FIAT represses bone matrix mineralization by interacting with ATF4 through its second leucine zipper.
We have characterized FIAT, a 66 kDa leucine zipper (LZ) protein that dimerizes with activating transcription factor 4 (ATF4) to form inactive dimers that cannot bind DNA. Computer analysis identifies three putative LZ motifs within the FIAT amino acid sequence. We have used deletion- and/or site-specific mutagenesis to individually inactivate these motifs in order to identify the functional LZ that mediates the FIAT-ATF4 interaction. Amino acids 194-222 that encode the FIAT LZ2 were deleted (mutant FIAT ZIP2 DEL). We inactivated each zipper individually by replacing two or three leucine residues within each zipper by alanine residues. The engineered mutations were L142A/L149A (mutant M1, first zipper), L208A/L215A/L222A (mutant M2, second zipper), and L441A/L448A (mutant M3, third zipper). MC3T3-E1 osteoblastic cells with an integrated 1.3 kb mouse osteocalcin gene promoter fragment driving expression of luciferase were transfected with expression vectors for ATF4 and the various FIAT deletion- or site-specific mutants. Inhibition of ATF4-mediated transcription was compared between wild-type (WT) and LZ FIAT mutants. The deletion mutant FIAT ZIP2 DEL and the sequence-specific M2 mutant did not interact with ATF4 and were unable to inhibit ATF4-mediated transcription. The M1 or M3 mutations did not affect the ability of FIAT to contact ATF4 or to inhibit its transcriptional activity. Stable expression of WT FIAT in osteoblastic cells inhibited mineralization, but not expression of the FIAT ZIP2 DEL and M2 mutants. This structure-function analysis reveals that FIAT interacts with ATF4 and modulates its activity through its second leucine zipper motif.
Yu
Vionnie W C
VW
Genetics Unit, Shriners Hospital for Children, Montreal, Quebec, Canada.
Gauthier
Claude
C
St-Arnaud
René
R
D051701:Activating Transcription Factor 4 / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001857:Bone Matrix / Q000378:metabolism*; D002113:Calcification, Physiologic; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D056970:Co-Repressor Proteins; D016350:Leucine Zippers; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D009687:Nuclear Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015675:Osteocalcin / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic; D014162:Transfection
D002352:Carrier Proteins; D056970:Co-Repressor Proteins; D009687:Nuclear Proteins; C502605:Txlng protein, mouse; D015675:Osteocalcin; D051701:Activating Transcription Factor 4