Tyr-317 Phosphorylation Increases Shc Structural Rigidity and Reduces Coupling of Domain Motions Remote from the Phosphorylation Site as Revealed by Molecular Dynamics Simulations

Abstract

Activated receptor tyrosine kinases bind the Shc adaptor protein through its N-terminal phosphotyrosine-binding (PTB) and C-terminal Src homology 2 (SH2) domains. After binding, Shc is phosphorylated within the central collagen-homology (CH) linker regionon Tyr-317, a residue remote to both the PTB and SH2 domains. Shc phosphorylation plays a pivotal role in the initiation of mitogenic signaling through the Ras/Raf/ MEK/ERK pathway, but it is unclear if Tyr-317 phosphorylation affects Shc-receptor interactions through the PTB and SH2 domains. To investigate the structural impact of Shc phosphorylation, molecular dynamics simulations were carried out using special-purpose Molecular Dynamics Machine-Grape computers. After a 1-nanosecond equilibration, atomic motions in the structures of unphosphorylated Shc and Shc phosphorylated on Tyr-317 were calculated during a 2-nanosecond period. The results reveal larger phosphotyrosine-binding domain fluctuations and more structural flexibility of unphosphorylated Shc compared with phosphorylated Shc. Collective motions between the PTB-SH2, PTB-CH, and CH-SH2 domains were highly correlated only in unphosphorylated Shc. Dramatic changes in domain coupling and structural rigidity, induced by Tyr-317 phosphorylation, may alter Shc function, bringing about marked differences in the association of unphosphorylated and phosphorylated Shc with its numerous partners, including activated membrane receptors.The Src homology and collagen domain protein (Shc) 1 is an adaptor protein that plays a pivotal role in the signal transfer from receptor tyrosine kinases (RTK) to downstream signaling pathways regulating cell survival, proliferation, and differentiation (1). After ligand-induced autophosphorylation, RTKs bind and phosphorylate Shc on selected tyrosine residues. The Shc protein consists of an amino-terminal phosphotyrosinebinding (PTB) domain, a central collagen-homology (CH) region, which contains the major site of tyrosine phosphorylation at Tyr-317 and a carboxyl-terminal Src homology 2 (SH2) domain (2). Phosphorylation of Tyr-317 results in the formation of a docking site for other signaling proteins, e.g. growth factor receptor binding protein 2 (Grb2).Current paradigms for signal transduction emphasize that large adaptor proteins, such as Shc, are organized as "beads on a string." A single bead is a domain, ranging in size from 50 to 120 amino acids; they are responsible for particular interactions with other proteins or phospholipids (3). A signaling protein containing several domains can simultaneously associate with two or more binding partners. For example, an adaptor protein can bind to RTKs using an SH2 or PTB domain and associate with a signaling partner by means of another domain. It may appear at first that the modular structure underlying signaling interactions implies that the phosphorylation of residues located outside specialized binding domains should not affect the structural flexibility and intramolecular motions of these domains. Consequently, it should n...

Molecular Dynamics Simulations Reveal that Tyr-317 Phosphorylation Reduces Shc Binding Affinity for Phosphotyrosyl Residues of Epidermal Growth Factor Receptor

“…The simulated systems of fully solvated Shc molecules include over 130,000 atoms. To specify the initial atom coordinates, we used our theoretical model structures of fulllength Y317-Shc and pY317-Shc derived previously (10). By assigning five different sets of initial velocities of the atoms, we sampled a vast conformational space and calculated the trajectories during 10 ns, resulting in an aggregate of 50 ns for each full-length system.…”

“…We used our theoretical model structure of Y317-Shc (PDB identification, 1WCP; p52 isoform of human Shc) (10) to specify the initial coordinates in MD simulations of full-length Y317-Shc molecules. The theoretical structure of the p52 isoform of human Shc (PDB identification, 1WCP) was modeled using two experimentally determined structures of the PTB and SH2 domains of Shc (PDB accession codes, 1shc and 1tce, respectively).…”

“…In a previous study, we derived a theoretical model structure of full-length Shc (Protein Data Bank (PDB) identification, 1WCP; Fig. 1), and investigated the dynamic properties of both phosphorylated Shc (pY317-Shc) and unphosphorylated Shc (Y317-Shc), using short-timescale (2-ns) molecular dynamics (MD) simulations (10). We found that Tyr-317 phosphorylation increases Shc structural rigidity and reduces coupling of domain motions between the PTB and SH2 domains, which might alter the Shc binding affinity for phosphorylated RTKs (10).…”

Scaffolding Protein Grb2-associated Binder 1 Sustains Epidermal Growth Factor-induced Mitogenic and Survival Signaling by Multiple Positive Feedback Loops

“…Ranges of kinetic parameters were constrained based on the literature data and in vivo measurements of the signaling kinetics. A number of model predictions were validated (11,35,37,39), and, in addition, our kinetic description of this pathway was tested and used by other groups (34,40,41).…”

Phosphorylation of threonine residues on Shc promotes ligand binding and mediates crosstalk between MAPK and Akt pathways in breast cancer cells

“…It has also been proposed that phosphorylation on Y317 introduces rigidity to the protein and limits the dynamic motions of the PTB and SH2 domains in molecular dynamics simulation studies (Suenaga et. al., 2009(Suenaga et. al., , 2004.…”