Smad6 is a Protein Kinase X Phosphorylation Substrate and is Required for HL-60 Cell Differentiation

To gain insight into the function of human protein kinase X (PrKX), a signal-transduction protein required for macrophage differentiation, we identified regulatory subunit I alpha of protein kinase A, T54 and Smad6 as partners for this protein using a yeast two-hybrid interaction screen. Interactions between PrKX and these proteins were substantiated by co-immunoprecipitation. Interaction between Smad6 and PrKX was also confirmed in human myeloid HL-60 cells following their phorbol 12-myristate 13-acetate (PMA)-induced differentiation into macrophages. In vitro phosphorylation assays demonstrated that PrKX phosphorylates Smad6 at a serine residue. Mutagenesis of this site resulted in abrogation of PrKX phosphorylation. Both PrKX and Smad6 were shown to be co-localized to the nuclear compartment of HL-60 cells during their macrophage differentiation where PrKX levels are induced and Smad6 protein levels remain relatively constant while levels of serine phosphorylation of Smad6 increase. By using in vitro electrophoretic mobility shift assays and in vivo chromatin immunoprecipitation, we also demonstrate that during macrophage differentiation Smad6 displays an increased binding to the human osteopontin, Id2, and Hex gene promoters, which correlates to an observed increased expression of these genes. Finally, vector-based RNA interference experiments established that both Smad6 and PrKX proteins are required for PMA-induced cell attachment and spreading.