Fig. 5: MAST4 regulates the translocation of DLX3 by mediating its phosphorylation near the NLS region.

a–d DLX3 localization in incisors from 6-week-old WT and Mast4 KO mice. a, b In the WT incisor, DLX3 localized to the nucleus (arrowheads) and cytoplasm. c, d In the Mast4 KO incisor, the abundance of DLX3 in the nucleus was lower than that in the WT incisor (blank arrows). e–g MAST4 and DLX3 localization in empty LPCX vector-transfected control (e), MAST4 PDZ-overexpressing (f), and MAST4 KO (g) mHAT9d cells. Immunocytochemistry was performed, and confocal z-stack images were acquired in ultra-high-resolution mode (Lightning mode in Leica operating software). 3D reconstructions of the images were generated. The yellow arrows indicate direct binding of MAST4 and DLX3 in (e). h Immunoprecipitation was conducted after transient transfection of both HA-MAST4 PDZ and HA-MAST4-Full into HEK293T cells. i Analysis of DLX3 expression using subcellular fractionation following transient transfection of full-length MAST4 in the HEK293T cell line. The expression of α-tubulin in the cytoplasm and Lamin B in the nucleus served as controls for the efficiency of subcellular fractionation. j Flag-DLX3 was immunoprecipitated, and the complexes were analyzed by western blotting. Note that DLX3 phosphorylation was increased in the presence of HA-MAST4-Full in the HEK293T cell line. k HA-MAST4 PDZ and various Dlx3 deletion mutants were cotransfected into HEK293T cells, followed by immunoprecipitation. Note that MAST4 bound to the C-terminus of the ND. l Schematic diagram showing the deletion of the entire Dlx3 sequence. Note that the NLS is located at aa 124–150. m HEK293T cells were transiently cotransfected with Flag-DLX3^WT, the DLX3^AAA mutant and HA-MAST4-Full. Flag-DLX3 was immunoprecipitated, and the complexes were analyzed by western blotting. Note that DLX3 phosphorylation was increased in the presence of MAST4. n Flag-DLX3^WT, the DLX3^AAA mutant and HA-MAST4-Full were transiently cotransfected into HEK293T cells, and subcellular fractionation was performed. Notably, compared with that of DLX3^WT, the proportion of the DLX3^AAA mutant in the cytoplasm was higher and was not regulated by MAST4 (mean ± SD, n = 3). NLS nuclear localization site, ND N-terminal domain, HD homeodomain, CD C-terminal domain. Scale bars; a–d, 40 μm; e–g, 5 μm. The data were representative of three independent experiments.