Phosphoproteins of cultured cerebellar granule cells and response to the differentiation-promoting stimuli NMDA, high K+ and ionomycin.

In order to investigate signalling pathways involved in the control of granule cell differentiation, survival and other functions by depolarization or activation of NMDA receptors we have characterized protein phosphorylation in cerebellar granule cells. Cultures of cerebellar granule cells were incubated with 32P orthophosphate and then challenged with NMDA, K+ or the Ca2+ ionophore ionomycin, agents which raise [Ca2+]i and stimulate differentiation and survival. Upon separation of labelled phosphoproteins by two-dimensional gel electrophoresis three differences were found in response to all of these agents. These were an increase in acidity of two phosphoproteins of 87 and 48 kDa (p87 and p48) and increased 32P-incorporation into a phosphoprotein of 120 kDa (p120). Treatment with PMA which stimulates neurite outgrowth but not survival affected p87 (increased its acidity) but not p48. The acidic shift of p87, therefore, is not sufficient to stimulate granule cell survival. The identification of p87 as the actin-binding MARCKS protein and the demonstration of its presence in neurites and growth cones of granule cells suggests that it may be involved in NMDA-stimulated neurite outgrowth. The phosphoproteins p120 and p48 may potentially be involved in events linking the rise in [Ca2+]i to increased granule cell survival or other aspects of granule cell differentiation.