Cell cycle-regulated phosphorylation of the human SIX1 homeodomain protein

Human SIX1 (HSIX1) is a member of the Six class of homeodomain proteins imp licated in muscle, eye, head, and brain development. To further understand the role of HSIX1 in the cell cycle and cancer, we developed an HSIX1-speci fic antibody to study protein expression at various stages of the cell cycl e. Our previous work demonstrated that HSIX1 mRNA expression increases as c ells exit S phase and that overexpression of HSIX1 can attenuate a DNA dama ge-induced G(2) cell cycle checkpoint. Overexpression of HSIX1 mRNA was obs erved in 44% of primary breast cancers and 90% of metastatic lesions. Now w t? demonstrate that HSIX1 is a nuclear phosphoprotein that becomes hyperpho sphorylated at mitosis in both MCF7 cells and in Xenopus extracts. The patt ern of phosphorylation observed in mitosis is similar to that seen by treat ing recombinant HSIX1 with casein kinase II (CK2) in vitro, Apigenin, a sel ective CK2 inhibitor, diminishes interphase and mitotic phosphorylation of HSIX1. Treatment of MCF7 cells with apigenin leads to a dose-dependent arre st at the G(2)/M boundary, implicating CK2, like HSIX1, in the G(2)/M trans ition. HSIX1 hyperphosphorylated in vitro by CK2 loses its ability to bind the MEF3 sites of the aldolase A promoter (pM), and decreased binding to pM is observed during mitosis, Because CK2 and HSIX1 have both been implicate d in cancer and in cell cycle control, we propose that HSIX1, whose activit y is regulated by CK2, is a relevant target of CK2 in G(2)/M checkpoint con trol and that both molecules participate in the same pathway whose dysregul ation leads to cancer.