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.