PCTAIRE-1 (also known as cyclin-dependent protein kinase (CDK) 16), is a Ser/Thr kinase that has been implicated in many cellular processes, including cell cycle, spermatogenesis, neurite outgrowth, and vesicle trafficking. Most recently, it has been proposed as a novel X-linked intellectual disability (XLID) gene, where loss-of-function mutations have been identified in human patients. The precise molecular mechanisms that regulate PCTAIRE-1 remained largely obscure, and only a few cellular targets/substrates have been proposed with no clear functional significance. We and others recently showed that cyclin Y binds and activates PCTAIRE-1 via phosphorylation and 14-3-3 binding. In order to understand the physiological role that PCTAIRE-1 plays in brain, we have performed a chemical genetic screen in vitro using an engineered PCTAIRE-1/cyclin Y complex and mouse brain extracts. Our screen has identified potential PCTAIRE-1 substrates (AP2-Associated Kinase 1 (AAK1), dynamin 1, and synaptojanin 1) in brain that have been shown to regulate crucial steps of receptor endocytosis, and are involved in control of neuronal synaptic transmission. Furthermore, mass spectrometry and protein sequence analyses have identified potential PCTAIRE-1 regulated phosphorylation sites on AAK1 and we validated their PCTAIRE-1 dependence in a cellular study and/or brain tissue lysates. Our results shed light onto the missing link between PCTAIRE-1 regulation and proposed physiological functions, and provide a basis upon which to further study PCTAIRE-1 function in vivo and its potential role in neuronal/brain disorders.