All Autophosphorylation Sites of Epidermal Growth Factor (EGF) Receptor and HER2/neu Are Located in Their Carboxyl-terminal Tails

Margolis, Ben; Lax, Irit; Kris, Richard; Dombalagian, Markar; Honegger, A M; Howk, Richard; Givol, David; Ullrich, A; Schlessinger, Joseph

doi:10.1016/s0021-9258(18)81674-x

Cited by 204 publications

(43 citation statements)

References 22 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…We have previously demonstrated that Grb2 binds to the autophosphorylated EGFR via its SH2 domain (36). The C-terminal tail of the EGFR contains at least five autophosphorylation sites (12,38,71) and has been shown to be the primary binding site for SH2 t. 1. Schematic representation of the intracellular domains of the wild-type and mutant EGFR.…”

Section: Resultsmentioning

confidence: 99%

“…We first analyzed the binding sites for Shc on the EGFR by using a dephosphorylation protection assay (55). Figure 5A shows the five tryptic fragments corresponding to the five autophosphorylation sites of the EGFR at Y-1173, Y-1148, Y-1086, Y-1068, and Y-992 (12,38,55,71). These phosphopeptide fragments were absent from the dephosphorylated EGFR preparation subjected to dephosphorylation by the cellular phosphatases (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…The following gradients were used: for Grb2-GST, from 0 to 10 min in 0% buffer B, from 10 to 50 min in a linear gradient to 40% buffer B, and from 50 to 51 min an increase to 60% buffer B; and for Shc SH2-GST, from 0 to 10 min in 0% buffer B, from 10 to 15 min in a linear gradient to 30% buffer B, from 15 to 55 min in a linear gradient to 50% buffer B, and from 55 to 56 min an increase to 80% buffer B. The assignment of tryptic fragments separated by HPLC to the autophosphorylation sites of the EGFR was described earlier (12,38,55,71). BlAcore measurements.…”

Section: Methodsmentioning

confidence: 94%

“…NIH 3T3 lines lacking endogenous EGFR (2.2 cells) were engineered to overexpress either wild-type or mutant EGFRs. Cell lines overexpressing the human EGFR or mutant EGFRs used in this study were HER14, Y1068F, Y1148F, Y1173F (21), CD63, and CD126 (38). Cells were maintained in Dulbecco's modified Eagle's medium containing 10% calf serum.…”

Section: Methodsmentioning

confidence: 99%

See 3 more Smart Citations

Hierarchy of Binding Sites for Grb2 and Shc on the Epidermal Growth Factor Receptor

Batzer

Rotin

Ureña

et al. 1994

Molecular and Cellular Biology

Self Cite

View full text Add to dashboard Cite

We analyzed the binding site(s) for Grb2 on the epidermal growth factor (EGF) receptor (EGFR), using cell lines overexpressing EGFRs containing various point and deletion mutations in the carboxy-terminal tail. Results of co-immunoprecipitation experiments suggest that phosphotyrosines Y-1068 and Y-1173 mediate the binding of Grb2 to the EGFR. Competition experiments with synthetic phosphopeptides corresponding to known autophosphorylation sites on the EGFR demonstrated that phosphopeptides containing Y-1068, and to a lesser extent Y-1086, were able to inhibit the binding of Grb2 to the EGFR, while a Y-1173 peptide did not. These findings were confirmed by using a dephosphorylation protection assay and by measuring the dissociation constants of Grb2's SH2 domain to tyrosine-phosphorylated peptides, using real-time biospecific interaction analysis (BIAcore). From these studies, we concluded that Grb2 binds directly to the EGFR at Y-1068, to a lesser extent at Y-1086, and indirectly at Y-1173. Since Grb2 also binds Shc after EGF stimulation, we investigated whether Y-1173 is a binding site for the SH2 domain of Shc on the EGFR. Both competition experiments with synthetic phosphopeptides and dephosphorylation protection analysis demonstrated that Y-1173 and Y-992 are major and minor binding sites, respectively, for Shc on the EGFR. However, other phosphorylation sites in the carboxy-terminal tail of the EGFR are able to compensate for the loss of the main binding sites for Shc. These analyses reveal a hierarchy of interactions between Grb2 and Shc with the EGFR and indicate that Grb2 can bind the tyrosine-phosphorylated EGFR directly, as well as indirectly via Shc.

show abstract

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 94%

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

Hierarchy of Binding Sites for Grb2 and Shc on the Epidermal Growth Factor Receptor

Batzer

Rotin

Ureña

et al. 1994

Molecular and Cellular Biology

Self Cite

View full text Add to dashboard Cite

show abstract

“…(Borgquist, 2008) (Paech, 1997 Taylor, 2002) Behrens, 2007;Paruthiyil, 2004) (Paruthiyil, 2004;Ström, 2004) (Borgquist, 2008;Chen, 2008) (Giangrande, 1999 (Tandon, 1990 (Menard, 2004;Tovey, 2004 Slamon, 1987 (Corzo, 2005;Sauer, 1999). (Guo, 2003;Rampaul, 2005;Chan, 1999 (Downward, 1984;Hsuan, 1989;Margolis, 1989;Walton, 1990;Levkowitz, 1999 (Sorkin, 1992 (Sorkin, 1992 (Pietras, 1998;Pegram, 1999) (Slamon, 1987 (Hudziak, 1989 (Baselga, 2006;Piccart-Gebhart, 2005;. øÛÙfiÛÔ, ÙÔ 50% ÙˆÓ fiÁÎˆÓ Ô˘ ˘ÂÚÂÎÊÚ¿˙Ô˘Ó HER2 ÂÌ-Ê•Ó›˙Ô˘Ó •ÓıÂÎÙÈÎfiÙËÙ• ÛÙÔ trastuzumab (Baselga, 2006;Piccart-Gebhart, 2005) (Bilancia, 2007;Engel, 2007;Meric-Bernstam, 2006;Nahta, 2006 (Bilancia, 2007;Engel, 2007;Meric-Bernstam, 2006;Nahta, 2006 (Franklin, 200...…”

mentioning

confidence: 99%

Συγκριτική Μορφολογική Ανοσοϊστοχημική Και Μοριακή Μελέτη Καρκινωμάτων Μαστού Με Τη Μέθοδο Των Μικροσυστοιχιών

Κολέτσα¹

View full text Add to dashboard Cite

Expression, synaptic localization, and developmental regulation of Ack1/Pyk1, a cytoplasmic tyrosine kinase highly expressed in the developing and adult brain

Ureña

Torre

Martı́nez

et al. 2005

J of Comparative Neurology

View full text Add to dashboard Cite

Cytosolic tyrosine kinases play a critical role both in neural development and in adult brain function and plasticity. Here we isolated a cDNA with high homology to human Ack1 and mouse Tnk2. This cDNA directs the expression of a 125-kD protein that can be autophosphorylated in tyrosines. Initially, this clone was named Pyk1 for proline-rich tyrosine kinase (Lev et al., 1995); however, since it corresponds to the mouse homolog of Ack1, here we called it Ack1/Pyk1. In this study we show that Ack1/Pyk1 mRNA and protein is highly expressed in the developing and adult brain. The highest levels of Ack1/Pyk1 expression were detected in the hippocampus, neocortex, and cerebellum. Electron microscopy studies showed that Ack1/Pyk1 protein is expressed in these regions both at dendritic spines and presynaptic axon terminals, indicating a role in synaptic function. Furthermore, we demonstrate that Ack1/Pyk1 mRNA levels are strongly upregulated by increased neural activity, produced by intraperitoneal kainate injections. During development, Ack1/Pyk1 was also expressed in the proliferative ventricular zones and in postmitotic maturing neurons. In neuronal cultures, Ack1/Pyk1 was detected in developing dendrites and axons, including dendritic tips and growth cones. Moreover, Ack1/Pyk1 colocalized with Cdc42 GTPase in neuronal cultures and coimmunoprecipitated with Cdc42 in HEK 293T cells. Altogether, our findings indicate that Ack1/Pyk1 tyrosine kinase may be involved both in adult synaptic function and plasticity and in brain development.

show abstract

All Autophosphorylation Sites of Epidermal Growth Factor (EGF) Receptor and HER2/neu Are Located in Their Carboxyl-terminal Tails

Cited by 204 publications

References 22 publications

Hierarchy of Binding Sites for Grb2 and Shc on the Epidermal Growth Factor Receptor

Hierarchy of Binding Sites for Grb2 and Shc on the Epidermal Growth Factor Receptor

Συγκριτική Μορφολογική Ανοσοϊστοχημική Και Μοριακή Μελέτη Καρκινωμάτων Μαστού Με Τη Μέθοδο Των Μικροσυστοιχιών

Expression, synaptic localization, and developmental regulation of Ack1/Pyk1, a cytoplasmic tyrosine kinase highly expressed in the developing and adult brain

Contact Info

Product

Resources

About