Phosphorylation of murine homeodomain protein Dlx3 by protein kinase C

The D1x3 homeodomain gene is expressed in terminally differentiated murine epidermal cells. As demonstrated for differentiation-specific granular mark ers, D1x3 is activated in primary mouse keratinocytes cultured in vitro by increasing the level of the extracellular Ca2+ This activation is mediated through a protein kinase C-dependent (PKC) pathway. In this study we invest igated whether PKC can modulate the activity of murine D1x3 protein. Using in vitro kinase assays, we show that PKC enzymes phosphorylate the D1x3 pro tein. Using keratinocyte nuclear extracts for the kinase reaction, we deter mined that D1x3 protein is phosphorylated, and the phosphorylation is inhib ited by the PKC-specific inhibitor GF109203X, suggesting that D1x3 is phosp horylated by PKC in vivo. Of the PKC isoforms present in the epidermis, we tested alpha, delta, epsilon and zeta, D1x3 is primarily phosphorylated by PKC alpha, By deletion and mutational analysis, we show that the serine res idue S-138, located in the homeodomain of D1x3 protein, was specifically ph osphorylated by PKC, The phosphorylation of purified D1x3 proteins by PKC p artially inhibited formation of complexes between D1x3 protein and DNA, The se results suggest that D1x3 protein can be directly phosphorylated by PKC and this affects the DNA binding activity of D1x3. (C) 2001 Published by El sevier Science B,V, on behalf of the Federation of European Biochemical Soc ieties.