The inhibitor-1 C terminus facilitates hormonal regulation of cellular protein phosphatase-1: functional implications for inhibitor-1 isoforms.
Inhibitor-1 (I-1) is a selective inhibitor of protein phosphatase-1 (PP1) and regulates several PP1-dependent signaling pathways, including cardiac contractility and regulation of learning and memory. The human I-1 gene has been spliced to generate two alternative mRNAs, termed I-1alpha and I-1beta, encoding polypeptides that differ from I-1 in their C-terminal sequences. Reverse transcription-PCR established that I-1alpha and I-1beta mRNAs are expressed in a developmental and tissue-specific manner. Functional analysis of I-1 in a Saccharomyces cerevisiae strain dependent on human I-1 for viability established that a novel domain encompassing amino acids 77-110 is necessary for PP1 inhibition in yeast. Expression of human I-1 in S. cerevisiae with a partial loss-of-function eukaryotic initiation factor-2alpha (eIF2alpha) kinase (Gcn2p) mutation permitted growth during amino acid starvation, consistent with the inhibition of Glc7p/PP1, the yeast eIF2alpha phosphatase. In contrast, human I-1alpha, which lacks amino acids 83-134, and I-1 with C-terminal deletions were significantly less effective in promoting yeast growth under starvation conditions. These data suggest that C-terminal sequences of I-1 enhance regulation of the eukaryotic eIF2alpha phosphatase. In vitro studies established that C-terminal sequences, deleted in both I-1alpha and I-1beta, enhance PP1 binding and inhibition. Expression of full-length and C-terminally truncated I-1 in HEK293T cells established the importance of the I-1 C terminus in transducing cAMP signals that promote eIF2alpha phosphorylation. This study demonstrates that multiple domains in I-1 target cellular PP1 complexes and establishes I-1 as a cellular regulator of eIF2alpha phosphorylation.
Weiser
Douglas C
DC
Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710, USA.
Sikes
Suzanne
S
Li
Shi
S
Shenolikar
Shirish
S
D017398:Alternative Splicing; D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D002460:Cell Line; D049109:Cell Proliferation; D002470:Cell Survival; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D015852:Eukaryotic Initiation Factor-2 / Q000378:metabolism*; D005091:Exons; D017353:Gene Deletion; D005982:Glutathione Transferase / Q000378:metabolism; D006728:Hormones / Q000378:metabolism*; D006801:Humans; D015151:Immunoblotting; D047908:Intracellular Signaling Peptides and Proteins; D051379:Mice; D008810:Mice, Inbred C57BL; D009154:Mutation; D009687:Nuclear Proteins; D010455:Peptides / Q000737:chemistry; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010765:Phosphorylase Phosphatase / Q000378:metabolism; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D020033:Protein Isoforms; D011494:Protein Kinases / Q000737:chemistry / Q000235:genetics; D054645:Protein Phosphatase 1; D017346:Protein Serine-Threonine Kinases; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry* / Q000502:physiology; D012333:RNA, Messenger / Q000378:metabolism; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins / Q000737:chemistry; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D015398:Signal Transduction; D013329:Structure-Activity Relationship; D013997:Time Factors; D014018:Tissue Distribution; D019892:eIF-2 Kinase / Q000378:metabolism
C087425:ANP32A protein, human; D000596:Amino Acids; D018076:DNA, Complementary; D015852:Eukaryotic Initiation Factor-2; D006728:Hormones; D047908:Intracellular Signaling Peptides and Proteins; D009687:Nuclear Proteins; D010455:Peptides; D020033:Protein Isoforms; D011506:Proteins; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D000242:Cyclic AMP; D005982:Glutathione Transferase; D011494:Protein Kinases; C070634:Eif2ak4 protein, mouse; C512912:GCN2 protein, S cerevisiae; D017346:Protein Serine-Threonine Kinases; D019892:eIF-2 Kinase; D017868:Cyclic AMP-Dependent Protein Kinases; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1; D010765:Phosphorylase Phosphatase