Phosphorylation of murine homeodomain protein Dlx3 by protein kinase C.
The Dlx3 homeodomain gene is expressed in terminally differentiated murine epidermal cells. As demonstrated for differentiation-specific granular markers, Dlx3 is activated in primary mouse keratinocytes cultured in vitro by increasing the level of the extracellular Ca(2+). This activation is mediated through a protein kinase C-dependent (PKC) pathway. In this study, we investigated whether PKC can modulate the activity of murine Dlx3 protein. Using in vitro kinase assays, we show that PKC enzymes phosphorylate the Dlx3 protein. Using keratinocyte nuclear extracts for the kinase reaction, we determined that Dlx3 protein is phosphorylated, and the phosphorylation is inhibited by the PKC-specific inhibitor GF109203X, suggesting that Dlx3 is phosphorylated by PKC in vivo. Of the PKC isoforms present in the epidermis, we tested alpha, delta, epsilon and zeta. Dlx3 is primarily phosphorylated by PKC alpha. By deletion and mutational analysis, we show that the serine residue S(138), located in the homeodomain of Dlx3 protein, was specifically phosphorylated by PKC. The phosphorylation of purified Dlx3 proteins by PKC partially inhibited formation of complexes between Dlx3 protein and DNA. These results suggest that Dlx3 protein can be directly phosphorylated by PKC and this affects the DNA binding activity of Dlx3.
Park
G T
GT
Laboratory of Skin Biology, NIAMS, National Institutes of Health, Bethesda, MD 20892, USA.
Denning
M F
MF
Morasso
M I
MI
D000595:Amino Acid Sequence; D000818:Animals; D000831:Animals, Newborn; D001665:Binding Sites / Q000502:physiology; D002454:Cell Differentiation; D002467:Cell Nucleus / Q000737:chemistry; D002478:Cells, Cultured; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors; D004247:DNA / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D018398:Homeodomain Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007211:Indoles / Q000494:pharmacology; D007527:Isoenzymes / Q000378:metabolism; D015603:Keratinocytes / Q000166:cytology / Q000378:metabolism; D008301:Maleimides / Q000494:pharmacology; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000032:analysis; D010766:Phosphorylation; D011233:Precipitin Tests; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D014157:Transcription Factors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
C088334:Distal-less homeobox proteins; D004791:Enzyme Inhibitors; D018398:Homeodomain Proteins; D007211:Indoles; D007527:Isoenzymes; D008301:Maleimides; D010446:Peptide Fragments; D014157:Transcription Factors; D004247:DNA; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; C070515:bisindolylmaleimide I