A constitutively active CKLiK mutant activates CREB and CREM independently of Ca⁺⁺ /calmodulin.

(A) Schematic representation of the predicted calmodulin-binding domain (CBD) and the autoinhibitory domain (AID) of CKLiK. Truncation mutants of these 2 domains are indicated. In CKLiK-309 the predicted CBD and in CKLiK-296 the predicted CBD and AID were deleted. (B) In vitro kinase assays. COS cells were transfected with 10 μg HA_CKLiK-WT (wt), HA_CKLiK-309 (309), or HA_CKLiK-296 (296) as indicated. Immunoprecipitations were performed and CREM phosphorylation was detected in the presence or absence of Ca⁺⁺/calmodulin as indicated. Expression of the different forms of CKLiK was detected by Western blotting of cell lysates with 12CA5 antibody. (C) Reporter assays. Cells were transfected with GAL4-CAT reporter construct, CREB-GAL4 (2 μg) and cotransfected with CKLiK-WT or mutated CKLiK-309 or CKLiK-296 (4 μg) as indicated. Eighteen hours before harvesting, cells were stimulated with (░) or without (■) 1 μmol/L ionomycin and reporter assays were performed. Data represent at least 3 independent experiments ± SEM and were corrected for transfection efficiency. (D) Active CKLiK is localized in the nucleus. Cells were grown on coverslips and transfected with CKLiK and active CKLiK-296 containing N-terminal enhanced green fluorescent protein (eGFP). Thirty-six hours after transfection cells were examined by fluorescence microscopy for CKLiK localization.