Molloy, Sean S. (1991) A study of the type II CA2+/calmodulin-dependent protein kinase on hippocampal neurons. Dissertation (Ph.D.), California Institute of Technology. http://resolver.caltech.edu/CaltechETD:etd-08232007-130039
Characterization of the type II Ca2+/calmodulin-dependent protein (CaM) kinase in vitro has revealed several intriguing physical and biochemical properties, including the induction of Ca2+-independent activity by autophosphorylation. This thesis describes our attempts to determine the importance of autophosphorylation to the regulation of the kinase in hippocampal neurons. In order to study the type II CaM kinase in these neurons, we established long-term cultures of rat hippocampal slices. We used these cultures to address several questions regarding the phosphorylation of the CaM kinase in the intact neurons, namely: 1) is the CaM kinase phosphorylated in the cultures under basal conditions, 2) if so, is phosphate incorporated into the sites previously characterized in vitro, 3) can phosphorylation of the CaM kinase be modulated in the neurons. Incubation of slice cultures with radiolabeled phosphate in situ showed that both the [alpha] and [beta] subunits of the kinase incorporate phosphate under basal conditions in intact neurons. Furthermore, HPLC analysis of tryptic fragments derived from [alpha] subunit radiolabeled in the cultures in situ revealed that the majority of phosphate was incorporated into Thr286 (the site which controls Ca2+-independent activity in vitro). Measurements of Ca2+-independent activity in homogenates showed that approximately one third of the kinase is autophosphorylated and constitutively active in the cultures. The proportion of Ca2+-independent enzyme in the cultures decreased by 80-90% following removal of external Ca2+. Application of the membrane permeant kinase inhibitors H7 and W7 also caused a substantial decrease in Ca2+-independent kinase activity, while the phosphatase inhibitor, okadaic acid, increased the proportion of Ca2+-independent kinase. Therefore, the resting level of Ca2+-independent CaM kinase apparently reflects a balance between continual Ca2+ dependent autophosphorylation and dephosphorylation by phosphatases. Homogenates of rat forebrains and hippocampi also had substantial levels of Ca2+-independent CaM kinase. These results suggest that the autophosphorylation mechanism acts to maintain a relatively high proportion of constitutively active kinase under conditions of low resting Ca2+ in neurons. This finding is in direct contrast to some models of kinase regulation in hippos pal neurons which predicted that the enzyme would only autophosphorylate following prolonged, synaptically driven increases in intracellular Ca2+. Furthermore, these studies indicate that pharmacological agents could either up or down regulate the level of constitutively active CaM kinase locally, at or near the synapse, by affecting the rate of autophosphorylation or dephosphorylation.
|Item Type:||Thesis (Dissertation (Ph.D.))|
|Degree Grantor:||California Institute of Technology|
|Thesis Availability:||Restricted to Caltech community only|
|Defense Date:||5 April 1991|
|Default Usage Policy:||No commercial reproduction, distribution, display or performance rights in this work are provided.|
|Deposited By:||Imported from ETD-db|
|Deposited On:||27 Aug 2007|
|Last Modified:||26 Dec 2012 02:58|
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