Loss of three major auto phosphorylation sites in the EGF receptor does not block the mitogenic action of EGF

Clark, Stella; Cheng, Dorothy J.; Hsuan, J. Justin; Haley, John D.; Waterfield, Michael D.

doi:10.1002/jcp.1041340313

Cited by 24 publications

(21 citation statements)

References 46 publications

(39 reference statements)

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…In similar experiments we found no stimulation of DNA synthesis by plateletderived growth factor or epidermal growth factor at concentrations from 2 to 225 ng/ml, respectively (data not shown), as might be expected because CHO-K1 has been reported to lack receptors for these two hormones (26,27). Characterization of Insulin Binding.…”

Section: Methodssupporting

confidence: 78%

The insulin receptor as a transmitter of a mitogenic signal in Chinese hamster ovary CHO-K1 cells.

Mamounas

Gervin

Englesberg

1989

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

Insulin is the only hormone required for continued growth of Chinese hamster ovary CHO-K1 cells in the defmed medium M-F12. When CHO-K1 cells are incubated in M-F12 without insulin for 48-72 hr, the cells accumulate in G1. In response to physiological concentrations of insulin an 18-fold increase in rate of DNA synthesis occurs due to cells entering S phase after an 8-to 10-hr lag; cell division begins after 24 hr. The inhibitory effect of actinomycin D and 5,6-dichlorobenzimidazole riboside indicates that RNA synthesis is required for progression to S phase. CHO-K1 cells possess insulin receptors, and the insulin effect results from insulin binding to its own receptor: (a) Binding occurs at physiological insulin concentrations with a half-maximal stimulation at 14 ng/ml. (it) At insulin concentrations used, insulin-like growth factor I and II (IGF-I and IGF-H) have little or no effect. The insulin family of hormones, including insulin and insulinlike growth factor I and II (IGF-I and IGF-II), play an essential role in human growth and development. These hormones generate transmembrane signals that regulate carbohydrate, lipid, and protein metabolism; glucose and amino acid transport; DNA synthesis; and cell division and morphological transformation. Because of the degree of similarity of structure and apparent function of these hormones and their receptors (insulin and IGF-I) and the cross-reactivity with regard to receptor binding, much effort has been expended to determine the actual mode of action of the individual hormones vis-a-vis their unique receptors. Both the insulin and IGF-I receptors seem to mediate some ofthe same acute responses that have usually been thought reserved for the insulin receptor (1). Both receptors possess liganddependent, tyrosine-specific protein kinase activity similar to receptors of other hormones and proteins of several oncogenes (2). The broad response elicited by IGF-II has usually been found to be mediated by the IGF-I and the insulin receptors (1). Recently, the receptor for IGF-II has also been found to be the receptor for mannose-6-phosphate (3). The IGF-I receptor is "normally" thought responsible for growth stimulation (1). The insulin stimulation of cell growth in culture has been assumed to be from insulin binding to the IGF-I receptor as a result of the use of insulin at greater than physiological insulin concentrations (4-8); however, this conclusion is open to question (9-15). We have developed a modified F12-defined medium (M-F12) for CHO-K1 cells in which insulin is the only hormone required for growth (16). On the basis that insulin stimulated growth in this medium at the nanogram range (1-10 ng/ml) characteristic of the levels found at normal physiological concentrations (17), we proposed that insulin at these concentrations is acting as its own receptor (16).In this paper we characterize the insulin receptor of CHO-K1 cells and present evidence that the insulin stimulation of DNA synthesis and cell division in this cell line results from insulin bindi...

show abstract

Section: Methodssupporting

confidence: 78%

The insulin receptor as a transmitter of a mitogenic signal in Chinese hamster ovary CHO-K1 cells.

Mamounas

Gervin

Englesberg

1989

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

show abstract

“…This plasmid, pRK7-o~, was cotransfected with a plasmid encoding neomycin-resistance into CHO cells. These cells were chosen since they lack EGF/TGF-ot receptors (Livneh et al, 1986;Clark et al, 1988) thus avoiding any interaction of transfected TGF-o~ with its receptor. CHO cells expressing the transfected TGF-oe precursor were obtained following selection in G418 and were named Ca cells.…”

Section: Expression Of Fuu-length and Truncated Tgfc~ Precursors In Cmentioning

confidence: 99%

Association of the transmembrane TGF-alpha precursor with a protein kinase complex.

Shum

Reeves

Kuo

et al. 1994

The Journal of Cell Biology

View full text Add to dashboard Cite

Abstract, A variety of growth factors including transforming growth factor-c~ (TGF-o0 are synthesized as transmembrane precursors. The short cytoplasmic domain of the transmembrane TGF-ot precursor lacks any apparent motif associated with signal transduction. However, the sequence conservation of this cytoplasmic domain and its abundance of cysteine residues, reminiscent of the cytoplasmic domains of CD4 and CD8, suggest a biological function. In this study, we showed that transmembrane TGF-c~ was rapidly internalized after interaction with a specific antibody and that this internalization was greatly decreased when the COOH-terminal 31 amino acids were removed. Chemical cross-linking experiments revealed two associated proteins of 86 and 106 kD which coimmunoprecipitated with the TGF-cx precursor. The association of p86 was dependent on the presence of the COOH-terminal cytoplasmic 31 amino acids of the TGF-ot precursor, whereas p106 still remained associated when this segment was deleted. In addition, p106 was tyrosine-phosphorylated and exposed on the cell surface. The protein complex associated with transmembrane TGF-ot displayed kinase activities towards tyrosine, serine, and threonine residues. These activities were not associated with transmembrane TGF-c~ when the COOH-terminal segment was truncated. The association of a protein kinase complex with transmembrane TGF-cx may provide the basic elements for a "reverse" mode of signaling through the cytoplasmic domain of this growth factor, which may lead to two-directional communication during ligandreceptor interaction.

show abstract

“…However, these tyrosine residues do not clearly define adaptor/effector protein binding sites of EGFR (Batzer et al, 1994;Soler et al, 1994b). When some or all of these autophosphorylation sites are mutated or deleted EGFR remains capable of activating the Ras/Erk pathway (Li et al, 1994;Soler et al, 1994a) and inducing mitogenesis in NIH 3T3 cells (Clark et al, 1988;Decker, 1993;Gotoh et al, 1994;Li et al, 1994;Soler et al, 1994a). NIH 3T3 cells were used for these studies because they were considered to be devoid of endogenous ErbB family members.…”

Section: Introductionmentioning

confidence: 99%

PC12 cell activation by epidermal growth factor receptor: role of autophosphorylation sites

Tyson

Larkin

Hamai

et al. 2003

Intl J of Devlp Neuroscience

View full text Add to dashboard Cite

PC12 cells have been used as a model system for neuronal differentiation due to their ability to alter their phenotype to a sympathetic neuron-like cell in response to nerve growth factor or fibroblast growth factor. Under some conditions, epidermal growth factor (EGF) can also induce PC12 cells to differentiate. To study signaling from the EGF receptor without the confounding effects of endogenous EGF receptors we generated a chimeric receptor comprised of the ectodomain of platelet-derived growth factor (PDGF) receptor in-frame with the transmembrane and cytoplasmic domains of EGF receptor, termed PER. Expression of PER in PC12 cells confers the ability of PDGF to induce differentiation whereas PDGF has no effect on untransfected PC12 cells. This response is kinase activity-dependent since a kinase-deficient mutant (K721M) fails to induce differentiation in response to PDGF. Mutation of five tyrosine residues that are autophosphorylated in response to EGF either individually or in combination had minimal effects on the ability of these receptors to induce morphological PC12 cell differentiation. The PER mutant with all five autophosphorylation sites mutated to phenylalanine (5YF) was equivalently capable of interacting with several important signaling molecules, including Shc, Grb2, Gab1, phospholipase Cgamma, and Cbl. Furthermore, both the phosphatidylinositol 3-kinase (PI3K)/Akt and Ras/Erk pathways were activated in a sustained manner when PER or 5YF-expressing cells were stimulated with PDGF. Our results show that the five autophosphorylation sites in the extra-kinase C-terminal domain of EGFR are not required for the ability of EGFR to induce morphological differentiation of PC12 cells.

show abstract

Loss of three major auto phosphorylation sites in the EGF receptor does not block the mitogenic action of EGF

Cited by 24 publications

References 46 publications

The insulin receptor as a transmitter of a mitogenic signal in Chinese hamster ovary CHO-K1 cells.

The insulin receptor as a transmitter of a mitogenic signal in Chinese hamster ovary CHO-K1 cells.

Association of the transmembrane TGF-alpha precursor with a protein kinase complex.

PC12 cell activation by epidermal growth factor receptor: role of autophosphorylation sites

Contact Info

Product

Resources

About