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VOL. 72 (4), 611-627, 2006 HEME RESPONSIVENESS IN VITRO IS A COMMON...

mouse HRI (as positive control), mouse GCN2, human PKR, and
Drosophila PERK. In order to achieve full activity of GCN2 and PKR,
they were assayed in the presence of Sindbis virus RNA (20) and
poly(I)·poly(C), respectively (Fig. 3A). Of great interest was the
finding that both the autokinase and eIF2a kinase activities of the
affinity-purified recombinant eIF2a kinases were inhibited by low

FIGURE 3. A, autokinase and eIF2a kinase activities of distinct eIF2a kinases are
inhibited by hemin. All four eIF2a kinases (mouse HRI and GCN2, human PKR

and Drosophila PERK) were expressed in HEK 293T cells as Myc-His fusion
proteins and purified by affinity chromatography. In vitro phosphorylation assays

were performed in the absence or presence of the indicated increasing
concentrations of hemin, as described under Material and Methods. The positions

of phosphorylated HRI, GCN2, PKR, PERK and eIF2a are indicated. B, eIF2a
phosphorylation was estimated by denstitometric analysis as described for Fig. 2.

C, eIF2a kinase activity of both S. pombe eIF2a kinases, SEK1 and SEK2, is
inhibited by hemin. All three eIF2a kinases (mouse HRI and S. pombe SEK1 and
SEK2) were expressed in E. coli, as previously reported. In vitro phosphorylation
assays were performed in the absence or presence of the indicated concentrations

of hemin. D, eIF2a phosphorylation was estimated and represented as in B.

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