Epidermal growth factor-receptor mutant lacking the autophosphorylation sites induces phosphorylation of Shc protein and Shc-Grb2/ASH association and retains mitogenic activity.

Gotoh, Noriko; Tojo, Arinobu; Muroya, Kenkoh; Hashimoto, Yuko; Hattori, Satoshi; Nakamura, Shugo; Takenawa, Tadaomi; Yazaki, Yoshio; Shibuya, Masabumi

doi:10.1073/pnas.91.1.167

Cited by 114 publications

(96 citation statements)

References 26 publications

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“…We also observed a similar age-related decrease in the association of the EGF receptor with the SH2 domain of the Shc protein, which recognizes tyrosine 1173, although the magnitude of the association was not as great as with the Shc-PTB domain (data not shown). The idea that reduced association of Shc with the EGF receptor due to defects in autophosphorylation sites with the effect of reducing mitogenic activity is supported by the data of Gotoh et al (42). A cell line containing a deletion mutant of the EGF receptor that lacked the autophosphorylation sites downstream of residue 1011 was used to show that EGF treatment could still result in stimulation of Shc phosphorylation but retained only approximately 20% of its mitogenic activity when measured as cell growth.…”

Section: Discussionsupporting

confidence: 52%

Age-dependent Decline in Mitogenic Stimulation of Hepatocytes

Palmer

Tuzon

Paulson

1999

Journal of Biological Chemistry

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The proliferative potential of the liver has been well documented to decline with age. However, the molecular mechanism of this phenomenon is not well understood. Cellular proliferation is the result of growth factor-receptor binding and activation of cellular signaling pathways to regulate specific gene transcription. To determine the mechanism of the age-related difference in proliferation, we evaluated extracellular signal-regulated kinase-mitogen-activated protein kinase activation and events upstream in the signaling pathway in epidermal growth factor (EGF)-stimulated hepatocytes isolated from young and old rats. We confirm the ageassociated decrease in extracellular signal-regulated kinase-mitogen-activated protein kinase activation in response to EGF that has been previously reported. We also find that the activity of the upstream kinase, Raf kinase, is decreased in hepatocytes from old compared with young rats. An early age-related difference in the EGF-stimulated pathway is shown to be the decreased ability of the adapter protein, Shc, to associate with the EGF receptor through the Shc phosphotyrosine binding domain. To address the mechanism of decreased Shc/ EGF receptor interaction, we examined the phosphorylation of the EGF receptor at tyrosine 1173, a site recognized by the Shc phosphotyrosine binding domain. Tyrosine 1173 of the EGF receptor is underphosphorylated in the hepatocytes from old animals compared with young in a Western blot analysis using a phosphospecific antibody that recognizes phosphotyrosine 1173 of the EGF receptor. These data suggest that a molecular mechanism underlying the age-associated decrease in hepatocyte proliferation involves an age-dependent regulation of site-specific tyrosine residue phosphorylation on the EGF receptor.

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Section: Discussionsupporting

confidence: 52%

Age-dependent Decline in Mitogenic Stimulation of Hepatocytes

Palmer

Tuzon

Paulson

1999

Journal of Biological Chemistry

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“…It also suggests that the kinase domain of the activated EGF receptor may not be responsible for Shc phosphorylation on Tyr-317, since although being very weakly phosphorylated following EGF stimulation, the GST-SHCY239/240F forms a complex with the activated EGFR. Previous studies have reported that Shc can undergo tyrosine phosphorylation in the absence of specific binding sites on the EGFR (42,43) and that phosphorylation on Tyr-317 does not depend on Shc binding to the activated EGFR (44). In addition, it has been shown that in v-Src or v-Sea transformed cells, Shc is phosphorylated and associates with Grb2 (45)(46)(47).…”

Section: Discussionmentioning

confidence: 99%

Differential Utilization of ShcA Tyrosine Residues and Functional Domains in the Transduction of Epidermal Growth Factor-induced Mitogen-activated Protein Kinase Activation in 293T Cells and Nerve Growth Factor-induced Neurite Outgrowth in PC12 Cells

Thomas

Bradshaw

1997

Journal of Biological Chemistry

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By transient expression of both truncated forms of p52 SHCA and those with point mutations in 293T cells, it has been shown that, in addition to Tyr-317, Tyr-239/240 is a major site of phosphorylation that serves as a docking site for Grb2⅐Sos1 complexes. In addition, analysis of epidermal growth factor (EGF)-induced activation of mitogen-activated protein kinase in 293T cells showed that the overexpression Shc SH2 or phosphotyrosine binding (PTB) domains of ShcA alone has a more potent negative effect than the overexpression of the forms of ShcA lacking Tyr-317 or Tyr 239/240 or both. In transiently transfected PC12 cells, the ShcA PTB domain and tyrosine phosphorylation in the CH1 domain, especially on Tyr-239/240, are crucial for mediating nerve growth factor (NGF)-induced neurite outgrowth. These findings suggest that the EGF and NGF (TrkA) receptor can utilize Shc in different ways to promote their activity. For EGF-induced mitogen-activated protein kinase activation in 293T cells, both Shc PTB and SH2 domains are essential for optimal activation, indicating that a mechanism independent of Grb2 engagement with Shc may exist. For NGF-induced neurite outgrowth in PC12 cells, Shc PTB plays an essential role, and phosphorylation on Tyr-239/240, but not on Tyr-317, is required.Activation of the Ras/MAP 1 kinase pathway, triggering proliferation and differentiation, is one of the major early events induced by receptor tyrosine kinases (reviewed in Ref. 1). Grb2 and Sos1 have been linked to the mediation of this activation (2, 3). Grb2 is a 23-kDa adaptor protein composed of an SH2-type phosphotyrosine recognition domain, flanked by two SH3 domains that recognize proline-rich sequences (reviewed in Ref. 4). Sos1 is a 170-kDa protein with a guanine nucleotide exchanging domain that can catalyze the conversion of inactive Ras, with bound GDP, into its active form, with bound GTP (reviewed in Ref. 5). Beside its catalytic domain, Sos1 contains a proline-rich region enabling it to couple with Grb2 (6). Activation of Ras can be achieved when cytosolic Sos translocates to the plasma membrane, where cellular Ras normally resides (7). This translocation is mediated by the recruitment of Grb2 onto the intracellular domain of an activated receptor tyrosine kinase, such as the epidermal growth factor receptor (EGFR) (8). Grb2 can dock through its SH2 domain with specific phosphotyrosine residues of the activated EGFR which subsequently allows relocalization of Sos1 to the plasma membrane level (reviewed in Ref. 9). Activated Ras allows a cascade of events starting with the activation of the serine kinase Raf that phosphorylates and activates a MAP kinase kinase (also denoted MEK), which in turn activates MAP kinase by dual phosphorylation on tyrosine and threonine residues (reviewed in Ref. 1).Although the EGFR can utilize Grb2 directly to mediate MAP kinase activation, it can also recruit Grb2 and Sos1 indirectly, which involves the Shc family of adaptor proteins (10, 11). The first member identified and the prototype of th...

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“…Neither Tyr-1173 nor Tyr-992 fulfills either the SH2 or SAIN domain-binding criteria, and their mode of interaction is unclear, although our results showing that mutation of the Pro in the IR NPXY motif reduces but does not eliminate SHC interaction, suggests that Tyr-1173 (NAGY) may also serve as a less efficient SAIN domain-binding site. Two other studies have suggested that mutation or deletion of all phosphorylated tyrosines in the EGF receptor showed phosphorylation of SHC and unimpaired mitogenic signaling (14,46). The explanation for these different results is unclear.…”

Section: Discussionmentioning

confidence: 99%

Phosphotyrosine-Dependent Interaction of SHC and Insulin Receptor Substrate 1 with the NPEY Motif of the Insulin Receptor via a Novel Non-SH2 Domain

Gustafson

Craparo

et al. 1995

Molecular and Cellular Biology

360

293

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The SHC proteins have been implicated in insulin receptor (IR) signaling. In this study, we used the sensitive two-hybrid assay of protein-protein interaction to demonstrate that SHC interacts directly with the IR. The interaction is mediated by SHC amino acids 1 to 238 and is therefore independent of the Src homology 2 domain. The interaction is dependent upon IR autophosphorylation, since the interaction is eliminated by mutation of the IR ATP-binding site. In addition, mutational analysis of the Asn-Pro-Glu-Tyr (NPEY) motif within the juxtamembrane domain of the IR showed the importance of the Asn, Pro, and Tyr residues to both SHC and IR substrate 1 (IRS-1) binding. We conclude that SHC interacts directly with the IR and that phosphorylation of Tyr-960 within the IR juxtamembrane domain is necessary for efficient interaction. This interaction is highly reminiscent of that of IRS-1 with the IR, and we show that the SHC IR-binding domain can substitute for that of IRS-1 in yeast and COS cells. We identify a homologous region within the IR-binding domains of SHC and IRS-1, which we term the SAIN (SHC and IRS-1 NPXY-binding) domain, which may explain the basis of these interactions. The SAIN domain appears to represent a novel motif which is able to interact with autophosphorylated receptors such as the IR.The recent identification and cloning of proteins which interact with receptor tyrosine kinases (RTKs) has allowed much insight into the molecular basis for RTK signal transduction (9,10,18,19,24,27,54). These effector proteins contain Src homology 2 (SH2) domains of approximately 100 amino acids which interact directly with phosphotyrosine-containing regions of each RTK (21, 35). Upon receptor autophosphorylation, these SH2 domain-containing proteins interact with the RTK. Unlike most RTKs, the insulin receptor (IR) and the related insulin-like growth factor 1 receptor (IGFIR) appear to interact with and phosphorylate an intermediate signaling protein termed IRS-1, for insulin receptor substrate 1 (17). After tyrosine phosphorylation by the IR, IRS-1 is thought to interact with a variety of SH2 domain-containing proteins, including the p85 subunit of phosphatidylinositol 3-kinase, GRB-2, and SH-PTP2 (Syp) (45, 52). These proteins presumably mediate some of the effects of the IR.Another substrate of the IR is the SH2 domain-containing protein SHC, so named because of its SH2 domain as well as its homology to collagen (36). Multiple SHC proteins exist, two of which (p52 and p46) result from the use of alternative translation start sites, while the origin of the p66 isoform is less clear. The SHC proteins have been implicated in mitogenic signaling by a variety of tyrosine kinases. These include the receptors for nerve growth factor (32), epidermal growth factor (EGF) (36), platelet-derived growth factor (PDGF) (59), and insulin (39). SHC has also been implicated in signaling by other classes of receptors, including the interleukin-2 receptor (40, 62) and the T-cell receptor (41). Other hormones which have been ...

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Epidermal growth factor-receptor mutant lacking the autophosphorylation sites induces phosphorylation of Shc protein and Shc-Grb2/ASH association and retains mitogenic activity.

Cited by 114 publications

References 26 publications

Age-dependent Decline in Mitogenic Stimulation of Hepatocytes

Age-dependent Decline in Mitogenic Stimulation of Hepatocytes

Differential Utilization of ShcA Tyrosine Residues and Functional Domains in the Transduction of Epidermal Growth Factor-induced Mitogen-activated Protein Kinase Activation in 293T Cells and Nerve Growth Factor-induced Neurite Outgrowth in PC12 Cells

Phosphotyrosine-Dependent Interaction of SHC and Insulin Receptor Substrate 1 with the NPEY Motif of the Insulin Receptor via a Novel Non-SH2 Domain

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