Phosphorylation of nerve growth factor receptor proteins in sympathetic neurons and PC12 cells. In vitro phosphorylation by the XXXX -independent protein kinase FA/GSK-3.

@entity8283 @entity268 @entity3208 @entity141 @entity10393 @entity1359 @entity11451 @entity2975 @entity563 @entity35 @entity1889 @entity8284 @entity855
We have examined phosphorylation of @entity11451 in cultured sympathetic neurons and PC12 cells. Dissociated @entity35 superior cervical ganglion neurons or PC12 cells were incubated with 32Pi to label cellular phosphoproteins. Membrane proteins were solubilized, and @entity11451 proteins were immunoprecipitated with the monoclonal antibody 192-IgG. Analysis by @entity3208 - @entity2975 gel electrophoresis and autoradiography showed that @entity11451 components of Mr = 80,000 and Mr = 210,000 were phosphorylated. Phosphorylation of neither species was affected by treating the cells with NGF or @entity1359 . When the 80,000-Da protein was subjected to complete trypsin proteolysis and then analyzed by reverse phase liquid chromatography, two 32P-labeled peptides were resolved. The more hydrophobic peptide accounted for most of the 32P and contained only @entity8283 ; the other peptide contained @entity8283 and @entity8284 . No @entity10393 was detected in the receptor proteins. When receptor molecules from nonlabeled PC12 cells were immunoprecipitated and then incubated in vitro with [gamma-32P] @entity855 and the @entity268 -independent protein kinase FA/GSK-3, phosphorylation occurred predominantly on @entity563 and to a lesser extent on @entity1889 . However, the immunoprecipitated receptor proteins neither autophosphorylated nor were they detectably phosphorylated by @entity268 -dependent protein kinase, casein kinase II, or protein kinase C (the @entity141 /phospholipid-dependent enzyme). We conclude that binding units of the @entity11451 are phosphorylated constitutively in at least two sites in intact cells and that they can be phosphorylated by FA/GSK-3 in vitro.
