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

terminus, the first kinase lobe (catalytic subdomains I to IV), the
kinase insert (amino acids 236-373), the second kinase lobe (catalytic
subdomains V to XI) and the carboxyl terminus (11). The data
reported here strongly suggest that the three unique regions in HRI
(the N- and C-terminus and the kinase insert) are not involved in the
regulation of HRI activity by heme. Furthermore, we report here that
the kinase insert domain of mouse HRI is required for kinase activity
but does not play a significant role in the heme regulation of HRI.
Thus, we also observed that the HRI Q4 mutant, that contains the
kinase insert domain of mouse GCN2 instead of its own domain, is
still heme-regulated. These findings demonstrate that the binding
domain responsible for the heme regulation of HRI is located within
its two kinase lobes and, therefore, raise the possibility that some
other eIF2a kinases may also be sensitive to inhibition by heme.

To test this possibility, we attempted to determine whether
members of the other known eIF2a kinase subfamilies had the ability
to be regulated by hemin in vitro. Here, we have shown that all
recombinant mouse GCN2, Drosophila PERK and human PKR
exhibit both the autokinase and the eIF2a kinase activities in vitro
and, strikingly, these two kinase activities are inhibited by low
concentrations of hemin. Our results indicate that the heme binding
to a still unknown but well conserved region, may block the binding
of ATP and thus inhibit both the autokinase and the eIF2a kinase
activities of all known eIF2a kinases in vitro.

By comparing the amino acid sequence of mouse HRI with all
other known eIF2a kinases (up to 20 members) we have found 11
invariant positions within the kinase domain that are absent in the
majority of other Ser/Thr protein kinases. These conserved residues
are as follows: R162, D189, Y229, W233, I380, Q381, C385, F447,
G458, L462 and P569 (Fig. 1B). It is of particular interest that three
of these residues (I380, Q381 and C385), uniquely conserved among
eIF2a kinases, are located in the kinase lobe that binds ATP. Thus,
according to sequence alignment with the catalytic subunit of cAMP-
dependent protein kinase (PKA-Ca) (15), and assuming that all Ser/
Thr protein kinases fold into topologically similar three-dimensional
core structures, it was predicted that the conserved sequence
LHIQMQLCE will form a very hydrophobic ß-strand (b5) in the small
kinase lobe and will connect the two lobes of the catalytic domain.

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