Resistance of Initiation Factor 2 (eIF-2α) Kinases to Staurosporine: An Approach for Assaying the Kinases in Crude Extracts

• Published in: Cellular signalling Vol. 11; no. 6; pp. 399 - 404
• Main Authors: Martı́n de la Vega, C, Garcı́a, A, Martı́n, M.E, Alcázar, A, Marin, O, Quevedo, C, Salinas, M
• Format: Journal Article
• Language: English
• Published: England Elsevier Inc 01-06-1999
• Subjects: Animals; eIF-2 Kinase - metabolism; eIF-2α Kinase; Eukaryotic initiation factor eIF-2; Neocortex - enzymology; Peptide phosphorylation; Rats; Staurosporine; Tissue Extracts; Translational control; eIF-2α Kinase; Peptide phosphorylation; Eukaryotic initiation factor eIF-2; Staurosporine; IFN–interferon; PI–phosphatase inhibitors; Translational control; HRI–heme-regulated translational inhibitor, (alternatively referred to as HCR, heme-controlled repressor, or HCI, heme-controlled inhibitor); dsRNA–double-stranded RNA; poly(I·C) or pIC–poly(I):poly(C); eIF-2α– the α subunit of eIF-2; eIF-2–eukaryotic initiation factor 2; PKR–double-stranded RNA activated protein kinase (also called p68 or DAI); PMS–postmitochondrial supernatant; 2 mM β-glycerophosphate, 2 mM sodium molybdate and 10 mM NaF
• ISSN: 0898-6568; 1873-3913
• DOI: 10.1016/S0898-6568(99)00009-1

We studied the effect of staurosporine on two well characterised mammalian eIF-2α kinases, the heme-regulated translational inhibitor (HRI), and interferon-inducible double-stranded RNA-activated protein kinase (PKR). Both pure eIF-2 and a synthetic peptide used to measure the activity of purified or immunoprecipitated enzymes (sequence ILLSELSRRRIRAI) were phosphorylated with purified enzymes and crude preparations of tissues or cells in the presence of the inhibitor. In the presence of 0.25 μM staurosporine (a concentration which completely inhibits a wide range of Ser/Thr protein kinases), the phosphorylation of eIF-2α by HRI and PKR was not inhibited. The lack of response of eIF-2α kinases to staurosporine allowed us to measure PKR activity in salt washed postmicrosomal supernatants without previous purification of the enzyme. In the presence of poly(I):poly(C), the PKR activator, we detected both an increased phosphorylation of eIF-2α and an increment in the autophosphorylation of PKR. We also confirmed an induction of PKR in cultured neuronal cells after treatment with interferon. The results obtained following phosphorylation of the synthetic peptide with crude extracts are less conclusive. Although its phosphorylation is specific because it displaces eIF-2 phosphorylation, and the presence of staurosporine prevents its phosphorylation by other serine/threonine kinases, it is a rather poor substrate for PKR.