Src kinase phosphorylates Notch1 to inhibit MAML binding.

Notch signaling is a form of intercellular communication which plays pivotal roles at various stages in development and disease. Previous findings have hinted that integrins and extracellular matrix may regulate Notch signaling, although a mechanistic basis for this interaction had not been identified. Here, we reveal that the regulation of Notch by integrins and extracellular matrix is carried out by Src family kinases (SFKs) working downstream of integrins. We identify a physical interaction between the SFK member, c-Src, and the Notch intracellular domain (NICD) that is enhanced by β3 integrin and the integrin binding ECM protein, MAGP2. Our results demonstrate that c-Src directly phosphorylates the NICD at specific tyrosine residues and that mutation of these phosphorylation sites increases Notch responsive transcriptional activity. Furthermore, we also find that phosphorylation of the NICD by SFKs attenuates Notch mediated transcription by decreasing recruitment of MAML to the Notch co-transcriptional complex. Finally, we also find that SFK activity decreases NICD half-life. Collectively, our results provide important mechanistic data that underlie the emerging role of Notch as a general sensor and responder to extracellular signals.