Epidermal growth factor (EGF) promotes phosphorylation at threonine-654 of the EGF receptor: possible role of protein kinase C in homologous regulation of the EGF receptor.

Whiteley, Brian; Glaser, Luis

doi:10.1083/jcb.103.4.1355

Cited by 87 publications

(37 citation statements)

References 46 publications

(71 reference statements)

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“…It has been recently shown that EGF itself promotes the phosphorylation of threonine 654 of its receptor, possibly through the activation of PKC (22,34). In the following experiments, we provide evidence that if PKC has been down regulated by chronic treatment with a phorbol ester (27), the EGF stimulatory effect on tyrosine autophosphorylation of the receptor is enhanced.…”

Section: Kinetics Of Tyrosine Phosphorylation Of Egf Receptor P-tyrsupporting

confidence: 49%

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Kinetics and regulation of the tyrosine phosphorylation of epidermal growth factor receptor in intact A431 cells.

Sturani¹,

Zippel²,

Toschi³

et al. 1988

Mol. Cell. Biol.

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We have previously reported that antibodies to phosphotyrosine recognize the phosphorylated forms of platelet-derived growth factor (PDGF) and epidermal growth factor (EGF) receptors (Zippel et al., Biochim. Biophys. Acta 881:54-61, 1986, and Sturani et al., Biochem. Biophys. Res. Commun. 137:343-350, 1986 The epidermal growth factor (EGF) receptor (26) is a transmembrane protein which contains the EGF-binding domain in its extracellular N-terminal region and a protein kinase domain in the intracellular portion (2, 32). Near the carboxy terminus, there are tyrosine residues which are specifically phosphorylated by the autokinase activity (13). When intact cells are exposed to EGF, autophosphorylation occurs primarily on tyrosine 1173. The significance of this self-phosphorylation has been debated (4, 14) and it has been suggested that it increases the tyrosine kinase activity toward other substrates (4).The EGF receptor is also a substrate of protein kinase C (PKC); addition of tumor promoters or of physiological activators of PKC to intact cells induces receptor phosphorylation on several serine and threonine residues, inhibits high-affinity binding of EGF, and reduces the protein kinase activity of the receptor (8,12,16,20). Moreover, it has recently been observed that EGF promotes the phosphorylation of the threonine 654 of its own receptor (22, 34), so that the possible role of PKC in homologous feedback regulation of EGF receptor has been suggested.The human carcinoma cell line A431 bears an unusually high number of EGF receptors, and it is largely used for studying their biochemistry and regulation (11,18,19,23,31). We report a study on kinetic and regulatory aspects of the tyrosine phosphorylation of EGF receptor in intact A431 cells. The tyrosine phosphorylation of the receptor has been studied by using azobenzyl phosphonate antibodies that specifically cross-react with phosphotyrosine (P-tyr) (10, * Corresponding author. 25). We have previously shown that these antibodies are a sensitive tool for detecting and quantitatively evaluating the tyrosine-phosphorylated form of platelet-derived growth factor (PDGF) (29, 38) and EGF receptors (30) in Western blots (immunoblots) of total cellular extracts. We have now determined the fraction of total EGF receptors phosphorylated on tyrosine and the cellular localization of the phosphorylated receptor molecules. Moreover, we have analyzed the conditions affecting receptor phosphorylation and dephosphorylation in intact A431 cells stimulated by EGF

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Section: Kinetics Of Tyrosine Phosphorylation Of Egf Receptor P-tyrsupporting

confidence: 49%

“…Moreover, it has recently been observed that EGF promotes the phosphorylation of the threonine 654 of its own receptor (22,34), so that the possible role of PKC in homologous feedback regulation of EGF receptor has been suggested.…”

mentioning

confidence: 99%

Kinetics and regulation of the tyrosine phosphorylation of epidermal growth factor receptor in intact A431 cells.

Sturani¹,

Zippel²,

Toschi³

et al. 1988

Mol. Cell. Biol.

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“…On the basis of these results, we propose that kinase C phosphorylation of Thr-654 provides a negative control mechanism for the regulation of the mitogenic response of cells to EGF. It was reported that in A431 cells EGF induces the phosphorylation of Thr-654 of the EGF receptor probably by activation of kinase C, potentially leading to a negative-feedback reaction to EGF action (28). This information together with the results presented here may provide an explanation for the well-documented inhibitory effect of EGF on A431 cell proliferation.…”

Section: Resultsmentioning

confidence: 50%

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

Livneh¹,

Dull²,

Berent³

et al. 1988

Mol. Cell. Biol.

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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.The mitogenic polypeptide epidermal growth factor (EGF) mediates its biological responses by activating an integral membrane glycoprotein denoted as the EGF receptor (reviewed in references 5 and 26; J. Schlessinger, Biochemistry, in press). Scatchard analysis of binding of 125I-EGF to intact cells reveals two affinity classes of EGF toward the receptor (16). The addition of the phorbol ester (TPA) to cells abolishes the high-affinity binding sites (4,16,18,27) and reduces the protein-tyrosine kinase activity of the receptor molecule (6, 9, 15). Moreover, TPA-induced kinase C phosphorylation of the EGF receptor occurs mainly on Thr-654 (7, 13), suggesting that the phosphorylation of this residue regulates the ligand-binding affinity and the kinase activity of the EGF receptor. The phosphorylation state of Thr-654 may also play a role in endocytosis as an Ala-654 mutant of the EGF receptor fails to internalize in response to TPA, while the native EGF receptor undergoes TPA-induced internalization (19). We have shown that mutations in the cytoplasmic domain of the EGF receptor can cause the loss of high-affinity EGF-binding sites (20,23). Hence, the so-called effect of TPA on EGF receptor internalization (2, 19, 26; Schlessinger, in press) most probably represents TPA-induced loss of high-affinity receptors rather than receptor internalization (26; Schlessinger, in press). The physiological role of the high-and low-affinity receptors is not known (26). Interestingly, growth factors other than EGF such as platelet-derived growth factor and bombesin, which bind to their own distinct membrane receptors, are also able to abolish the high-affinity state of the EGF r...

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“…For example, phosphorylation of serine and threonine residues within the cytoplasmic domains of the insulin (67,68) and epidermal growth factor receptors (69 -75) diminishes their affinity for insulin and epidermal growth factor, respectively. With respect to the epidermal growth factor receptor, these effects mimic those involved in regulation of the receptor by heterologous ligands (76) such as platelet-derived growth factor (77) and those induced through a negative feedback pathway triggered by binding of epidermal growth factor itself (78).…”

Section: Activation Of Pk-c By Elevated Glucose Concentrations Leads mentioning

confidence: 99%

Regulation of the Ligand Binding Activity of the Human Very Low Density Lipoprotein Receptor by Protein Kinase C-dependent Phosphorylation

Sakthivel¹,

Zhang²,

Strickland³

et al. 2001

Journal of Biological Chemistry

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The very low density lipoprotein receptor (VLDL-R) binds and internalizes several ligands, including very low density lipoprotein (VLDL), urokinase-type plasminogen activator:plasminogen activator inhibitor type 1 complexes, lipoprotein lipase, and the 39-kDa receptor-associated protein that copurifies with the low density lipoprotein receptor-related protein/␣ 2 -macroglobulin receptor. Although several agonists regulate VLDL-R mRNA and/or protein expression, post-transcriptional regulation of receptor activity has not been described. Here, we report that the ligand binding activity of the VLDL-R in THP-1 monocytic cells, endothelial cells, smooth muscle cells, and VLDL-R-transfected HEK 293 cells is diminished after treatment with phorbol 12-myristate 13-acetate. This response was blocked by inhibitors of protein kinase C (PK-C), including a specific inhibitor of the PK-C ␤II isoform, and was associated with phosphorylation of serine residues in the cytoplasmic domain of the receptor. Culture of endothelial cells in the presence of high glucose concentrations, which stimulate diacylglycerol synthesis and PK-C ␤II activation, also induced a PK-C-dependent loss of VLDL-R ligand binding activity. Taken together, these studies demonstrate that the ligand binding activity of the VLDL-R is regulated by PK-C-dependent phosphorylation and that hyperglycemia may diminish VLDL-R activity. The very low density lipoprotein receptor (VLDL-R)1 is a member of the low density lipoprotein receptor (LDL-R) family, which encompasses several structurally related proteins such as the LDL receptor-related protein/␣ 2 -macroglobulin receptor, gp330 (megalin), and apoE receptor 2 (1-4) among others. Members of this family are characterized by a cytoplasmic domain NPXY sequence that mediates ligand internalization through clathrin-coated pits (5) and an extracellular domain comprised of cysteine-rich complement-type and epidermal growth factor precursor-like repeats that regulate ligand binding specificity (2). Except for the presence of an additional complement-type repeat in its extracellular domain, the domain structure of the VLDL-R is identical to that of the LDL-R (6 -8). Despite this homology, however, the ligand binding specificity of these receptors differs. Though both receptors bind apoE-containing lipoproteins (6, 7, 9, 10), only the LDL receptor binds lipoproteins containing apoB-100 (6, 7), whereas the VLDL-R binds several additional ligands such as lipoprotein lipase (9), (11) urokinase-type plasminogen activator:plasminogen activator inhibitor type 1 (u-PA:PAI-1) complexes (11,12), and Lp(a) (13). Finally, the 39-kDa receptor-associated protein (RAP), which co-purifies with (14, 15) and binds with high affinity to the LDL receptor-related protein/␣ 2 -macroglobulin receptor (16 -18), binds tightly to the VLDL-R (19) but with only low affinity to the 20).Differences in the tissue distribution of the LDL and VLDL receptors also suggest non-overlapping physiologic functions. Although the LDL-R is highly expressed in liver...

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Epidermal growth factor (EGF) promotes phosphorylation at threonine-654 of the EGF receptor: possible role of protein kinase C in homologous regulation of the EGF receptor.

Cited by 87 publications

References 46 publications

Kinetics and regulation of the tyrosine phosphorylation of epidermal growth factor receptor in intact A431 cells.

Kinetics and regulation of the tyrosine phosphorylation of epidermal growth factor receptor in intact A431 cells.

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

Regulation of the Ligand Binding Activity of the Human Very Low Density Lipoprotein Receptor by Protein Kinase C-dependent Phosphorylation

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