(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.