1 research outputs found
Incorporation of Phosphorylated Tyrosine into Proteins: <i>In Vitro</i> Translation and Study of Phosphorylated IκB‑α and Its Interaction with NF-κB
Phosphorylated
proteins play important roles in the regulation of many different
cell networks. However, unlike the preparation of proteins containing
unmodified proteinogenic amino acids, which can be altered readily
by site-directed mutagenesis and expressed <i>in vitro</i> and <i>in vivo</i>, the preparation of proteins phosphorylated
at predetermined sites cannot be done easily and in acceptable yields.
To enable the synthesis of phosphorylated proteins for <i>in
vitro</i> studies, we have explored the use of phosphorylated
amino acids in which the phosphate moiety bears a chemical protecting
group, thus eliminating the negative charges that have been shown
to have a negative effect on protein translation. Bis-<i>o</i>-nitrobenzyl protection of tyrosine phosphate enabled its incorporation
into DHFR and IκB-α using wild-type ribosomes, and the
elaborated proteins could subsequently be deprotected by photolysis.
Also investigated in parallel was the re-engineering of the 23S rRNA
of Escherichia coli, guided by the
use of a phosphorylated puromycin, to identify modified ribosomes
capable of incorporating unprotected phosphotyrosine into proteins
from a phosphotyrosyl-tRNA<sub>CUA</sub> by UAG codon suppression
during <i>in vitro</i> translation. Selection of a library
of modified ribosomal clones with phosphorylated puromycin identified
six modified ribosome variants having mutations in nucleotides 2600–2605
of 23S rRNA; these had enhanced sensitivity to the phosphorylated
puromycin. The six clones demonstrated some sequence homology in the
region 2600–2605 and incorporated unprotected phosphotyrosine
into IκB-α using a modified gene having a TAG codon in
the position corresponding to amino acid 42 of the protein. The purified
phosphorylated protein bound to a phosphotyrosine specific antibody
and permitted NF-κB binding to a DNA duplex sequence corresponding
to its binding site in the IL-2 gene promoter. Unexpectedly, phosphorylated
IκB-α also mediated the exchange of exogenous DNA into
an NF-κB–cellular DNA complex isolated from the nucleus
of activated Jurkat cells