Neurotrophic activity of the Antennapedia homeodomain depends on its specific DNA-binding properties.
In previous reports we have demonstrated that the 60-aa peptide corresponding to the homeodomain of the Drosophila protein Antennapedia (pAntp) translocates through the membrane of neurons in culture, accumulates in neuronal nuclei, and promotes neurite growth. To analyze the importance of specific pAntp DNA-binding properties in this phenomenon we have constructed three mutant versions of pAntp that differ in their ability to translocate through the membrane and to bind specifically the cognate sequence for homeodomains present in the promoter of HoxA5. We demonstrate that removing two hydrophobic residues of the third helix inhibits pAntp internalization and suppresses its neurotrophic activity. We also show that pAntp neurotrophic activity is lost when mutations are introduced in positions preserving its penetration and nuclear accumulation but abolishing its capacity to bind specifically the cognate DNA-binding motif for homeoproteins. Our results strongly suggest that pAntp neurotrophicity requires both its internalization and its specific binding to homeobox cognate sequences. We propose that homeoproteins might regulate important events in the morphological differentiation of the postmitotic neuron.
Le Roux
I
I
Centre National de la Recherche Scientifique Unité Recherche Associée 1414, Ecole Normale Supérieure, Paris, France.
Joliot
A H
AH
Bloch-Gallego
E
E
Prochiantz
A
A
Volovitch
M
M
D000818:Animals; D051943:Antennapedia Homeodomain Protein; D001483:Base Sequence; D002462:Cell Membrane / Q000378:metabolism; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D004330:Drosophila / Q000196:embryology / Q000235:genetics / Q000378:metabolism*; D029721:Drosophila Proteins; D004625:Embryo, Nonmammalian / Q000378:metabolism; D004926:Escherichia coli; D005801:Genes, Homeobox; D018398:Homeodomain Proteins; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D016501:Neurites / Q000737:chemistry / Q000378:metabolism; D009474:Neurons / Q000737:chemistry / Q000378:metabolism*; D009687:Nuclear Proteins / Q000378:metabolism; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D015183:Restriction Mapping; D013379:Substrate Specificity; D014157:Transcription Factors
D051943:Antennapedia Homeodomain Protein; C497974:Antp protein, Drosophila; D004268:DNA-Binding Proteins; D029721:Drosophila Proteins; D018398:Homeodomain Proteins; D009687:Nuclear Proteins; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D014157:Transcription Factors; D004247:DNA