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The 66-kDa Shc isoform is a negative regulator of the epidermal growth factor-stimulated mitogen-activated protein
1Department of Physiology & Biophysics, The University of Iowa, Iowa City, Iowa 52242, USA.
Abstract:
In addition to tyrosine phosphorylation of the 66-, 52-, and 46-kDa Shc isoforms, epidermal growth factor (EGF) treatment of Chinese hamster ovary cells expressing the human EGF receptor also resulted in the serine/threonine phosphorylation of approximately 50% of the 66-kDa Shc proteins. The serine/threonine phosphorylation occurred subsequent to tyrosine phosphorylation and was prevented by pretreatment of the cells with the MEK-specific inhibitor PD98059. Surprisingly, only the gel-shifted 66-kDa Shc isoform (serine/threonine phosphorylated) was tyrosine phosphorylated and associated with Grb2. In contrast, only the non-serine/threonine-phosphorylated fraction of 66-kDa Shc was associated with the EGF receptor. To assess the relationship between the three Shc isoforms in EGF-stimulated signaling, the cDNA encoding the 66-kDa Shc species was cloned from a 16-day-old mouse embryo library. Sequence alignment confirmed that the 66-kDa Shc cDNA resulted from alternative splicing of the primary Shc transcript generating a 110-amino acid extension at the amino terminus. Co-immunoprecipitation of Shc and Grb2 from cells overexpressing the 52/46-kDa Shc isoforms versus the 66-kDa Shc species directly demonstrated a competition of binding for a limited pool of Grb2 proteins. Furthermore, expression of the 66-kDa Shc isoform markedly accelerated the inactivation of ERK following EGF stimulation. Together, these data indicate that the serine/threonine phosphorylation of 66-kDa Shc impairs its ability to associate with the tyrosine-phosphorylated EGF receptor and can function in a dominant-interfering manner by inhibiting EGF receptor downstream signaling pathways.
Insights
Epidermal growth factor (EGF) triggers serine/threonine phosphorylation of 66-kDa Shc, which impairs its EGF receptor binding and inhibits downstream signaling. This suggests a dominant-interfering role for phosphorylated 66-kDa Shc in EGF pathways.
Area of Science:
- Cellular signaling pathways
- Receptor tyrosine kinase signaling
- Protein phosphorylation
Background:
- Shc proteins are key adaptors in epidermal growth factor receptor (EGFR) signaling.
- EGFR activation leads to tyrosine phosphorylation of Shc isoforms.
- The role of serine/threonine phosphorylation of Shc, particularly the 66-kDa isoform, remains less understood.
Purpose of the Study:
- To investigate the functional consequences of serine/threonine phosphorylation on the 66-kDa Shc isoform.
- To elucidate the interplay between different Shc isoforms and their binding partners in EGFR signaling.
- To determine the role of 66-kDa Shc serine/threonine phosphorylation in regulating downstream signaling.
Main Methods:
- Treatment of Chinese hamster ovary cells expressing human EGFR with EGF.
- Analysis of Shc protein phosphorylation using Western blotting and co-immunoprecipitation.
- Cloning and sequencing of the 66-kDa Shc cDNA.
- Assessment of Shc-Grb2 and Shc-EGFR interactions.
- Measurement of ERK inactivation following EGF stimulation.
Main Results:
- EGF treatment induced both tyrosine and serine/threonine phosphorylation of 66-kDa Shc.
- Serine/threonine phosphorylation of 66-kDa Shc occurred after tyrosine phosphorylation and was MEK-dependent.
- Serine/threonine phosphorylated 66-kDa Shc preferentially associated with Grb2, while non-phosphorylated 66-kDa Shc associated with EGFR.
- Overexpression of 66-kDa Shc competed with 52/46-kDa Shc isoforms for Grb2 binding.
- 66-kDa Shc expression accelerated ERK inactivation.
Conclusions:
- Serine/threonine phosphorylation of 66-kDa Shc impairs its association with the activated EGFR.
- Phosphorylated 66-kDa Shc acts in a dominant-interfering manner, inhibiting EGFR downstream signaling.
- Alternative splicing generating the 66-kDa Shc isoform and its subsequent phosphorylation create a regulatory mechanism within the EGFR pathway.