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SATURNINO HERRERO Y COLS. AN. R. ACAD. NAC. FARM.

described (11). Bacterial lysates were diluted (1/100) in 20 mM Tris-

HCl (pH 7.4) and assayed for their ability to phosphorylate eIF2a as
described below.

TABLE I. Sequences of mutagenic HRI oligonucleotides

Construct Mutagenic primer *

HRI C409A 5’-GTGGACGAAGCTGCTGCTCCCTATGTTATGGC (sense)
HRI C550A 5’-GCCATAACATAGGGAGCAGCAGCTTCGTCCAC (antisense)
HRI 138-580 5’-CCCTCAGTAAAAGGGCTCCGGTGCAAGCC (sense)
5’-GGCTTGCACCGGAGCCCTTTTACTGAGGG (antisense)
5’-CAGAGTGAGCTTTTTTAAACAACTGGAAATG (sense)
5’-CATTTCCAGTTGTTTAAAAAAGCTCACTCTG (antisense)

* Underlined codons correspond to the indicated amino acid mutations and the generated
stop codon. The changed nucleotides are marked in bold.

Cell Culture and Transfection

HEK 293T cells were grown and transfected as previously
described (20). The plasmids used were the pcDNA3.1/Myc-His
vectors (Invitrogen) containing the coding region of mouse GCN2
(pcMGCN2-WT) (5), mouse HRI (pcMHRI-WT) (11), human PKR
(provided by Dr. J. Gil), Drosophila melanogaster PERK (13), or rat
?PKC (21) in frame with a C-terminal tag encoding the myc epitope
and a polyhistidine metal-binding peptide.

Affinity Purification and in vitro Phosphorylation Assays

Affinity purification of proteins was performed as previously
described (20). The eluted proteins from the metal affinity resin as
well as the E. coli lysates containing the HRI wild type and mutant
proteins were assayed for their ability to phosphorylate eIF2a as
reported previously (12, 22), with modifications as described. In a
total volume of 20 µl, 5 µl of kinase fractions were incubated with
various concentrations of hemin (0-4 µM) for 30 min at 30º C in the
kinase buffer (20 mM Tris-HCl (pH 7.6), 2.5 mM MgCl2, 2.5 mM

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