Regulation of double-stranded RNA-activated eukaryotic initiation factor 2 alpha kinase by type 2 protein phosphatase...

Protein synthesis initiation in reticulocyte lysates is inhibited by low concentrations (1-20 ng/ml) of double-stranded RNA (ds RNA) due to the activation of a ds RNA-dependent cAMP-independent protein kinase (ds I) that phosphorylates the alpha subunit of the eukaryotic initiation factor eIF-2. In lysates, ds I is present in the latent inactive form and is associated with the ribosome complement. Latent ds I is solubilized by extraction with high-salt buffers and can be purified in its latent form. Activation of purified latent ds I requires ds RNA and ATP and is accompanied by the ds RNA-dependent autophosphorylation of a polypeptide doublet of 70,000 and 72,000 daltons ("70k/72k"), which represent different phosphorylated states of the same polypeptide. These are phosphorylated in the sequence 70k-->72k; increased phosphorylation of 72k is associated with increased ds I activation. Lysates (or Sepharose 6B ribosomes) treated with ds RNA display a similar ds I phosphoprotein profile, and this is accompanied by the phosphorylation of endogenous eIF-2alpha (38,000 daltons). Delayed (32)P pulses in ds RNA-inhibited lysates indicate that the phosphates on ds I and eIF-2alpha turn over. Under defined conditions, activated ds I in lysates is selectively dephosphorylated by endogenous protein phosphatase(s), and this is accompanied by the dephosphorylation of eIF-2alpha. Similarly, purified activated ds I is rapidly dephosphorylated by unfractionated lysate protein phosphatase(s) and by type 2 protein phosphatase but not by type 1 protein phosphatase. The dephosphorylation of ds I occurs in the sequence 72k-->70k and is correlated with ds I inactivation. The heat-stable protein phosphatase inhibitor-2, which selectively blocks type 1 protein phosphatase, does not significantly affect the dephosphorylation of ds I by type 2 protein phosphatase or by unfractionated lysate phosphatases. The data support the conclusion that a ds I phosphatase activity with type 2 characteristics is involved in the regulation of ds I activity.