The Regions of the Fe65 Protein Homologous to the Phosphotyrosine Interaction/Phosphotyrosine Binding Domain of Shc Bind the Intracellular Domain of the Alzheimer's Amyloid Precursor Protein

Francesca Fiore,Nicola Zambrano,Giuseppina Minopoli,Virgilio Donini,Angela Duilio,Tommaso Russo

doi:10.1074/jbc.270.52.30853

Abstract

Fe65 is a protein mainly expressed in several districts of the mammalian nervous system. The search of protein sequence data banks revealed that Fe65 contains two phosphotyrosine interaction (PID) or phosphotyrosine binding (PTB) domains, previously identified in the Shc adaptor molecule. The two putative PID/PTB domains of Fe65 were used to construct glutathione S-transferase-Fe65 fusion proteins. Co-precipitation experiments demonstrated that the Fe65 PID/PTB domains interacted with several proteins of apparent molecular mass 135, 115, 105, and 51 kDa. The region of Fe65 containing the PID/PTB domains was used as a bait to screen a human brain cDNA library in yeast by the two-hybrid system. Three different cDNA clones were isolated, two of which contain overlapping segments of the cDNA encoding the COOH terminus of the Alzheimer's beta-amyloid-precursor protein (APP), that represents the short intracellular domain of this membrane protein. The third clone contains a cDNA fragment coding for the COOH terminus of the human counterpart of a mouse beta-amyloid-like precursor protein. The alignment of the three APP encoding cDNA fragments found in the screening suggests that the region of APP involved in the binding is centered on the NPTY sequence, which is analogous to that present in the intracellular domains of the growth factor receptors interacting with the PID/PTB domain of Shc.

Highlights

In combination, in a wide range of proteins [2,3,4]
The Fe65 PID/phosphotyrosine binding (PTB) Domains Bind to Several Proteins—In order to evaluate if Fe65 PID/PTB domains interact with cellular proteins, we expressed and purified from E. coli a Glutathione S-transferase (GST)
Fe65 fusion protein in which GST was fused to the Fe65 region from amino acid 312 to 612, which contains two elements that can be aligned with the PID/PTB domain present in Shc

Summary

Introduction

In combination, in a wide range of proteins [2,3,4]. Shc is one of the members of this machinery; it is involved in the coupling of the activated tyrosine kinase growth factor receptors to the Ras activation pathway [5, 6], through at least two other proteins, Grb, which binds to tyrosine-phosphorylated Shc [6], and Sos, which, upon binding to Grb, acts on Ras as a GDP/GTP exchange factor [7]. The analysis of sequence data banks for proteins showing similarities with this PID/PTB domain of Shc allowed the identification of a group of proteins sharing with Shc a region of about 160 amino acids with common predicted structural features and several conserved motifs [12] This group of proteins includes the Fe65 protein. The corresponding mRNA is mainly expressed in the brain and in several districts of the nervous system, including the ganglia of the somatic and visceral nervous system and the gangliar structures of sense organs [14] It encodes a protein of about 90 kDa, present both in the cytoplasm and in the nucleus, which contains a domain highly homologous to the retroviral integrases [15, 16]. The screening of a human brain cDNA expression library in yeast, based on the two-hybrid system, allowed us to demonstrate that this region binds the intracellular domain of the Alzheimer’s ␤-amyloid precursor protein (APP)

Methods

Results

Conclusion