Mechanism of action of an eukaryotic initiation factor-2 (eIF-2) associated 67 kDa glycoprotein (p67) and an eIF-2 kinase (dsI).
Mechanism of regulation of eIF-2 alpha-subunit phosphorylation by dsI and p67 was studied. The results are as follows: (1) At low dsI concentration, p67 protected equimolar concentration of eIF-2. (2) At high dsI concentration, dsI efficiently phosphorylated eIF-2 alpha-subunit even when equimolar concentrations of both p67 and eIF-2 were present. Significantly increased p67 concentration was necessary to protect eIF-2 alpha-subunit at high dsI concentration. (3) dsI was also phosphorylated as it phosphorylated eIF-2 alpha-subunit. p67 inhibited both eIF-2 alpha-subunit and dsI phosphorylation similarly. (4) Although the [32P]-labelled dsI formed during the reaction could be effectively chased upon subsequent addition of excess unlabelled eIF-2 and ATP, the [32P] labelled eIF-2 formed under identical conditions, retained most of the radioactivity. (5) dsI coimmunoprecipitated with three subunit eIF-2 and p67 inhibited this coimmunoprecipitation reaction. It has been proposed: Three subunit eIF-2 and free p67 are in equilibrium with eIF-2 bound to p67 and, eIF-2.p67 complex is resistant to dsI phosphorylation. Activated dsI is already phosphorylated. At high concentration, dsI(P) can bind to free three subunit eIF-2 and form eIF-2.dsI(P) complex. dsI(P) in this complex then transfers its phosphoryl residue to eIF-2 and forms eIF-2 alpha(P) in an irreversible reaction. In a subsequent reaction, unphosphorylated dsI is autophosphorylated using [gamma 32P]-ATP and the cycle continues. Inhibition of eIF-2 alpha-subunit phosphorylation by p67 blocks this phosphorylation cycle and consequent dsI phosphorylation.
Chakraborty
A
A
Department of Chemistry, University of Nebraska, Lincoln 68588-0304.
Saha
D
D
Bose
A
A
Hileman
R E
RE
Chatterjee
M
M
Gupta
N K
NK
D000626:Aminopeptidases; D015852:Eukaryotic Initiation Factor-2 / Q000737:chemistry*; D006023:Glycoproteins / Q000737:chemistry* / Q000378:metabolism; D063208:Methionyl Aminopeptidases; D008970:Molecular Weight; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry*; D019892:eIF-2 Kinase
D015852:Eukaryotic Initiation Factor-2; D006023:Glycoproteins; D017346:Protein Serine-Threonine Kinases; D019892:eIF-2 Kinase; D000626:Aminopeptidases; C081311:METAP2 protein, human; D063208:Methionyl Aminopeptidases