The regulation of protein synthesis by the availability of heme in reticulocytes is well established. However, the mechanism by which heme regulates translational initiation is not clear. In this study, we have examined the heme regulation directly on the homogeneous heme-regulated eIF-2A kinase (HRI), which is activated during heme deficiency. We found that HRI purified as a hemoprotein with the characteristic Soret band of hemoprotein at 424 nm. This HRI was an active autokinase and eIF-2A kinase, and its kinase activities were inhibited by submicromolar concentrations of hemin with an apparent K i of 0.5 µM. Homogeneous HRI was a homodimer, and its activities could not be inhibited by incubation with purified inactive K199R HRI in vitro. Our results suggest that there are two distinct types of hemebinding sites in the HRI homodimer. The binding of heme to the first site is stable, while the binding of heme to the second site is responsible for the rapid downregulation of HRI activity by heme. These results indicate that HRI binds heme and serves as a sensor of the availability of heme to coordinate the balanced synthesis of globins and heme in erythroid cells.Keywords : protein kinase ; heme ; hemoprotein ; translation ; initiation.Protein synthesis in reticulocytes is regulated by the availability of heme. During heme deficiency, protein synthesis is inhibited at the level of initiation, as a result of the activation of the heme-regulated eukaryotic initiation factor 2A (eIF-2A) kinase (HRI) (reviewed in [1Ϫ3]). Phosphorylation of the A subunit of eIF-2 at the Ser51 residue by activated HRI results in the formation of the eIF-2(AP)/eIF-2B complex and renders eIF-2B non-functional. eIF-2B is required for the exchange of GTP for GDP bound to eIF-2 in the recycling of eIF-2 for another round of initiation. Since eIF-2B is limiting, phosphorylation of a fraction of eIF-2 is sufficient to shut-off protein synthesis.In addition to HRI, there are two other eIF-2A kinases that have been studied extensively (reviewed in [4]); these are human and mouse double-stranded RNA-dependent eIF-2A kinase (PKR), and yeast and Drosophila [5] GCN2 protein kinase. These three eIF-2A kinases share extensive homology in the kinase catalytic domains [5Ϫ8], and phosphorylate eIF-2A at Ser51 [9,10]. However, the regulatory mechanisms of these three eIF-2A kinases are very different, involving heme for HRI, dsRNA for PKR and the condition of amino acid starvation for GCN2. Both HRI and PKR can functionally substitute for GCN2 in the GCN4 translational control in yeast [11]. The autophosphorylation, eIF-2A phosphorylation and the inhibition of protein synthesis by purified HRI is inhibited by incubation with hemin [28]. In addition, the binding of hemin to a highly purified HRI has been demonstrated [29]. We have shown that hemin promotes intersubunit disulfide-bond formation in HRI [30], and that this disulfide-bond formation in HRI correlates with the maintenance of protein synthesis, the reversal of the inhibition of protein synthesi...

We showed that CCh-treatment leads to changes in muscarinic M(2), M(3), and M(4) receptor transcripts as well as M(2) and M(3) protein levels. We also found that CCh decreased proliferation of K562 cells in a time dependent manner, an effect prevented by atropine. These results suggest that CCh modulates K562 chronic myelogenous leukemic cells proliferation through muscarinic acetylcholine receptors.

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Activating mutations of the G proteins, Gsalpha (gsp) and Gi2alpha (gip) have been reported in subsets of pituitary tumors. The objective of the study was to assess the frequency of gsp and gip mutations in pituitary tumors from Turkish patients and to investigate the possibility of mutations of protein kinase A catalytic subunit (PKAC) that activates the downstream effectors of adenylyl cyclase. PCR-amplified genomic DNA was analyzed for the presence of mutations in codons 201 and 227 of Gsalpha, codon 179 and 205 of Gi2alpha and codon 196 of PKAC, by single strand conformation polymorphism analysis, allele-specific oligonucleotide hybridization and DNA sequencing. Twenty-two patients from Turkey, 15 females and 7 males were investigated; 7 somatotroph adenomas, 7 clinically non-functioning tumors, 7 prolactinomas and 1 corticotroph adenoma. G protein mutations were identified in 6 of 22 (27.3%) pituitary tumors. Four tumors (3/7 somatotroph adenomas, 43%, 1/7 clinically non-functioning tumor) demonstrated gsp mutations at codon 201 arginine to cysteine and one recurrent somatotroph adenoma demonstrated a mutation of the Gi2alpha gene at codon 193 lysine to arginine. One tumor exhibited a C to T variation in the intervening sequence between codons 179 and 205 of the Gi2alpha gene. No mutations at codon 227 of Gsalpha, codons 179 and 205 of Gi2alpha and codon 196 of the PKAC gene were identified.

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