Expression of Calmodulin‐Dependent Phosphodiesterase, Calmodulin‐Dependent Protein Phosphatase, and Other Calmodulin‐Binding Proteins in Human SMS‐KCNR Neuroblastoma Cells

Abstract: Calmodulin (CaM)‐dependent enzymes, such as CaM‐dependent phosphodiesterase (CaM‐PDE), CaM‐dependent protein phosphatase (CN), and CaM‐dependent protein kinase II (CaM kinase II), are found in high concentrations in differentiated mammalian neurons. In order to determine whether neuroblastoma cells express these CaM‐dependent enzymes as a consequence of cellular differentiation, a series of experiments was performed on human SMS‐KCNR neuroblastoma cells; these cells morphologically differentiate in response to retinoic acid and phorbol esters [12‐O‐tetradecanoylphorbol 13‐acetate (TPA)]. Using biotinylated CaM overlay procedures, immunoblotting, and protein phosphorylation assays, we found that SMS‐KCNR cells expressed CN and CaM‐PDE, but did not appear to have other neuronal CaM‐binding proteins. Exposure to retinoic acid, TPA, or conditioned media from human HTB‐14 glioma cells did not markedly alter the expression of CaM‐binding proteins; 21‐day treatment with retinoic acid, however, did induce expression of novel CaM‐binding proteins of 74 and 76 kilodaltons. Using affinity‐purified polyclonal antibodies, CaM‐PDE immunoreactivity was detected as a 75‐kilodalton peptide in undifferentiated cells, but as a 61‐kilodalton peptide in differentiated cells. CaM kinase II activity and subunit autophosphorylation was not evident in either undifferentiated or neurite‐bearing cells; however. CaM‐dependent phosphatase activity was seen. Immunoblot analysis with affinity‐purified antibodies against CN indicated that this enzyme was present in SMS‐KCNR cells regardless of their state of differentiation. Although SMS‐KCNR cells did not show a complete pattern of neuronal CaM‐binding proteins, particularly because CaM kinase II activity was lacking, they may be useful models for examination of CaM‐PDE and CN expression. It is possible that CaM‐dependent enzymes can be used as sensitive markers for terminal neuronal differentiation.