Developmental changes in protein phosphorylation in chicken forebrain. II. Calmodulin stimulated phosphorylation.

The development of calmodulin stimulated protein phosphorylation, with particular reference to calmodulin-stimulated protein kinase II (CMK II), was investigated in 3 subcellular fractions of chicken forebrain: cytosol (S3), crude synaptic plasma membranes (P2-M) and occluded cytosol (P2-S). Changes in the level of calmodulin-stimulated phosphorylation of endogenous proteins occurred over a protracted time course and were not complete until after day 52 post-hatching. By day 15 post-hatching, calmodulin-stimulated phosphoproteins characteristic of embryonic fractions had all disappeared and those characteristic of adult tissue were present but not necessarily at their mature levels. The levels of CMK II were estimated from the autophosphorylation of the alpha-subunit which was the only phosphoprotein present at 53,000 Da in the 3 fractions. Overall, calmodulin-stimulated phosphorylation and CMK II levels were low in embryonic brain and high in adult brain but two specific changes in CMK II were observed during development: (1) although CMK II concentrations increased in both membrane and cytosolic fractions until day 23 the kinase was predominantly cytoplasmic (approximately 75%) until day 23, after which it became increasingly membrane bound so that by day 52 post-hatching the majority of CMK II was present in the synaptic membrane fraction, and (2) the relative concentrations of the alpha- and beta-subunits changed from an alpha:beta-value of approximately 1:1 in the 19 day embryo to approximately 1:2 by 15 days post-hatch after which no further change was seen. The occurrence of major changes in the calmodulin stimulated protein phosphorylation system for up to 6-8 weeks after synapse formation is completed in the forebrain, provides further support for the existence of a synapse maturation phase of neuronal differentiation which is distinct from synapse formation. This phase involves only a specific subset of the developmental changes occurring in the calmodulin-stimulated phosphorylation system.