PKCα is a key mediator of the neuronal differentiation controlled by NGF and ATP. However, its downstream signaling pathways remain to be elucidated. To identify the signaling partners of PKCα, we analyzed proteins coimmunoprecipitated with this enzyme in PC12 cells differentiated with NGF and ATP and compared them with those obtained with NGF alone or growing media. Mass spectrometry analysis (LC-MS/MS) identified plectin, peripherin, filamin A, fascin, and β-actin as potential interacting proteins. The colocalization of PKCα and its interacting proteins increased when PC12 cells were differentiated with NGF and ATP. Peripherin and plectin organization and the cortical remodeling of β-actin were dramatically affected when PKCα was down-regulated, suggesting that all three proteins might be functional targets of ATP-dependent PKCα signaling. Taken together, these data demonstrate that PKCα is essential for controlling the neuronal development induced by NGF and ATP and interacts with the cytoskeletal components at two levels: assembly of the intermediate filament peripherin and organization of cortical actin.