Interactions of the heme-regulated eIF-2 alpha kinase with heat shock proteins in rabbit reticulocyte lysates.
Inhibition of protein synthesis in rabbit reticulocyte lysates occurs in response to a variety of conditions including heme deficiency, addition of oxidants, and heat stress. The inhibition of translation is due to the activation of a heme-regulated protein kinase (HRI) which specifically phosphorylates the alpha-subunit of the eukaryotic initiation factor eIF-2. In this report, immunoadsorption with monoclonal antibodies (mAbs) and Western blot analysis were used to investigate the interaction of HRI, the 90-kDa heat shock protein (hsp 90), hsp 70, and the EC1 antigen in rabbit reticulocyte lysates under protein synthesizing conditions. The data indicate that hsp 90, hsp 70, and the EC1 antigen interact with HRI in rabbit reticulocyte lysate. The EC1 antigen is a protein that has been demonstrated to be associated with several steroid hormone receptor-hsp 90 complexes and reacts with the KN 382/EC1 mAb (EC1). The association of HRI with hsp 90 and the EC1 antigen in the reticulocyte lysate was found to be dependent on the presence of hemin at a concentration of 5 microM or higher; little HRI was coadsorbed by the 8D3 anti-hsp 90 mAb or the EC1 mAb in the absence of hemin. Hsp 70 remains associated with HRI in the absence of hemin, suggesting that hsp 90 and 70 may bind to HRI at different sites. The immunological properties of the hsp 70 associated with HRI indicate that it may be the constitutively express heat shock cognate protein (hsc 73). The results suggest that the association of HRI with hsp 90 and the EC1 antigen may be in a dynamic equilibrium, in which complex formation is either facilitated or stabilized by the presence of hemin, and supports the notion that these proteins in conjunction with hsp 70 may play a role in regulating HRI activity or activation in situ.
Department of Biochemistry, Oklahoma State University, Stillwater 74078-0454.
R L
RL
Matts
Z
Z
Xu
J K
JK
Pal
J J
JJ
Chen
D000818:Animals; D000911:Antibodies, Monoclonal; D015153:Blotting, Western; D002474:Cell-Free System; D002846:Chromatography, Affinity; D006360:Heat-Shock Proteins / Q000097:blood* / Q000302:isolation & purification; D006418:Heme / Q000378:metabolism*; D011494:Protein Kinases / Q000097:blood* / Q000302:isolation & purification; D011817:Rabbits; D012156:Reticulocytes / Q000378:metabolism*; D019892:eIF-2 Kinase
D000911:Antibodies, Monoclonal; D006360:Heat-Shock Proteins; D006418:Heme; D011494:Protein Kinases; D019892:eIF-2 Kinase