The Characterization of a Novel Putative Signalling Protein and its Interaction with Tyrosine 1253 of the NEU/ERBB2 Receptor Tyrosine Kinase

Abstract

The Neu/ErbB2 receptor tyrosine kinase has been implicated in the induction of mammary tumourigenesis. Both Ras-dependent and independent signalling downstream of activated Neu is believed to mediate cellular signalling that contributes cellular transformation in oncogenic Neu. Signalling from five 𝘣𝘰𝘯𝘢 𝘧𝘪𝘥𝘦 autophosphorylation sites (termed sites A through E) in the carboxyl tail of the receptor mediate these signals to the cytosol. Of the four positive regulatory sites (B, C, D, E), only three (B, C, D,) signal through known signalling molecules. Site E, (Y1253) in Neu, though known to interact with DOKR is essentially an orphan site. The discovery of a 34kD protein capable of associating to peptides corresponding to site E prompted investigations into the nature of site E signalling. Mass spectrometric analyses revealed that the 34kD protein is 2,4-dienoyl-CoA reductase (DECR1), a lipid metabolism protein typically localized to the mitochondria. Investigations into this protein reveal that DECR1 is capable of associating with Neu site E in a tyrosine phosphorylation and sequence specific manner. Furthermore, analyses with site E second-site mutants (YE[APEY], YE[DPEY], YE[NAEY] and YE[NDEY]) show that DECR1 associates in a manner consistent to a PTB domain-containing protein. Analyses of amino acid sequence demonstrate the presence of a putative Bcl-domain in DECR1 suggestive of a role in apoptosis. Experiments into the apoptotic activity of DECR1 proved inconclusive. The examination of sub-cellular localization of Neu and DECR1 showed that the two proteins are found in both the mitochondrial and plasma membrane fractions. These results demonstrate that DECR1 may be a veritable binding partner for Neu Y1253.