Growth arrest and DNA damage-inducible protein GADD34 assembles a novel signaling complex containing protein phosphatase 1 and inhibitor 1

The growth arrest and DNA damage-inducible protein, GADD34, was identified by its interaction with human inhibitor 1 (I-1), a protein kinase A (PKA)-a ctivated inhibitor of type 1 protein serine/threonine phosphatase (PPI), in a yeast two-hybrid screen of a human brain cDNA library. Recombinant GADD3 4 (amino acids 233 to 674) bound both PKA-phosphorylated and unphosphorylat ed I-1(1-171). Serial truncations mapped the C terminus of I-1 (amino acids 142 to 171) as essential for GADD34 binding. In contrast, PKA phosphorylat ion was required for PPI binding and inhibition by the N-terminal I-1(1-80) fragment. Pulldowns of GADD34 proteins expressed in HEK293T cells showed t hat I-1 bound the central domain of GADD34 (amino acids 180 to 483). By com parison, affinity isolation of cellular GADD34/PP1 complexes showed that PP I bound near the C terminus of GADD34 (amino acids 483 to 619), a region th at shows sequence homology with the virulence factors ICP34.5 of herpes sim plex virus and NL-S of avian sarcoma virus. While GADD34 inhibited PP1-cata lyzed dephosphorylation of phosphorylase a, the GADD34-bound PP1 was an act ive eIF-2 alpha phosphatase. In brain extracts from active ground squirrels , GADD34 bound both I-1 and PP1 and eIF-2 alpha was largely dephosphorylate d. In contrast, the I-1/GADD34 and PP1/GADD34 interactions were disrupted i n brain from hibernating animals, in which eIF-2 alpha was highly phosphory lated at serine-51 and protein synthesis was inhibited. These studies sugge sted that modification of the I-1/GADD34/PP1 signaling complex regulates th e initiation of protein translation in mammalian tissues.