Abstract
The hemin-controlled inhibitor, or HCI, is a cyclic nucleotide-independent protein kinase which specifically phosphorylates the alpha subunit of eukaryotic initiation factor 2 (eIF-2) leading to potential limitations in functional eIF-2 and decreases in protein synthesis initiation. We have recently demonstrated that hemin regulates eIF-2 alpha kinase activity by promoting formation of the inactive heterodimer between HCI and the 90-kDa heat shock protein (hsp90). The formation of the inactive form of HCI by hemin is prevented by treatment with sulfhydryl reagents such as N-ethylmaleimide or when hsp90 is previously phosphorylated (Méndez, R., Moreno, A., and de Haro, C. (1992) J. Biol. Chem. 267, 11500-11507). In this report, using isoelectric focusing and Western blot analyses with antibodies against a synthetic HCI peptide, we have demonstrated that HCI was also phosphorylated when a heme-reversible HCI preparation was preincubated with ATP. Furthermore, our results indicate that casein kinase II (CK II) is the enzyme involved in the regulation of HCI. Thus, the synthetic peptide RRREEETEEE, which is a specific substrate for CK II, acts as a competitive inhibitor of HCI and hsp90 phosphorylation and, at the same time, inhibits the activation of HCI, whereas a synthetic peptide which corresponds to residues 45-59 of the alpha subunit of eIF-2, including the Ser51 phosphorylated by HCI, only inhibits competitively the phosphorylation of eIF-2 alpha. In addition, treatments which modify hsp90 disabling the formation of the inactive dimer with HCI make unnecessary the presence of CK II for activation of HCI. The data strongly suggest that hemin promotes formation of an inactive HCI.hsp90 dimer preventing phosphorylation by CK II. Interestingly, the addition of the CK II peptide substrate also prevents the activation of HCI in a heme-deficient reticulocyte lysate. We hypothesize, therefore, that under physiological conditions, CK II activity appears to be necessary for activation of HCI.
Highlights
From the Centro de Biologia Molecular “Seuero Ochoa,” Consejo Superior de Inuestigaciones Cientifcas, Uniuersidad Autonoma de Madrid, Canto Blanco, 28049 Madrid, Spain
11500-11507).In this report, using isoelectric focusing 41-59 in eIF-2aderived from rat and human[7] and the yeast and Western blotanalyseswith antibodiesagainst a syn- Saccharomyces cerevisiae [8].These data suggest that yeast thetic HCI peptide, we have demonstrated that HCI was eIF-2a may be phosphorylated on Ser5’ to regulate transalso phosphorylatedwhen a heme-reversibleHCI prepa- lation and,Dever et a l . [9] have recently reported that ration was preincubated with ATP
I1 (CK 11)is the enzyme involved in the regulation of cells, provided that a serine residue is present at position 51
Summary
From the Centro de Biologia Molecular “Seuero Ochoa,” Consejo Superior de Inuestigaciones Cientifcas, Uniuersidad Autonoma de Madrid, Canto Blanco, 28049 Madrid, Spain. In an earlier paper[17], we reported that hemin promotes formation of an inactive dimer (HCI.hsp90) and that it is activated by treatment with sulfhydryl reagents such as NEM or when the heme-reversible HCI is previously phossubstrate. Antibodies against SyntheticHCI Peptide-Based on the HCI cDNA coding sequence that Chen et al [10]obtained, a syntheticpeptide corresponding to amino acids 263-276 was made with a n additional cysteine residue in its NH,-terminal end. This peptide was coupled to keyhole limpet hemocyanin as described [25]. This HCI preparatiotnibodies as described [27].In all cases, the nonimmune controlsdid not was only activated by incubation withATP, under reducing conditions. precipitate either HCI or hsp (data not shown)
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