Tyr721 regulates specific binding of the CSF-1 receptor kinase insert to PI 3′-kinase SH2 domains: a model for SH2-mediated receptor-target interactions.

Abstract

Efficient binding of active phosphatidylinositol (PI) 3′‐kinase to the autophosphorylated macrophage colony stimulating factor receptor (CSF‐1R) requires the noncatalytic kinase insert (KI) region of the receptor. To test whether this region could function independently to bind PI 3′‐kinase, the isolated CSF‐1R KI was expressed in Escherichia coli, and was inducibly phosphorylated on tyrosine. The tyrosine phosphorylated form of the CSF‐1R KI bound PI 3′‐kinase in vitro, whereas the unphosphorylated form had no binding activity. The p85 alpha subunit of PI 3′‐kinase contains two Src homology (SH)2 domains, which are implicated in the interactions of signalling proteins with activated receptors. Bacterially expressed p85 alpha SH2 domains complexed in vitro with the tyrosine phosphorylated CSF‐1R KI. Binding of the CSF‐1R KI to PI 3′‐kinase activity, and to the p85 alpha SH2 domains, required phosphorylation of Tyr721 within the KI domain, but was independent of phosphorylation at Tyr697 and Tyr706. Tyr721 was also critical for the association of activated CSF‐1R with PI 3′‐kinase in mammalian cells. Complex formation between the CSF‐1R and PI 3′‐kinase can therefore be reconstructed in vitro in a specific interaction involving the phosphorylated receptor KI and the SH2 domains of p85 alpha.

Identification of a second Grb2 binding site in the v-Fms tyrosine kinase

“…In agreement with previous reports (Reedijk et al, 1992;Joos et al, 1996), such speci®c binding was indeed observed, provided that the GST-KI-Fms protein was phosphorylated ( Figure 3c). As expected, some Grb2 was detected in the GST-pY-KI-Fms fraction (van der Geer and Hunter, 1993).…”

“…Several autophosphorylation sites of the Fms molecules have been mapped. These include Y543 in the JX domain (Joos et al, 1996), Y696, Y705, and Y720 (corresponding to Y697, Y706 and Y721 in the mouse c-Fms) in the KI domain, and Y807 in the K2 domain (Tapley et al, 1990;van der Geer and Hunter, 1990Roussel et al, 1990;Reedijk et al, 1992;Trouliaris et al, 1995a,b;Joos et al, 1996). Which are the cellular proteins that bind to these autophosphorylation sites?…”

“…Y543 mediates association with a p55 polypeptide of yet unknown function (Joos et al, 1996). The short segment of the KI domain encompassing Y696, Y705, and Y720, provides binding sites for Grb2 (van der Geer and Hunter, 1993), STAT1 (Novak et al, 1996), and the p85 subunit of phosphatidyl-inositol-3' (PI-3') kinase (Reedijk et al, 1992), respectively. Furthermore, p120RasGAP was shown to bind to Y807 of v-Fms in phosphorylation-dependent manner (Trouliaris et al, 1995b).…”

New insights into protein‐tyrosine kinase receptor signaling complexes

“…One of the best studied sites is that which mediates interactions with the SH2 domains of the p85 subunit of the PI 3-kinase. This enzyme has been shown to associate with the human PDGF P-receptor via tyrosines 740 and 75 1, which lie in the kinase insert region of this receptor (Fantl et al, 1992;Kashishian et al, 1992); with the murine CSF-1 receptor via tyrosine 721 (Reedijk et al, 1992); with tyrosine 315 of polyoma middle T antigen in the mT/pp60C-S" transforming complex (Talmage et al, 1989); and with several phosphorylated tyrosines on the insulin receptor substrate, IRS-1 Yonezawa et al, 1992). All of these tyrosines lie within the consensus sequence YxxM (where x can be a wide range of possible residues).…”

Specific and common activities of the FLT3 and KIT tyrosine kinase receptors revealed by the use of cultured mast cells

“…It is not known whether this results from either the lack of PLC␥ activity or from the different location, at the C-terminus sequence of the SH2-p85-PI3Ј kinase binding site (Tyr 958) of FLT3 64 compared to other class III RTK. [65][66][67] Alternatively, because this phosphorylated tyrosine residue is also a docking site for the GRB2 adaptator molecule (Beslu et al manuscript in preparation), competitive association could titrate this site for one of the substrate and hide its associated function.…”