Differential and regulated binding of cAMP-dependent protein kinase and protein kinase C isoenzymes to gravin in human model neurons: Evidence that gravin provides a dynamic platform for the localization for kinases during neuronal development.
The membrane cortex has an important role in generating and maintaining spatially and functionally distinct domains in neurons. As a tool to functionally characterize molecules of the membrane cortex, we generated novel monoclonal antibodies against a fraction enriched for components of the neuronal membrane skeleton. We obtained two antibodies against the kinase-anchoring protein gravin. Gravin was strongly up-regulated during differentiation of human model neurons (NT2-N neurons) and was enriched at the inner peripheral cortex in close proximity to the plasma membrane where its localization primarily depended on association with membranes. In differentiated neurons, gravin colocalized in putative signaling complexes with protein kinase C (PKCbetaII) and partially with PKCalpha and cAMP-dependent protein kinase (PKA). Colocalization with PKCepsilon was not observed. PKCbetaII, PKCalpha, and PKA but not PKCepsilon coprecipitated with gravin indicating physical interaction. Binding of gravin to PKCalpha required the presence of Ca2+ and was increased after inhibition of PKC. In contrast, binding of PKCbetaII and PKA were independent of Ca2+ and PKC inhibition. Activation of PKC decreased binding of PKCalpha to gravin, decreased its association with the plasma membrane, and reduced the mean size of gravin particles. Taken together the data suggest that gravin provides a dynamic platform to localize kinases in an isoenzyme-specific and activation-dependent manner at specific sites in neurons.
Piontek
Jörg
J
Department of Neurobiology, IZN, University of Heidelberg, INF 345, 69120 Heidelberg, Germany.
Brandt
Roland
R
D054758:A Kinase Anchor Proteins; D000911:Antibodies, Monoclonal / Q000378:metabolism; D002118:Calcium / Q000737:chemistry / Q000378:metabolism; D018797:Cell Cycle Proteins; D002454:Cell Differentiation; D002462:Cell Membrane / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D003902:Detergents / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D005786:Gene Expression Regulation; D006801:Humans; D007150:Immunohistochemistry; D008856:Microscopy, Fluorescence; D016253:Microscopy, Immunoelectron; D008954:Models, Biological; D009474:Neurons / Q000201:enzymology* / Q000378:metabolism*; D011233:Precipitin Tests; D011485:Protein Binding; D020033:Protein Isoforms; D011493:Protein Kinase C / Q000737:chemistry* / Q000378:metabolism; D051744:Protein Kinase C-epsilon; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism; D015398:Signal Transduction; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013347:Subcellular Fractions / Q000378:metabolism; D014162:Transfection; D015854:Up-Regulation
D054758:A Kinase Anchor Proteins; C491162:AKAP12 protein, human; D000911:Antibodies, Monoclonal; D018797:Cell Cycle Proteins; D003902:Detergents; D020033:Protein Isoforms; D011506:Proteins; D017868:Cyclic AMP-Dependent Protein Kinases; C497255:PRKCE protein, human; D011493:Protein Kinase C; D051744:Protein Kinase C-epsilon; D002118:Calcium