Presenilin1/γ‐secretase promotes the EphB2‐induced phosphorylation of ephrinB2 by regulating phosphoprotein associated with glycosphingolipid‐enriched microdomains/Csk binding protein

Abstract

Reverse signaling through the ephrinB ligands is important for several morphogenetic events, such as axon guidance, neuronal plasticity, spine maturation, and synaptogenesis. Signaling is initiated by binding of EphB receptors to ephrinB ligands, stimulating their tyrosine phosphorylation via an unclear mechanism. Here we show that this mechanism involves presenilin1 (PS1)/γ-secretase regulation of phosphoprotein associated with glycosphingolipid-enriched microdomains/Csk binding protein (PAG/Cbp), an adaptor protein that controls the activity of Src kinases. Using immunoprecipitation and Western blot of mouse primary neuronal and human embryonic kidney (HEK293) cell extracts overexpressing PAG/Cbp, we show that EphB2 induces tyrosine dephosphorylation of PAG/Cbp in a γ-secretase-dependent manner. In these cells, PAG/Cbp dephosphorylation is promoted by the PS1/γ-secretase-produced fragment of ephrinB2 cleavage (ephrinB2/CTF2), which forms complexes with PAG/Cbp when introduced exogenously. EphB2-induced tyrosine phosphorylation of ephrinB2 depends on PAG/Cbp because EphB2 cannot increase ephrinB2 phosphorylation in cells treated with anti-PAG siRNA or in PAG/Cbp-knockout (KO) cells. Furthermore, in contrast to WT PS1, familial Alzheimer disease (FAD) PS1 mutants expressed in PS1-KO mouse embryonic fibroblasts inhibited both the EphB2-induced dephosphorylation of PAG/Cbp and the phosphorylation of ephrinB2. PS1 FAD mutations may thus inhibit the function of ephrinB in the brain, promoting neurodegeneration in Alzheimer disease.

Nephric duct insertion requires EphA4/EphA7 signaling from the pericloacal mesenchyme

“…This specific tyrosine phosphorylation site has been described to activate downstream Src family or associated kinases as a result of active ephrin B reverse signaling (Palmer et al, 2002;Wu et al, 2011;Georgakopoulos et al, 2011). In the wild type, we found phospho-specific staining in membranes of epithelial cells of the ND, the cloaca and endothelial cells at E10.5, whereas strongly reduced phospho-specific staining was detectable in the ND and the cloaca in the DKO situation (Fig.…”

The role of proteases in regulating Eph/ephrin signaling

“…ADAM10 is a prime example, where regulation likely occurs at multiple levels. Thus formation of a ligand-receptor complex across cell-cell junctions enables recognition by the ADAM10 substrate-binding domain, while the ensuing receptor activation likely leads to conformational changes in both Eph EphB-stimulated metalloprotease/ g-secretase sequential cleavage, controlling cytoskeletal changes in neural cells 98,124 Ephrin-B and calcium/NMDA activation promotes distinct ectodomain/ g-secretase cleavage events in neural cells 106 Contributes to the formation and maintenance of dentritic spines via activation of the Rac signaling pathway 133 Serine proteases RHBDL2 ephrin-B3 Exogenous co-expression degrades ephrin, physiological relevance unclear 142 Neuropsin EphB2…”

“…98 A later study showed involvement of the Csk binding protein (PAG/Cbp), an adaptor protein that controls the activity of Src kinases. 124 Ephrin-B2-soluble CTF2 forms a complex with PAG/Cbp promoting Src activation, 124 which in other contexts has been shown to control various cellular events such as cell proliferation, survival, and migration 125 most likely via remodelling of the actin cystoskeleton 126 ( Fig. 4A; Table 1).…”

“…Inside the cell, ephrin-B2-CTF promotes tyrosine dephosphorylation of (PAG/Cbp), and relieves Csk-inhibition of Src kinases, which in turn facilitates tyrosine phosphorylation of ephrin-B2. 98,124 Ligandbinding to EphB2 also promotes receptor ectodomain cleavage, endocytosis, and g-secretase cleavage, to produce soluble Eph cytoplasmic domain (bottom cell). 106 This regulated intramembrane proteolysis in both cells contributes to cytoskeletal regulation, migration, proliferation and survival.…”

Intramembranous processing by γ‐secretase regulates reverse signaling of ephrin‐B2 in migration of microglia

“…This type of regulated intramembranous proteolysis (RIP) has previously been demonstrated for ephrin-B2 (Georgakopoulos et al 2006), and later also for EphB2 (Litterst et al 2007;Xu et al 2009) and EphA4 (Inoue et al 2009). The γ-secretase mediated cleavage of ephrin-B2 upon EphB2 receptor binding regulates activity of Src in murine embryonic fibroblasts (MEF) (Georgakopoulos et al 2011;Waschbusch et al 2009). …”