Neurobiological processes are modulated by linking neuronal activity to phosphorylation-based signalling to influence protein function. The identity, timing and interdependence of phosphoproteins and protein kinase activation, and mechanistic consequences was largely unresolved. We compared global activity-dependent phospho-signalling in synaptosomes with signalling in cultured hippocampal neurons, resulting in the identification of 1,917 and 7,070 unique phosphopeptides that contain activity-dependent phosphorylation sites, respectively. We monitored phospho-signalling in synaptosomes for 15 min after the stimulation, which had not been attempted at a large scale. We identified distinct patterns of presynaptic phospho-regulation across the time course that may constitute co-regulated protein networks. We also computationally predicted the protein kinases responsible. Proteins with strong signalling responses across multiple patterns were identified as potential signal integrators. The active zone scaffold protein, bassoon, was the major target of phospho-signalling, both in the number of targeted phosphorylation sites and the magnitude of change within the entire data set. These two properties of the phosphoproteomics data were combined with measures of neuronal importance from genetic data to highlight significant phospho-signalling substrates. This work constitutes a large data resource for neuronal phospho-signalling.