Abstract
Distinct waves of SHC1 phosphorylation and interactors temporally define EGFR signaling output.
Major finding: Distinct waves of SHC1 phosphorylation and interactors temporally define EGFR signaling output.
Concept: Sequential recruitment of adaptor proteins switches the EGF-dependent pathways activated by SHC1.
Impact: Scaffold proteins may both spatially and temporally orchestrate receptor tyrosine kinase signaling.
Scaffold proteins associate with the intracellular domains of receptor tyrosine kinases (RTK) and spatially regulate signaling by provide docking sites for adaptor proteins and other downstream effectors. Using a quantitative, linear mass spectrometry–based approach to evaluate phosphorylation and binding partners of the scaffold protein SHC1, which mediates EGF receptor (EGFR) signaling, Zheng and colleagues also found evidence that scaffold proteins can temporally regulate RTK signaling output. Analysis of SHC1 phosphorylation sites at 16 time points during the first 90 minutes of EGF stimulation revealed that phosphorylation of SHC1 tyrosine residues occurs rapidly in response to EGF. A distinct group of proteins binding SHC1 with similar kinetics, including the adaptor protein GRB2, were primarily involved in RAS- and phosphoinositide 3-kinase (PI3K)/AKT-mediated growth and survival signaling. Subsequent waves of serine and threonine phosphorylation included feedback phosphorylation of serine 29 by AKT, which temporally correlated with the recruitment of a second group of SHC1-interacting proteins that included the tyrosine phosphatase PTPN12. Dephosphorylation of SHC1 by PTPN12 and subsequent GRB2 displacement then facilitated the recruitment of a delayed wave of GRB2-independent SHC1-interacting proteins. Proteins in this third group were enriched for those with roles in cytoskeletal reorganization, cell migration, trafficking, and inhibitors of mitogenic signaling pathways induced by the first wave of SHC1 interactors. One of these proteins, the pseudokinase SGK269, was required for the recruitment of several other proteins in the group and induced morphologic changes associated with cell invasiveness, suggesting that SGK269 replaces GRB2 as an adaptor protein to mediate the activation of different cellular pathways downstream of EGFR. Together, these observations reveal how the dynamics of SHC1 phosphorylation and interactions mediate the consequences of EGFR signaling over time and raise the possibility that other scaffold proteins may act similarly to control the output of activated RTKs.
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