The Fe65 adaptor protein interacts through its PID1 domain with the transcription factor CP2/LSF/LBP1.
The neural protein Fe65 possesses three putative protein-protein interaction domains: one WW domain and two phosphotyrosine interaction/phosphotyrosine binding domains (PID1 and PID2); the most C-terminal of these domains (PID2) interacts in vivo with the Alzheimer's beta-amyloid precursor protein, whereas the WW domain binds to Mena, the mammalian homolog of Drosophila-enabled protein. By the interaction trap procedure, we isolated a cDNA clone encoding a possible ligand of the N-terminal PID/PTB domain of Fe65 (PID1). Sequence analysis of this clone revealed that this ligand corresponded to the previously identified transcription factor CP2/LSF/LBP1. Co-immunoprecipitation experiments demonstrated that the interaction between Fe65 and CP2/LSF/LBP1 also takes place in vivo between the native molecules. The localization of both proteins was studied using fractionated cellular extracts. These experiments demonstrated that the various isoforms of CP2/LSF/LBP1 are differently distributed among subcellular fractions. At least one isoform, derived from alternative splicing (LSF-ID), is present outside the nucleus; Fe65 was found in both fractions. Furthermore, transfection experiments with an HA-tagged CP2/LSF/LBP1 cDNA demonstrated that Fe65 interacts also with the nuclear form of CP2/LSF/LBP1. Considering that the analysis of Fe65 distribution in fractionated cell extracts demonstrated that this protein is present both in nuclear and non-nuclear fractions, we examined the expression of Fe65 deletion mutants in the two fractions. This analysis allowed us to observe that a small region N-terminal to the WW domain is phosphorylated and is necessary for the presence of Fe65 in the nuclear fraction.
Dipartimento di Biochimica e Biotecnologie Mediche, Università degli Studi di Napoli Federico II, via S. Pansini 5, 80131 Napoli, Italy.
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Zambrano
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Minopoli
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de Candia
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Russo
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002352:Carrier Proteins / Q000378:metabolism; D002458:Cell Fractionation; D002460:Cell Line; D003001:Cloning, Molecular; D003598:Cytoskeletal Proteins; D004268:DNA-Binding Proteins / Q000378:metabolism*; D008840:Microfilament Proteins; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000737:chemistry*; D009687:Nuclear Proteins / Q000737:chemistry* / Q000378:metabolism; D010766:Phosphorylation; D016601:RNA-Binding Proteins; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017384:Sequence Deletion / Q000235:genetics; D014157:Transcription Factors / Q000378:metabolism*; D014162:Transfection / Q000235:genetics
C486243:Apbb1 protein, mouse; C486242:Apbb1 protein, rat; D002352:Carrier Proteins; D003598:Cytoskeletal Proteins; D004268:DNA-Binding Proteins; C102176:Enah protein, mouse; D008840:Microfilament Proteins; D009419:Nerve Tissue Proteins; D009687:Nuclear Proteins; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; C501905:Tfcp2 protein, mouse; D014157:Transcription Factors