The effects of activation of protein kinase C (PKC) on intermediate filaments (IFs) of different cell types were investigated. In primary cultures of murine or rabbit spinal cord, or human fibroblasts, brief exposure to activators of PKC led to the following changes in IFs: (1) Disassembly of GFAP-filaments in astrocytes and of vimentin in fibroblasts and fragmentation of the neurofilament (NF) network in neuronal cell bodies (occurred within 30 minutes). The distribution of PKC isoforms was investigated in these cell types and no IF-associated isoform was found. Disassembly of IFs may be explained by direct phosphorylation of N-terminal domains of IFs by PKC. (2) Activation of PKC led also to changes similar to those occurring in various motor neuron diseases: Focal swellings of proximal neurites of motor neurons, but not other types of neurons (within hours), and hyperphosphorylation of C-terminal domains of NF proteins (persisting over days). NF phosphorylation was determined by increased immunoreactivity with antibodies SMI31 and SMI34 recognizing NF proteins (NF-M & NF-H) when C-terminal KSP repeat domains are in intermediate and hyperphosphorylated states, respectively. Increased SMI34 labeling was prevented by pretreatment with the selective NMDA receptor antagonist, D-2-amino-5-phosphonovaleric acid (APV), but not by the AMPA/kainate antagonist, CNQX, or the metabotropic glutamate receptor antagonist, MCPG, indicating a cooperative effect of NMDA receptor activation in PKC-induced NF hyperphosphorylation. Since PKC does not phosphorylate C-terminal domains of NF proteins, involvement of other kinases was investigated. Hyperphosphorylation of NFs induced by PKC was prevented by the specific Ca$ sp{2+}$/calmodulin-dependent protein kinase II (CaMKII) inhibitor, KN-62, but not by its analogue, KN-04. The following mechanism is proposed for NF hyperphosphorylation in motor neuron diseases: Activation of PKC, as a nonspecific response to injury, acts cooperati / The results of the present thesis together demonstrate multiple effects of PKC activation on modulation of IFs that might contribute to dysfunction of cells in certain diseases.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:QMM.40341 |
| Date | January 1996 |
| Creators | Doroudchi, Mohammad Mehdi |
| Contributors | Durham, Heather D. (advisor) |
| Publisher | McGill University |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Format | application/pdf |
| Coverage | Doctor of Philosophy (Department of Physiology.) |
| Rights | All items in eScholarship@McGill are protected by copyright with all rights reserved unless otherwise indicated. |
| Relation | alephsysno: 001529664, proquestno: NN19720, Theses scanned by UMI/ProQuest. |
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