PLD1 induced by EGF in PLD1-transfected COS-7 cells. EGF-induced PLD1 phosphorylation occurred primarily in the caveolin-enriched membrane (CEM) fraction, and the kinetics of PLD1 phosphorylation in the CEM were strongly correlated with PLD1 phosphorylation in the total membrane. Interestingly, EGF-induced PLD1 phosphorylation and activation and the coimmunoprecipitation of PLD1 with caveolin-1 and the EGF receptor in the CEM were significantly attenuated in the palmitoylation-deficient C240S/C241S mutant, which did not localize to the CEM. Immunocytochemical analysis revealed that wild-type PLD1 colocalized with caveolin-1 and the EGF receptor and that phosphorylated PLD1 was localized exclusively in the plasma membrane, although some PLD1 was also detected in vesicular structures. Transfection of wild-type PLD1 but not of C240S/C241S mutant increased EGF-induced raf-1 translocation to the CEM and ERK phosphorylation. This study shows, for the first time, that EGF-induced PLD1 phosphorylation and activation occur in the CEM and that the correct localization of PLD1 to the CEM via palmitoylation is critical for EGF signaling.

Cloning and characterization of a gene encoding pig epidermal growth factor

1991 ◽  
Vol 6 (1) ◽  
pp. 63-70 ◽  
Author(s):  
J. C. Pascall ◽  
D. S. C. Jones ◽  
S. M. Doel ◽  
J. M. Clements ◽  
M. Hunter ◽  
...  
Keyword(s):  
Amino Acid ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Amino Acid Sequence ◽  
Egf Receptor ◽  
Predicted Amino Acid Sequence ◽  
Recombinant Polypeptide ◽  
Gene Encoding ◽  
Epidermal Growth ◽  
Direct Amplification

ABSTRACT A portion of the pig epidermal growth factor (EGF) gene has been isolated and characterized. The nucleotide sequencies of exons 20 and 21, which encode the EGF region of the precursor protein, show 85% similarity with the human EGF gene sequence. In addition, conservation of the intron—exon boundaries between the two species was generally observed. Although the pig exon 21 appeared to lack a single nucleotide at its 5′ end relative to the human gene, sequences obtained by direct amplification of the genomic DNA around the 5′ end of this exon using the polymerase chain reaction, and from a pig EGF cDNA recombinant isolated from a kidney library, indicated that the deletion was probably a cloning artifact. Comparison of the predicted amino acid sequence of pig EGF with that of EGF from other species, as well as with several other polypeptides which bind to the EGF receptor, indicated conservation of Gly18, Tyr37, Gly39 and Arg41 in addition to all six cysteine residues and Leu47, which are known to be critical for biological activity. A synthetic gene encoding the predicted amino acid sequence of pig EGF was expressed in yeast. The recombinant polypeptide was shown to compete with 125I-labelled mouse EGF for binding to cells and to stimulate DNA synthesis in quiescent monolayers of Swiss 3T3 cells.

scholarly journals A Tale of Two Cbls: Interplay of c-Cbl and Cbl-b in Epidermal Growth Factor Receptor Downregulation

2008 ◽  
Vol 28 (9) ◽  
pp. 3020-3037 ◽  
Author(s):  
Steven Pennock ◽  
Zhixiang Wang
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Egf Receptor ◽  
Growth Factor Receptor ◽  
Wild Type ◽  
Receptor Downregulation ◽  
Epidermal Growth ◽  
Precise Role ◽  
Second Peak

ABSTRACT The precise role of Cbl in epidermal growth factor (EGF) receptor (EGFR) endocytosis and trafficking remains to be fully uncovered. Here, we showed that mutant EGFR1044, which was truncated after residue 1044, did not associate with c-Cbl and was not ubiquitinated initially in response to EGF but was internalized with kinetics similar to those of wild-type EGFR. This finding indicates that c-Cbl-mediated ubiquitination is not required for EGF-induced EGFR endocytosis. We also showed that the previously identified internalization-deficient mutant receptor EGFR1010LL/AA bound to c-Cbl and was fully ubiquitinated in response to EGF, which indicates that c-Cbl binding and ubiquitination are not sufficient for EGFR internalization. We next investigated EGFR trafficking following EGFR internalization. We found that c-Cbl disassociation from EGFR occurred well in advance of EGFR degradation and that this event was concurrent with the selective dephosphorylation of EGFR at Y1045. This finding suggests that once EGFR is ubiquitinated, continual Cbl association is not required for EGFR degradation. Because EGFR1044 is ubiquitinated and degraded similarly to wild-type EGFR, we examined the role of another prominent Cbl homologue, Cbl-b, and found that Cbl-b was associated with both EGFR and EGFR1044. Further study showed that Cbl-b bound to EGFR at two regions: one in the C-terminal direction from residue 1044 and one in the N-terminal direction from residue 958. Moreover, Cbl-b association with EGFR rose markedly following a decrease in c-Cbl association, corresponding to a second peak of EGFR ubiquitination occurring later in EGFR trafficking. Using RNA interference to knock down both c-Cbl and Cbl-b, we were able to abolish EGFR downregulation. This knockdown had no affect on the rate of EGF-induced EGFR internalization. We found that the two Cbls accounted for total receptor ubiquitination and that while c-Cbl and Cbl-b are each alone sufficient to effect EGFR degradation, both are involved in the physiological, EGF-mediated process of receptor downregulation. Furthermore, these data ultimately reveal a previously unacknowledged temporal interplay of two major Cbl homologues with the trafficking of EGFR.

scholarly journals Roles of JAKs in activation of STATs and stimulation of c-fos gene expression by epidermal growth factor.

1996 ◽  
Vol 16 (1) ◽  
pp. 369-375 ◽  
Author(s):  
D W Leaman ◽  
S Pisharody ◽  
T W Flickinger ◽  
M A Commane ◽  
J Schlessinger ◽  
...  
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Kinase Activity ◽  
Egf Receptor ◽  
Regulatory Element ◽  
Kinase Domain ◽  
Minor Role ◽  
Wild Type ◽  
Epidermal Growth ◽  
A Minor

The tyrosine kinase JAK1 and the transcription factors STAT1 and STAT3 are phosphorylated in response to epidermal growth factor (EGF) and other growth factors. We have used EGF receptor-transfected cell lines defective in individual JAKs to assess the roles of these kinases in STAT activation and signal transduction in response to EGF. Although JAK1 is phosphorylated in response to EGF, it is not required for STAT activation or for induction of the c-fos gene. STAT activation in JAK2- and TYK2-defective cells is also normal, and the tyrosine phosphorylation of these two kinases does not increase upon EGF stimulation in wild-type or JAK1-negative cells. In cells transfected with a kinase-negative mutant EGF receptor, there is no STAT activation in response to EGF and c-fos is not induced, showing that the kinase activity of the receptor is required, directly or indirectly, for these two responses. The data do not support a role for any of the three JAK family members tested in STAT activation and are consistent with a JAK-independent pathway in which the intrinsic kinase domain of the EGF receptor is crucial. Furthermore, data from transient transfection experiments in HeLa cells, using c-fos promoters lacking the STAT regulatory element c-sis-inducible element, indicate that this element may play only a minor role in the induction of c-fos by EGF in these cells.

Release of a phorbol ester-induced mitogenic block by mutation at Thr-654 of the epidermal growth factor receptor

1988 ◽  
Vol 8 (6) ◽  
pp. 2302-2308
Author(s):  
E Livneh ◽  
T J Dull ◽  
E Berent ◽  
R Prywes ◽  
A Ullrich ◽  
...  
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Ligand Binding ◽  
Binding Affinity ◽  
Egf Receptor ◽  
Wild Type ◽  
3T3 Cells ◽  
Kinase C ◽  
Epidermal Growth ◽  
Nih 3T3

The tumor promoter phorbol ester (TPA) modulates the binding affinity and the mitogenic capacity of the epidermal growth factor (EGF) receptor. Moreover, TPA-induced kinase C phosphorylation occurs mainly on Thr-654 of the EGF receptor, suggesting that the phosphorylation state of this residue regulates ligand-binding affinity and kinase activity of the EGF receptor. To examine the role of this residue, we prepared a Tyr-654 EGF receptor cDNA construct by in vitro site-directed mutagenesis. Like the wild-type receptor, the mutant receptor exhibited typical high- and low-affinity binding sites when expressed on the surface of NIH 3T3 cells. Moreover, TPA regulated the affinity of both wild-type and mutant receptors and stimulated receptor phosphorylation of serine and threonine residues other than Thr-654. The addition of TPA to NIH 3T3 cells expressing a wild-type human EGF receptor blocked the mitogenic capacity of EGF. However, this inhibition did not occur in cells expressing the Tyr-654 EGF receptor mutant. In the latter cells, EGF was able to stimulate DNA synthesis even in the presence of inhibitory concentrations of TPA. While phosphorylation of sites other than Thr-654 may regulate ligand-binding affinity, the phosphorylation of Thr-654 by kinase C appears to provide a negative control mechanism for EGF-induced mitogenesis in mouse NIH 3T3 fibroblasts.

scholarly journals Mutation of proline-1003 to glycine in the epidermal growth factor (EGF) receptor enhances responsiveness to EGF.

1994 ◽  
Vol 5 (7) ◽  
pp. 739-746 ◽  
Author(s):  
S M Schuh ◽  
E P Newberry ◽  
M A Dalton ◽  
L J Pike
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Kinase Activity ◽  
Egf Receptor ◽  
Biological Effects ◽  
Soft Agar ◽  
Mitogen Activated Protein Kinase ◽  
Protein Kinase Activity ◽  
Egf Receptors ◽  
Epidermal Growth

We have shown previously that the epidermal growth factor (EGF) receptor is phosphorylated at Ser-1002 and that this phosphorylation is associated with desensitization of the EGF receptor. Ser-1002 is followed immediately by Pro-1003, a residue that may promote the adoption of a specific conformation at this site or severe as a recognition element for the interaction of the EGF receptor with other proteins. To examine these possibilities, we have mutated Pro-1003 of the EGF receptor to a Gly residue and have analyzed the effect of this mutation on EGF-stimulated signaling. Cells expressing the P1003G EGF receptors exhibited higher EGF-stimulated autophosphorylation and synthetic peptide phosphorylation compared to cells expressing wild-type EGF receptors. In addition, the ability of EGF to stimulate PI 3-kinase activity and mitogen-activated protein kinase activity was enhanced in cells expressing the P1003G EGF receptor. Cells expressing P1003G receptors also demonstrated an increased ability to form colonies in soft agar in response to EGF. These results indicate that mutation of Pro-1003 leads to a potentiation of the biological effects of EGF. The findings are consistent with the hypothesis that Pro-1003 plays a role in a form of regulation that normally suppresses EGF receptor function.

scholarly journals Release of a phorbol ester-induced mitogenic block by mutation at Thr-654 of the epidermal growth factor receptor.

1988 ◽  
Vol 8 (6) ◽  
pp. 2302-2308 ◽  
Author(s):  
E Livneh ◽  
T J Dull ◽  
E Berent ◽  
R Prywes ◽  
A Ullrich ◽  
...  
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Ligand Binding ◽  
Binding Affinity ◽  
Egf Receptor ◽  
Wild Type ◽  
3T3 Cells ◽  
Kinase C ◽  
Epidermal Growth ◽  
Nih 3T3

The tumor promoter phorbol ester (TPA) modulates the binding affinity and the mitogenic capacity of the epidermal growth factor (EGF) receptor. Moreover, TPA-induced kinase C phosphorylation occurs mainly on Thr-654 of the EGF receptor, suggesting that the phosphorylation state of this residue regulates ligand-binding affinity and kinase activity of the EGF receptor. To examine the role of this residue, we prepared a Tyr-654 EGF receptor cDNA construct by in vitro site-directed mutagenesis. Like the wild-type receptor, the mutant receptor exhibited typical high- and low-affinity binding sites when expressed on the surface of NIH 3T3 cells. Moreover, TPA regulated the affinity of both wild-type and mutant receptors and stimulated receptor phosphorylation of serine and threonine residues other than Thr-654. The addition of TPA to NIH 3T3 cells expressing a wild-type human EGF receptor blocked the mitogenic capacity of EGF. However, this inhibition did not occur in cells expressing the Tyr-654 EGF receptor mutant. In the latter cells, EGF was able to stimulate DNA synthesis even in the presence of inhibitory concentrations of TPA. While phosphorylation of sites other than Thr-654 may regulate ligand-binding affinity, the phosphorylation of Thr-654 by kinase C appears to provide a negative control mechanism for EGF-induced mitogenesis in mouse NIH 3T3 fibroblasts.

scholarly journals Large deletions in the cytoplasmic kinase domain of the epidermal growth factor receptor do not affect its laternal mobility.

1986 ◽  
Vol 103 (2) ◽  
pp. 327-331 ◽  
Author(s):  
E Livneh ◽  
M Benveniste ◽  
R Prywes ◽  
S Felder ◽  
Z Kam ◽  
...  
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Diffusion Coefficients ◽  
Egf Receptor ◽  
Lateral Diffusion ◽  
Cytoplasmic Domain ◽  
Protein Kinase Activity ◽  
Lateral Mobility ◽  
Cos Cells ◽  
Epidermal Growth

The lateral diffusion coefficients of various epidermal growth factor (EGF) receptor mutants with increasing deletions in their carboxy-terminal cytoplasmic domain were compared. A full size cDNA construct of human EGF receptor and different deletion constructs were expressed in monkey COS cells. The EGF receptor mutants expressed on the cell surface of the COS cells were labeled with rhodamine-EGF, and the lateral diffusion coefficients of the labeled receptors were determined by the fluorescence photo-bleaching recovery method. The lateral mobilities of three deletion mutants, including a mutant that has only nine amino acids in the cytoplasmic domain, are all similar (D approximately equal to 1.5 X 10(-10) cm2/s) to the lateral mobility of the "wild-type" receptor, which possess 542 cytoplasmic domain of EGF receptor, including its intrinsic protein kinase activity and phosphorylation state, are not required for the restriction of its lateral mobility.

The structure and function of the epidermal growth factor receptor studied by using antisynthetic peptide antibodies

1985 ◽  
Vol 226 (1242) ◽  
pp. 127-134 ◽  
Keyword(s):  
Amino Acids ◽  
Epidermal Growth Factor Receptor ◽  
Amino Acid ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Amino Acid Sequence ◽  
Egf Receptor ◽  
Growth Factor Receptor ◽  
Binding Domain ◽  
Epidermal Growth

The human epidermal growth factor receptor has been purified and partial amino acid sequence obtained. A synthetic oligonucleotide was used to select complementary DNA clones from placental and A431 clone banks. The nucleotide sequence of a 5.8 kilobase transcript was determined and used to predict the total amino acid sequence of the receptor. We have predicted a model for the receptor which has an external ligand binding domain of 621 amino acids, a transmembrane region of 23 amino acids, and a cytoplasmic domain of 542 amino acids having protein tyrosine kinase activity. The kinase autophosphorylation sites have been mapped onto the primary amino acid sequence. Analysis of protein sequence databases have shown that the erb -B oncogene of avian erythroblastosis virus has acquired part of the avian EGF receptor gene. The hypothesis has been proposed that transformation by this virus is the result of expression of a truncated EGF receptor which lacks the majority of the EGF binding domain and delivers a continuous proliferation signal to transformed cells. We describe here the production of polyclonal and monoclonal antibodies to selected synthetic peptides from the EGF receptor and v-erb B sequences. Antisera to sequences encompassing the three major sites of autophosphorylation and the putative ATP binding site all recognize the native EGF receptor molecule. We have used these reagents to test our model of EGF receptor structure and v-erb B function.

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