Modification of the cytoskeleton by protein kinases may play an important role in neuronal regeneration and plasticity. A type II calcium/calmodulin-dependent kinase (CaM kinase II) is a proposed mediator of cytoskeletal function due to its ability to phosphorylate the microtubule-associated protein (MAP-2) which is co-localized with the kinase in dendrites. The predominant form of CaM kinase II in adult rat forebrain is a 500-730 kDa holoenzyme containing two subunits (50 kDa/60 kDa) in a 3/1 molar ratio. Suggestive evidence indicates that several isoforms of the kinase are present in brain and under developmental control. MAP-2 consists of three homologous proteins (a/b/c) that are also developmentally regulated. All three MAP-2 variants promote tubulin assembly into microtubules, whereas only the high-molecular weight MAP-2a/b additionally promote stabilization of the dendritic cytoskeleton by interacting with other cellular elements. The primary goal of this proposal is to examine the relationship between isoforms of CaM kinase II and MAP a/b/c substrates in order to gain insights into the mechanism in which CaM kinase II regulates cytoskeletal dynamics. Two model systems will be sued to compare the expression of kinase isozymes and MAP-2 variants in neurons and neuron-like cells in culture. (a) several unique isoforms of CaM kinase II present in rat brain will be separated using cation-exchange chromatography. Developmental differences in these isoforms and MAP-2 variants will be examined. (b) The neuroblastoma/ glioma cell line (NG108-15) contains a unique isoform of CaM kinase II and kinase is induced during cellular differentiation. Differentiation of cells is characterized by development of electrical excitability, ability to form functional synapses, and elaboration of cytoskeleton-filled neurites. Therefore, this cell line is a promising model system to study kinase regulation of cytoskeleton. The following questions will be addressed: (1) What are the isoforms of CaM kinase II responsible for the phosphorylation of immature and mature variants of the microtubule-associated proteins MAP-2a, MAP-2b, MAP-2c? (2) Are these isozymes and their respective cytoskeletal protein substrates developed concurrently in intact rat brains? (3)Is the co-development of these isozymes and respective cytoskeletal substrates observed in cultured NG108 cells?
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