Phosphorylation of murine homeodomain protein Dlx3 by protein kinase C

Abstract

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.

Fig. 1

Dlx3 is phosphorylated by PKC. A: Bacterially expressed Dlx was immunoprecipitated with anti-His antibody and incubated with 5 μg of nuclear extract of mouse keratinocytes. The PKC inhibitor GF was added in the reaction at 1 μM (lane 3) and 10 μM concentrations (lane 4). The PKA inhibitor H-89 was also added in the reaction (lane 5). Samples were subjected to SDS–PAGE, and transferred to a PVDF membrane. The amount of Dlx3 protein in each reaction was determined by Western blotting analysis (lower panel) using the T7 tag antibody (Novagen). The membrane was exposed to X-ray film for autoradiography. Arrows indicate the Dlx3 protein. B: Dlx3 protein (100 ng) was incubated for 30 min at 37°C in the presence or absence of PKC and γ-ATP. Dlx3 protein was also incubated with casein kinase II (CKII). Samples were then separated by electrophoresis on SDS–PAGE. The amount of Dlx3 protein used in each reaction was determined by Western blot analysis (lower panel). C: Dlx3 protein was incubated in the presence or absence of PKC isoforms α, δ, ɛ, ζ (Calbiochem) in the presence of γ-ATP. The amount of Dlx3 protein used in each reaction is shown in the Western blot in the lower panel using the T7 tag antibody (Novagen). The bottom panel shows the autophosphorylation of each PKC isoform, demonstrating the specific activity of each PKC isoform.

Fig. 2

Phosphorylation by PKC within the homeodomain of Dlx3. A: Schematic representation of Dlx3 deletions. B: In vitro PKC phosphorylation of Dlx3 proteins described in A. Each mutant protein was incubated with γ-ATP in the presence or absence of PKC. Each sample was subjected to SDS–PAGE and transferred to a PVDF membrane. The upper panel shows the results of autoradiography. The amount of each protein is shown in the bottom panel by Western blot.

Fig. 3

Determination of the PKC phosphorylation site in the homeodomain of Dlx3. A: Schematic representation of the amino acids in the homeodomain region (underlined). Three serine residues and three threonine residues are in this homeodomain region. B: Spectra from mass spectrometry analysis of non-phosphopeptide (left panel) and phosphopeptide (right panel) of the Dlx3 homeodomain region (phosphorylated by PKC in vitro). The numbers on each peak represent the molecular weight of each proteolytic peptide. The sequence of each peptide was identified based on the molecular weight. The arrow indicates the new peak that by MW corresponds to the 1513.80 peak plus the phosphate group. C: Each mutant Dlx3 protein was incubated with γ-ATP in the presence or absence of PKC. Each sample was subjected to SDS–PAGE and transferred to a PVDF membrane. The upper panel presents the results of autoradiography. The amount of protein in each reaction was analyzed by Western blotting (lower panel).

Fig. 4

PKC phosphorylation modulates Dlx3 function. A: Increasing amounts of GST-Dlx3 fusion protein from 50 ng to 200 ng and unlabeled ATP were incubated for 30 min at 37°C in the presence or absence of PKCα (Calbiochem). Samples were incubated with radiolabeled oligonucleotides encoding the consensus Dlx3 binding site for gel retardation assay. Arrowhead indicates the complex formed by GST-Dlx3 and probe. B: The shifted bands were scanned and plotted against protein concentration.