The α subunit of eukaryotic protein synthesis initiation factor (eIF‐2α) is phosphorylated at a single serine residue (Ser51) by two distinct and well‐characterised protein kinase, the haem‐controlled repressor (HCR) and the double‐stranded RNA‐activated inhibitor (dsI). The sequence adjacent to Ser51 is rich in basic residues (Ser51‐Arg‐Arg‐Arg‐Ile‐Arg) suggesting that they may be important in the substrate specificity of the two kinases, as is the case for several other protein kinases. A number of proteins and synthetic peptides containing clusters of basic residues were tested as substrates for HCR and dsI. Both kinases were able to phosphorylate histones and protamines ar multiple sites as judged by two‐dimensional mapping of the tryptic phosphopeptides. These data also showed that the specificities of the two kinases were different from one another and from the specificities of two other protein kinases which recognise basic residues, cAMP‐dependent protein kinase and protein kinase C. In histones, HCR phosphorylated only serine residues while dsI phosphorylated serine and threonine. Based on phosphoamino acid analyses and gel filtration of tryptic fragments, dsI was capable of phosphorylating both ‘sites’ in clupeine Y1 and salmine A1, whereas HCR acted only on the N‐terminal cluster of serines in these protamines. The specificities of HCR and dsI were further studied using synthetic peptides with differing configurations of basic residues. Both kinases phosphorylated peptides containing C‐terminal clusters of arginines on the ‘target’ serine residue, provided that they were present at positions + 3 and/or + 4 relative to Ser51. However, peptides containing only N‐terminal basic residues were poor and very poor substrates for dsI and HCR, respectively. These findings are consistent with the disposition of basic residues near the phosphorylation site in eIF‐2α and show that the specificities of HCR and dsI differ from other protein kinases whose specificities have been studied.
|Number of pages||9|
|Journal||European Journal of Biochemistry|
|Publication status||Published - Feb 1991|
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