CaMKII regulates retinoic acid receptor transcriptional activity and the differentiation of myeloid leukemia cells
Jutong Si, LeMoyne Mueller, Steven J. Collins
Jutong Si, LeMoyne Mueller, Steven J. Collins
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Research Article Oncology

CaMKII regulates retinoic acid receptor transcriptional activity and the differentiation of myeloid leukemia cells

Abstract

Retinoic acid receptors (RARs) are members of the nuclear hormone receptor family and regulate the proliferation and differentiation of multiple different cell types, including promyelocytic leukemia cells. Here we describe a biochemical/functional interaction between the Ca2+/calmodulin–dependent protein kinases (CaMKs) and RARs that modulates the differentiation of myeloid leukemia cells. We observe that CaMKIIγ is the CaMK that is predominantly expressed in myeloid cells. CaMKII inhibits RAR transcriptional activity, and this enzyme directly interacts with RAR through a CaMKII LxxLL binding motif. CaMKIIγ phosphorylates RARα both in vitro and in vivo, and this phosphorylation inhibits RARα activity by enhancing its interaction with transcriptional corepressors. In myeloid cell lines, CaMKIIγ localizes to RAR target sites within myeloid gene promoters but dissociates from the promoter upon retinoic acid–induced myeloid cell differentiation. KN62, a pharmacological inhibitor of the CaMKs, enhances the terminal differentiation of myeloid leukemia cell lines, and this is associated with a reduction in activated (autophosphorylated) CaMKII in the terminally differentiating cells. These observations reveal a significant cross-talk between Ca2+ and retinoic acid signaling pathways that regulates the differentiation of myeloid leukemia cells, and they suggest that CaMKIIγ may provide a new therapeutic target for the treatment of certain human myeloid leukemias.

Authors

Jutong Si, LeMoyne Mueller, Steven J. Collins

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Figure 4

Phosphorylation of RARα by CaMKIIγ.

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Phosphorylation of RARα by CaMKIIγ. (A) In vitro–translated RARα was sub...
(A) In vitro–translated RARα was subjected to an in vitro kinase reaction with in vitro–translated CaMKII. (B) The 4 consensus CaMK sites (R-x-x-S/T) within the ligand-binding domain of RARα are arbitrarily labeled 1–4. The (K-x-x-S/T) CaMK consensus site within the RARα hinge domain is labeled site 0. (C) A Gst-RARα fusion protein was constructed that harbors nonphosphorylatable alanine mutations at consensus CaMK sites 1–4 [Gst-RARm(1,2,3,4)]. This Gst-RARα fusion protein and the parental WT Gst-RARα were subjected to in vitro kinase reactions using CaMKIIγ immunoprecipitated from HL60 cells. The lower band corresponds to the immunoprecipitated CaMKIIγ that is autophosphorylated during this reaction. (D) A Gst-RARα fusion fragment harboring RARα amino acids 135–291 (RARα-WT) as well as the same fusion protein that is mutated (T→A) at RARα site 0 (RARα-T209A,T210A) was subjected to an in vitro kinase reaction using immunoprecipitated HL60 CaMKIIγ. The radiolabeled upper bands represent CaMKIIγ that is autophosphorylated during this reaction. (E) 293 cells were transfected with the indicated expression vectors and then metabolically labeled with [32P]orthophosphate. After 4 hours, anti-HA immunoprecipitates were electrophoresed on an agarose gel, and then radioautography was performed. CaMKIIca is the constitutively active CaMKIIα. Anti-HA Western blots (bottom row) served as a control for the amount of immunoprecipitated HA-RAR from each transfectant. (F) HL60R cells were electroporated with the βRARE-tk-LUC reporter (15 μg) together with expression vectors harboring the WT or the indicated CaMKII (T209,T210) site–mutated RARα. Six hours after RA addition (1 μM), relative luciferase activity was determined.