Page 72 - 72_04

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

All distinct eIF2a kinases share extensive homology in the kinase
catalytic domain. Apart from the 12 conserved subdomains found in
most protein kinases, they have additional characteristic features,
including an insert region between subdomains IV and V and a
conserved sequence in subdomain V that distinguishes them from
other serine/threonine kinases (15). Especially notable is the presence
of this conserved motif, essentially LY/HIQME/QY/LC, located N-
terminal to subdomain V in all of the known members (up to 20) of
this kinase family, suggesting that this motif might contain a putative
substrate-specific recognition domain (16).

HRI is regulated by heme, and it seems that heme binds directly
to HRI and blocks kinase activity (17). In fact, it has been shown
that HRI is a hemoprotein with two distinct heme-binding sites
(17, 18). Both of them, the N-terminus and the kinase insertion,
which are unique to HRI, appear to be involved in the heme
regulation of HRI (18). Furthermore, HRI is among six hemoproteins
that have a putative heme regulatory motif (HRM) (19); HRI contains
two of these motifs that are not present in other eIF2a kinases,
although the role of the HRMs in the heme regulation of full-length
HRI remains uncertain. Thus, the heme-binding site responsible for
the reversible heme regulation of HRI must be determined in order
to understand the mechanism of the regulation of HRI by heme.

In this study we have examined the effects of several deletions
and point mutations in specific regions of HRI on its activity by
in vitro assays. We have also tested these mutants of HRI for
responsiveness to heme. Our findings indicate that none of three
unique regions in HRI, the N-terminus, the kinase insertion and
the C-terminus are required for the regulation of HRI by heme.
Furthermore, we have found that both the autokinase and the eIF2a
kinase activities of the other members of the eIF2a kinase family,
such as PKR, GCN2 and PERK, were also sensitive to hemin in vitro.
We conclude that the heme-binding site responsible for the reversible
heme regulation is located within the eIF2a kinase domain and
this conserved motif(s) is characteristic of all known members of the
eIF2a kinase family.

614