Purchase this article with an account.
C. Arruti, F.R. Zolessi, A. Toledo; Retinal Neuroblasts Have a Cdk–phosphorylated MARCKS Form Associated to Plasma Membrane Microdomains . Invest. Ophthalmol. Vis. Sci. 2005;46(13):5141.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Purpose: To identify the kinase responsible for the neuron specific MARCKS phosphorylation at Ser 25 as well as to determine the subcellular region where this phospho MARCKS variant is located. Methods: A monoclonal antibody that binds to an epitopic region containing Ser 25 (S25) was used for Western blot analysis and indirect immunoflorescence detection on cultured chick embryo retina cells, treated or not with protein kinase inhibitors. Fluorescent cholera toxin was used to localize GM1. Triton X100 resistant membrane domains were isolated by sucrose gradient centrifugation. Results: S25 is located at the consensus sequence S–P–X–K, which matches the phosphorylation motif for Cdk1, Cdk2 and Cdk5. Accordingly roscovitin treatments of differentiating retina neuroblasts provoked an important decrease in MARCKS phosphorylation whereas inhibitors of other proline–directed kinases had no effect. While MARCKS protein was always found at the neuroblast periphery and at the cell cytoplasm, S25p–MARCKS appeared as clusters confined to the cell periphery. These clusters were positive with cholera toxin labelling, suggesting they are GM1 rich regions. In addition, the acutely extracted cholesterol by methyl–ß–cyclodextrin (MCD) provoked an enlargement of cluster sizes in cultured retina neuroblasts. S25p–MARCKS was found in detergent resistant membrane fractions. Conclusions: A fraction of total MARCKS present in neuroblasts would be phosporylated in vivo by Cdk5 and this phosphorylated form, S25p–MARCKS, would be associated to plasma membrane in microdomain regions.
This PDF is available to Subscribers Only