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)-activated inhibitor of type 1 protein serine/threonine phosphatase (PP1), in a yeast two-hybrid screen of a human brain cDNA library. Recombinant GADD34 (amino acids 233 to 674) bound both PKA-phosphorylated and unphosphorylated 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 phosphorylation was required for PP1 binding and inhibition by the N-terminal I-1(1-80) fragment. Pulldowns of GADD34 proteins expressed in HEK293T cells showed that I-1 bound the central domain of GADD34 (amino acids 180 to 483). By comparison, affinity isolation of cellular GADD34/PP1 complexes showed that PP1 bound near the C terminus of GADD34 (amino acids 483 to 619), a region that shows sequence homology with the virulence factors ICP34.5 of herpes simplex virus and NL-S of avian sarcoma virus. While GADD34 inhibited PP1-catalyzed dephosphorylation of phosphorylase a, the GADD34-bound PP1 was an active eIF-2alpha phosphatase. In brain extracts from active ground squirrels, GADD34 bound both I-1 and PP1 and eIF-2alpha was largely dephosphorylated. In contrast, the I-1/GADD34 and PP1/GADD34 interactions were disrupted in brain from hibernating animals, in which eIF-2alpha was highly phosphorylated at serine-51 and protein synthesis was inhibited. These studies suggested that modification of the I-1/GADD34/PP1 signaling complex regulates the initiation of protein translation in mammalian tissues.
Connor
J H
JH
Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710, USA.
Weiser
D C
DC
Li
S
S
Hallenbeck
J M
JM
Shenolikar
S
S
D000818:Animals; D000943:Antigens, Differentiation; D017209:Apoptosis; D001921:Brain / Q000378:metabolism; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D002460:Cell Line; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004249:DNA Damage; D018076:DNA, Complementary / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004722:Endoribonucleases; D015723:Gene Library; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D007700:Kinetics; D008957:Models, Genetic; D010749:Phosphoprotein Phosphatases; D010766:Phosphorylation; D010940:Plant Proteins / Q000378:metabolism*; D011233:Precipitin Tests; D011485:Protein Binding; D054645:Protein Phosphatase 1; D011506:Proteins / Q000737:chemistry* / Q000502:physiology*; D016601:RNA-Binding Proteins / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012589:Sciuridae; D015398:Signal Transduction; D020798:Two-Hybrid System Techniques
D000943:Antigens, Differentiation; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D047908:Intracellular Signaling Peptides and Proteins; C083693:PP1 protein, Physcomitrella patens; D010940:Plant Proteins; D011506:Proteins; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; C021042:protein phosphatase inhibitor-1; D017868:Cyclic AMP-Dependent Protein Kinases; D004722:Endoribonucleases; C087519:PPP1R15A protein, human; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1; C097081:PPP1R8 protein, human