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

threonine protein kinases, are regulated in vitro by hemin, suggesting
that the putative heme-regulatory motifs are shared by all members
of the eIF2a kinase family.

DISCUSSION

The molecular mechanism(s) by which hemin regulates
mammalian HRI has yet to be determined. Furthermore, the precise
heme-binding domain responsible for rapid regulation of HRI by
heme is not characterized. Some previous reports have suggested
that purified HRI is a homodimer and a hemoprotein with two
distinct heme-binding sites. Binding of heme to an N-terminal
domain (amino acids 1-138) of HRI is stable and autonomous,
whereas binding to a kinase-insertion domain (amino acids 241-406)
is dynamic and may be responsible for the reversible heme regulation
of HRI (18). It should be noted that this reported kinase-insertion
domain contains not only the entire mouse kinase insert sequence
unique to HRI but also includes the kinase subdomain V and the
adjacent amino acids (LHIQMQLC), a highly conserved motif among
eIF2a kinases. Additionally, HRI is among six hemoproteins that
have a putative heme regulatory motif (HRM) where Cys at position
2 seems to be essential (19). HRI contains two of these HRMs, which
are not present in the other eIF2a kinases. The data reported here
indicate that the HRMs do not play an essential role in the heme-
responsiveness of HRI. Thus, Cys-409 and Cys-550 were mutated
individually to alanine and the entire HRM1 was deleted with no
apparent effect on either the eIF2a kinase activity or the heme
responsiveness of the HRI mutants. Furthermore, two novel eIF2a
kinases from S. pombe, highly similar to HRI, show heme
responsiveness in vitro although amino acid sequence analyses
revealed that they lacked any of this HRM.

Comparison of the deduced amino acid sequences of mammalian
HRI with the other eIF2a kinases reveals the existence in HRI of
three unique regions, very different in sequence and size, in addition
to the two kinases lobes containing the 12 conserved catalytic
subdomains characteristic of all eukaryotic Ser/Thr protein kinases.
Thus, mouse HRI comprises these five domains (Fig. 1B): the amino

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