Protein Kinase C-Mediated Phosphorylation of the Leukemia-Associated HOXA9 Protein Impairs Its DNA Binding Ability and Induces Myeloid Differentiation

Abstract

HOXA9 expression is a common feature of acute myeloid leukemia, and high-level expression is correlated with poor prognosis. Moreover, HOXA9 overexpression immortalizes murine marrow progenitors that are arrested at a promyelocytic stage of differentiation when cultured and causes leukemia in recipient mice following transplantation of HOXA9 expressing bone marrow. The molecular mechanisms underlying the physiologic functions and transforming properties of HOXA9 are poorly understood. This study demonstrates that HOXA9 is phosphorylated by protein kinase C (PKC) and casein kinase II and that PKC mediates phosphorylation of purified HOXA9 on S204 as well as on T205, within a highly conserved consensus sequence, in the N-terminal region of the homeodomain. S204 in the endogenous HOXA9 protein was phosphorylated in PLB985 myeloid cells, as well as in HOXA9-immortalized murine marrow cells. This phosphorylation was enhanced by phorbol ester, a known inducer of PKC, and was inhibited by a specific PKC inhibitor. PKC-mediated phosphorylation of S204 decreased HOXA9 DNA binding affinity in vitro and the ability of the endogenous HOXA9 to form cooperative DNA binding complexes with PBX. PKC inhibition significantly reduced the phorbol-ester induced differentiation of the PLB985 hematopoietic cell line as well as HOXA9-immortalized murine bone marrow cells. These data suggest that phorbol ester-induced myeloid differentiation is in part due to PKC-mediated phosphorylation of HOXA9, which decreases the DNA binding of the homeoprotein.

This study was supported by the Department of Veterans Affairs (C.L. and H.J.L.), NIH grant RO1CA80029 (C.L.), and NIH grant RO1 GM55814001A2 (C.L.).

We thank Cynthia Wolberger for helpful conversations, Alan Pollock for advice on use of the retroviral packaging line, Steve Fong and Sophia Rosenfeld for technical assistance, and the UCSF Cancer Center cell sorting facility for performing FACS.