Incorporation of Phosphorylated Tyrosine into Proteins: In Vitro Translation and Study of Phosphorylated IκB-α and Its Interaction with NF-κB

Abstract

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 in vitro and in vivo, the preparation of proteins phosphorylated at predetermined sites cannot be done easily and in acceptable yields. To enable the synthesis of phosphorylated proteins for in vitro 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-o-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_CUA by UAG codon suppression during in vitro 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.

Figure 1

Structures of tyrosine (1), phosphotyrosine (2), o-nitrobenzylphosphotyrosine (3), and bis(o-nitrobenzyl)phosphotyrosine (4).

Figure 2

(A) Translation of DHFR from wild-type (lane 1) and modified (TAG codon in protein position 10) genes in the absence (lane 2) and in the presence of tyrosyl-tRNA_CUA (lane 3), bis(o-nitrobenzyl)phosphotyrosyl-tRNA_CUA (lane 4), o-nitrobenzylphosphotyrosyl-tRNA_CUA (lane 5), and phosphotyrosyl-tRNA_CUA (lane 6). (B) Translation of IκB-α from wild-type (lane 1) and modified (TAG codon in protein position 42) genes in the absence of an activated tRNA_CUA (lane 2) and in the presence of bis(o-nitrobenzyl)-phosphotyrosyl-tRNA_CUA (lane 3) and phosphotyrosyl-tRNA_CUA (lane 4).

Figure 3

(A) Analysis of wild-type IκB-α (lane 1) and the caged IκB-α protein having a bis-o-nitrobenzyl protected phosphate group before (lane 2) and after (lane 3) 3 min of UV irradiation. The samples were analyzed for reactivity to a polyclonal antibody to phosphotyrosine and for ³⁵S-methionine content. (B) Analysis of the caged IκB-α protein having a bis-o-nitrobenzyl protected phosphate group before, and as a function of the time of UV irradiation. Aliquots were taken at predetermined times and analyzed for reactivity to a polyclonal antibody to phosphotyrosine and for ³⁵S-methionine content.

Figure 4

Analysis of the phosphopuromycin sensitivity of BL-21(DE-3) cells having modified ribosomes. The assays were carried out in the presence of 2.5 μg/mL of erythromycin to partially block the activity of endogenous wild-type ribosomes.

Figure 5

Comparison of the in vitro translation yields of phosphorylated IκB-α obtained using S-30 preparations containing representative modified vs wild-type E. coli ribosomes. The IκB-α samples obtained from wild-type (wt; lanes 1) and modified (TAG codon in protein position 42) genes in the absence (no; lanes 2) and in the presence of phosphotyrosyl-tRNA_CUA (pTyr; lanes 3) were analyzed by 12% SDS-polyacrylamide gel electrophoresis.

Figure 6

Immunoblotting analysis of IκB-α samples obtained by in vitro translation from the wild-type gene and from the modified (TAG codon in protein position 42) gene in the presence of phosphotyrosyl-tRNA_CUA (pTyr) and purified by Ni-NTA chromatography. Analysis of samples by 4%–12% SDS-polyacrylamide gradient gel electrophoresis using (A) phosphorimager detection of ³⁵S-methionine and (B) anti-phosphorylated IκB-α (pTyr42) rabbit polyclonal IgG.

Figure 7

MALDI MS/MS analysis of the tryptic peptides (amino acids 39–47) from (A) wild-type and (B) phosphorylated IκB-α samples.

Figure 8

Purification of NF-κB (as a complex with cellular DNA) from extracts of Jurkat cells (A) without initial cell activation and (B) following cell activation with PMA + A23187. The extracts were fractionated using DEAE-Sepharose CL-6B. Each fraction was incubated with ³²P-labeled double-stranded DNA corresponding to the NF-κB binding site in the IL-2 gene promoter. Samples were separated by 6% native polyacrylamide gel electrophoresis and analyzed using a phosphorimager. Lane 1, crude extract; lane 2, flow through from DEAE-Sepharose CL-6B column; lanes 3–8, eluates resulting from wash with 100, 200, 300, 400, 500, and 600 mM NaCl, respectively. Formation of the NF-κB–³²P-labeled DNA complex is envisioned by (partial) chemical exchange for cellular DNA. (C) Comparison of the amount of purified NF-κB isolated from unactivated vs activated Jurkat cells after purification by DEAE-Sepharose CL-6B (300 mM NaCl wash). Samples were separated by 6% native polyacrylamide gel electrophoresis and analyzed using a phosphorimager. Lane 1, purified NF-κB isolated from extracts of unactivated Jurkat cells; lane 2, purified NF-κB isolated from extracts of Jurkat cells activated with PMA + A23187.

Figure 9

(A) Assay of NF-κB (as a complex with cellular DNA) treated with radiolabeled DNA. Samples were separated by 6% native polyacrylamide gel electrophoresis and analyzed using a phosphor-imager. Lane 1, binding of DNA to NF-κB purified from unactivated Jurkat cells; lane 2, binding of DNA to purified NF-κB from Jurkat cells activated with PMA + A23187; lane 3, binding of DNA to purified NF-κB from Jurkat cells activated with PMA + A23187 in the additional presence of 20 ng of wild-type IκB-α; lane 4, binding of DNA to purified NF-κB from Jurkat cells activated with PMA + A23187 in the additional presence of 20 ng of IκB-α phosphorylated on Tyr42; lane 5, binding of DNA to purified NF-κB from Jurkat cells activated with PMA + A23187 in the additional presence of 20 ng of IκB-α phosphorylated on Tyr42, and 200 ng of NF-κB (p50) polyclonal rabbit IgG. (B) Rationalization of the change in intensity of the observed bands.

Figure 10

Binding of nonphosphorylated IκB-α (IκB-α wt) and phosphorylated IκB-α (IκB-α 42-pTyr) to NF-κB. (A) Analysis (native 8% polyacrylamide gel) of the purified lysate from activated Jurkat cells (Figure 8B, lane 5) in complex with ³⁵S-labeled wild-type and phosphorylated IκB-α prepared by in vitro protein synthesis. Lanes 1 and 8, ³⁵S-labeled protein not treated with lysate; lanes 2 and 5, ³⁵S-labeled proteins treated with lysate; lanes 3 and 6, ³⁵S-labeled proteins treated with lysate diluted 2-fold with 50 mM Tris–HCl, pH 7.4; lanes 4 and 7, ³⁵S-labeled protein treated with lysate diluted 4-fold with 50 mM Tris–HCl, pH 7.4. (B) Quantification of the binding illustrated in the gel in panel A for ³⁵S-labeled wild-type (blue) and phosphorylated (orange) IκB-α. C, control not treated with lysate; 1, purified lysate present at full strength; 0.5, lysate present at 2-fold dilution; 0.25, lysate present at 4-fold dilution.

Figure 11

Time-dependent binding of NF-κB and ³²P end-labeled DNA. (A) Samples were analyzed by 6% native polyacrylamide gel electrophoresis and quantified using a phosphorimager. Upper panel, binding of NF-κB and DNA without phosphorylated IκB-α; lower panel, binding of NF-κB and DNA in the presence of 20 ng of phosphorylated IκB-α. Lane 1, incubation for 0 min; lane 2, incubation for 0.5 min; lane 3, incubation for 1 min; lane 4, incubation for 3 min. (B) Relative intensity of NF-κB–DNA complexes analyzed by 6% native polyacrylamide gel electrophoresis. The intensity of the NF-κB–DNA complex in the reaction of NF-κB and DNA without phosphorylated IκB-α at 0.5 min was defined as 100%.

Scheme 1

Synthesis of the pdCpA Ester of Bis(o-nitrobenzyl)phosphotyrosine and Bis(o-nitrobenzyl)phosphotyrosyl-tRNA_CUA

Scheme 2

Synthesis of Phosphopuromycin