CALCIUM METABOLISM AND PROTEIN PHOSPHORYLATION IN NEURONAL SYSTEMS

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Analysis of phosphorylation of neurofilament proteins in mammalian nervous system has been examined using a neurofilament enriched preparation isolated from bovine spinal cord. The protein kinase activities in this preparation were studied using a variety of endogenous and exogenous substrates as well as specific activators and inhibitors. All three neurofilament subunits (NF-L-M, and H) and an 18KD protein were phosphorylated by endogenous protein kinases in this preparation. Neither activators or inhibitors of cAMP-dependent or cAMP-dependent or Ca ions/calmodulin dependent kinases altered this labeling pattern. However, staurosporine, a potent inhibitor of protein kinase C, selectively inhibited 32-P incorporation into the 18KD protein, but not the neurofilament proteins (NF). Using various exogenous substrates, it was possible to demonstrate that this preparation did contain significant Calcium/calmodulin protein kinase II and PKC-like protein kinase activities which could be inhibited by mastoparan and staurosporine, respectively. High salt extraction of the NF preparation was used to separate the kinase activities. The kinase activity in the pellet, which contained virtually all the NF proteins, phosphorylated the 18KD protein and H1-histone but not the NF proteins or casein. The soluble fraction could phosphorylate all the NF proteins and casein, but not H1-histone. These data indicate that the principal NF protein kinase activity in the bovine NF preparations appears to be due to a casein-like protein-kinase(s). Dephosphorylation of neuroflament proteins appeared to accelerate their degradation by Calpain. This suggests that the protein phosphorylation may affect the proteolytic degradation of the neurofilament proteins. The ionic mechanisms of IP-3-induced Calcium release was investigated in rat brain microsomes. IP-3-stimulated Calcium release was found to be insensitive to a variety of Calcium channel blockers; however, the K channel blockers blocked this release. Moreover, addition of IP- 3 increased 86-Rb influx into the microsomes and was sensitive to K channel blockers, suggesting that IP-3-induced Calcium release requires an opposite flow of K ions, and modulation of K channels by their channel blockers amy underlie the inhibition of IP-3- induced Ca ions release from brain microsomes.