Conformational changes accompany phosphorylation of the epidermal growth factor receptor C‐terminal domain

• Published in: Protein science 2005-11, Vol.14 (11), p.2793-2803
• Main Authors: Lee, Nam Y., Koland, John G.
• Format: Article
• Language: English
• Subjects: Adenosine Triphosphate - analogs & derivatives; Adenosine Triphosphate - chemistry; A‐loop, activation loop; BFP, blue fluorescence protein; Catalytic Domain; EGFR, epidermal growth factor receptor; EGFR‐CT, C‐terminal domain of EGFR; ErbB; Fluorescence Resonance Energy Transfer; Fluorescent Dyes - chemistry; FRET; FRET, fluorescence resonance energy transfer; HER; ICD, intracellular domain; Light; Luminescent Proteins - chemistry; Phosphorylation; Protein Structure, Tertiary; protein tyrosine kinase; PTK, protein tyrosine kinase; Receptor, Epidermal Growth Factor - chemistry; Receptor, Epidermal Growth Factor - metabolism; Scattering, Radiation; SH2, Src homology‐2; TNP‐ATP, 2′(3′)‐O‐(2,4,6‐trinitrophenyl)‐adenosine 5′‐triphosphate; Δ1022, C‐terminal truncation of EGFR at residue 1022; Δ976, C‐terminal truncation of EGFR at residue 976
• ISSN: 0961-8368; 1469-896X
• DOI: 10.1110/ps.051630305

The precise regulation of epidermal growth factor receptor (EGFR) signaling is crucial to its function in cellular growth control. Various studies have suggested that the C‐terminal phosphorylation domain, itself a substrate for the EGFR kinase activity, exerts a regulatory influence upon it, although the molecular mechanism for this regulation is unknown. The fluorescence resonance energy transfer (FRET) technique was employed to examine how C‐terminal domain conformational changes in the context of receptor activation and autophosphorylation might regulate EGFR enzymatic activity. A novel FRET reporter system was devised in which recombinant purified EGFR intracellular domain (ICD) proteins of varying C‐terminal lengths were site‐specifically labeled at their extreme C termini with blue fluorescent protein (BFP) and a fluorescent nucleotide analog, 2′(3′)‐O‐(2,4,6‐trinitrophenyl)‐adenosine 5′‐triphosphate (TNP‐ATP), binding at their active sites. This novel BFP/TNP‐ATP FRET pair demonstrated efficient energy transfer as evidenced by appreciable BFP‐donor quenching by bound TNP‐ATP. In particular, a marked reduction in energy transfer was observed for the full‐length BFP‐labeled EGFR‐ICD protein upon phosphorylation, likely reflecting its movement away from the active site. The estimated distances from the BFP module to the TNP‐ATP‐occupied active site for the full‐length and C‐terminally truncated proteins also reveal the possible folding geometry of this domain with respect to the kinase core. The present studies demonstrate the first use of BFP/TNP‐ATP as a FRET reporter system. Furthermore, the results described here provide biophysical evidence for phosphorylation‐dependent conformational changes in the C‐terminal phosphorylation domain and its likely interaction with the kinase core.