Phosphorylation of the Drosophila engrailed protein at a site outside its homeodomain enhances DNA binding.
The engrailed gene encodes a homeodomain-containing phosphoprotein that binds DNA. Here, we show that engrailed protein is posttranslationally modified in embryos and in embryo-derived cultured cells but is essentially unmodified when expressed in Escherichia coli. Engrailed protein produced by bacteria can be phosphorylated in nuclear extracts prepared from Drosophila embryos, and phosphotryptic peptides from this modified protein partly reproduce two-dimensional maps of phosphotryptic fragments obtained from metabolically labeled engrailed protein. The primary embryonic protein kinase modifying engrailed protein is casein kinase II (CK-II). Analysis of mutant proteins revealed that the in vitro phosphoacceptors are mainly clustered in a region outside the engrailed homeodomain and identified serines 394, 397, 401, and 402 as the targets for CK-II phosphorylation. CK-II-dependent phosphorylation of an N-truncated derivative of engrailed protein purified from bacteria increased its DNA binding 2-4-fold.
Bourbon
H M
HM
Department of Biochemistry and Biophysics, University of California, San Francisco 94143, USA.
Martin-Blanco
E
E
Rosen
D
D
Kornberg
T B
TB
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D004330:Drosophila / Q000235:genetics / Q000378:metabolism*; D029721:Drosophila Proteins; D015180:Electrophoresis, Gel, Two-Dimensional; D018398:Homeodomain Proteins / Q000096:biosynthesis / Q000378:metabolism*; D007301:Insect Hormones / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010748:Phosphopeptides / Q000737:chemistry / Q000302:isolation & purification; D010750:Phosphoproteins / Q000096:biosynthesis / Q000378:metabolism*; D010766:Phosphorylation; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D014157:Transcription Factors / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*
D017931:DNA Primers; D029721:Drosophila Proteins; C063459:En protein, Drosophila; D018398:Homeodomain Proteins; D007301:Insect Hormones; D010446:Peptide Fragments; D010748:Phosphopeptides; D010750:Phosphoproteins; D011994:Recombinant Proteins; D014157:Transcription Factors