Evidence for a Requirement for Both Phospholipid and Phosphotyrosine Binding via the Shc Phosphotyrosine-Binding Domain In Vivo

Abstract

The adapter protein Shc is a critical component of mitogenic signaling pathways initiated by a number of receptors. Shc can directly bind to several tyrosine-phosphorylated receptors through its phosphotyrosinebinding (PTB) domain, and a role for the PTB domain in phosphotyrosine-mediated signaling has been well documented. The structure of the Shc PTB domain demonstrated a striking homology to the structures of pleckstrin homology domains, which suggested acidic phospholipids as a second ligand for the Shc PTB domain. Here we demonstrate that Shc binding via its PTB domain to acidic phospholipids is as critical as binding to phosphotyrosine for leading to Shc phosphorylation. Through structure-based, targeted mutagenesis of the Shc PTB domain, we first identified the residues within the PTB domain critical for phospholipid binding in vitro. In vivo, the PTB domain was essential for localization of Shc to the membrane, as mutant Shc proteins that failed to interact with phospholipids in vitro also failed to localize to the membrane. We also observed that PTB domain-dependent targeting to the membrane preceded the PTB domain's interaction with the tyrosine-phosphorylated receptor and that both events were essential for tyrosine phosphorylation of Shc following receptor activation. Thus, Shc, through its interaction with two different ligands, is able to accomplish both membrane localization and binding to the activated receptor via a single PTB domain.The adapter protein Shc is a critical regulator of downstream signaling events that lead to such diverse biological processes as neuronal differentiation (20,34,35,55), lymphocyte proliferation (31), cell survival mediated by cytokines and integrins (11,64), and cellular transformation via polyoma virus middle T antigen (3, 7). Shc mediates these effects, at least in part,

Constitutive activation of the Ras/MAP kinase pathway and enhanced TCR signaling by targeting the Shc adaptor to membrane rafts

“…In this respect, although a small fraction of Shc has been found associated to total cellular membranes in T-cells (Ravichandran et al, 1997), the capacity of Shc to interact with phospholipids through its PTB domain does not appear to permit a stable localization of Shc at the plasma membrane, although it might contribute to stabilizing Shc in a membrane-proximal localization following recruitment to activated receptors, as reported for the PH domain of Sos (Chen et al, 1997). Interestingly, ablation of the two Grb-2 binding sites results in complete abrogation of the constitutive e ects elicited by membrane targeted Shc, suggesting that these e ects depend on successful activation of the Ras pathway by Grb-2/Sos recruitment, and not on additional Grb-2-independent pathways involving Shc interaction with other signaling proteins.…”

Internalized Epidermal Growth Factor Receptors Participate in the Activation of p21 in Fibroblasts

“…Upon formation of a receptor-Shc⅐Grb2⅐Sos complex, an interaction between the Sos pleckstrin homology domain and a specific membrane lipid would be stabilized by analogy to the effect of Sos localization on its association with Ras-GDP. This complex might then allow the Shc phosphotyrosine-binding domain to rapidly exchange its phosphotyrosine ligand for a lipid (51), generating an assembly that could exist transiently as an independent species. The Sos pleckstrin homology domain and Shc phosphotyrosine-binding domain both bind PIP 2 in vitro (52,53), and our previous results suggest that activated EGFR only has access to this lipid at the plasma membrane.…”

SHC and SHIP phosphorylation and interaction in response to activation of the FLT3 receptor

“…This might be related to the inability of the FLT3 receptor to form a complex with SHC (our personal observation), in contrast to other RTKs such as the EGF receptor, which binds to SHC through both the PTB and SH2 domains. 28 The specific requirements for SHC phosphorylation upon FLT3 activation are clearly different from what has been described upon activation by IL3 26 where SHC must not only be recruited to the membrane in a phospholipid/PTB-dependent manner, but must also be able to contact the tyrosine phosphorylated ␤ c chain of the IL3 receptor, also through its PTB domain. One can suggest that FLT3-dependent SHC phosphorylation is solely dependent upon its recruitment to the membrane via phospholipids.…”

“…In addition, the SHC PTB domain is able to bind acidic membrane phospholipids, in particular phosphatidylinositol-4-phosphate (Ptdlns(4)P). 25 The F198 → V mutation in the SHC PTB domain specifically affects binding to phosphotyrosines but not phospholipids [25][26][27] and is not necessary for SHC phosphorylation (Figure 4). Similarly, an inactivating mutation in the SH2 domain of SHC does not preclude SHC phosphorylation.…”