Oligonucleotide DNA probes were used to determine the distribution of mRNAs encoding the alpha- and beta-subunits of Ca2+/calmodulin-dependent protein kinase type II (CaM-KII) in developing rat brain. The regional and temporal distribution of these mRNAs closely paralleled the distribution and developmental appearance previously reported for their respective protein subunits. alpha-Subunit mRNA was barely detectable in sagittal sections at 4 d postnatal but increased as much as 10-fold in frontal cortex by day 16. beta-Subunit mRNA, on the other hand, was readily detected at 4 d postnatal and changed only slightly during development. Telencephalic structures exhibited the highest levels of CaM-KII mRNA and the brain stem displayed the least. alpha-Subunit mRNA was not observed in cerebellar granule cells and was barely detectable in Purkinje cells, while the beta-mRNA was easily detected in both neuronal types. mRNAs for both alpha- and beta-subunits were present in many neuronal cell bodies; however, only the alpha-subunit mRNA was localized to molecular layers of the hippocampus and lamina I of the frontal cortex. These layers of neuropil are relatively cell sparse and contain extensive dendritic arborizations and synaptic contacts. Since polyribosomes have been observed near hippocampal dendritic spines, the localization of alpha-subunit mRNA to dendrites of pyramidal and dentate granule cells suggests that this subunit is synthesized in situ at postsynaptic sites. The co-localization of translational machinery and high concentrations of CaM-KII in postsynaptic elements suggests an important relationship between alpha-subunit synthesis and the maintenance and plasticity of postsynaptic structures.

A calcium/calmodulin-dependent protein kinase termed CaM-kinase

We have analyzed Ca2+/calmodulin-dependent protein kinase II (CaM-kinase II) localization, activity, and endogenous protein substrates during differentiation and synaptogenesis in cultured hippocampal neurons. Primary cultures from hippocampi from 18 d embryonic rats are composed primarily of pyramidal neurons, with minimal contamination by nonneuronal cells. We have used monoclonal (Mab) and affinity-purified polyclonal antibodies that recognize either or both of the subunits of CaM-kinase II in order to localize the enzyme at progressive stages of neuronal differentiation. Diffuse but specific binding, determined by indirect immunofluorescence analyses, was first detected in cell bodies and growth cones of pyramidal neurons after 4 d in culture. Immunoreactivity increased during the next 3 d of culture, at which time fluorescent, labeling was patchy along neuritic processes. By 10 d, intensely fluorescent, discrete spots were observed along processes and on cell bodies. Astrocyte cultures prepared from newborn rat cortex showed no detectable immunofluorescence with anti-CaM-kinase II antibodies. Cytosolic and particulate fractions from cultured pyramidal neurons and astrocytes were analyzed using immunoblot, in vitro phosphorylation, 2-dimensional gel electrophoresis, and phosphopeptide mapping techniques. Although pure astrocyte cultures contained low levels of Ca2+/CaM-stimulated protein kinase activity, they did not display detectable levels of immunoreactive 50 kDa subunit nor 50 and 60 kDa phosphoproteins analogous to the autophosphorylated subunits of CaM-kinase II. Immunoblot analysis detected the 60 kDa kinase subunit in particulate and cytosolic fractions from 2 d neurons. By contrast, the 50 kDa subunit of CaM-kinase II was not detected in cytosolic or particulate fractions of pyramidal neurons before 4 d in culture. In 2 d pyramidal neuron cultures, only low levels of Ca2+/CaM-stimulated protein phosphorylation were observed. Ca2+/CaM-dependent phosphorylation of 10 d pyramidal cell proteins was 3-5-fold greater than that of 2 d cultures, and included major phosphoproteins of 48, 50, 56, 58/60, 80-86, 90, 120, 138, 175, and 190 kDa. Phosphopeptide maps of 58/60 and 50 kDa phosphoproteins gave patterns very similar to those of the autophosphorylated 60 and 50 kDa subunits, respectively, of purified CaM-kinase II. A phosphoprotein doublet of 83 kDa was identified as synapsin I. Developmental changes in Ca2+/CaM-dependent phosphorylation in pyramidal neuron cultures were very similar to those previously described in subcellular fractions from postnatal rat forebrain.

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