Gα(13) Stimulates the Tyrosine Phosphorylation of Ric-8A

The G12 family of heterotrimeric G proteins is defined by their α-subunits, Gα(12) and Gα(13). These α-subunits regulate cellular homeostasis, cell migration, and oncogenesis in a context-specific manner primarily through their interactions with distinct proteins partners that include diverse effector molecules and scaffold proteins. With a focus on identifying any other novel regulatory protein(s) that can directly interact with Gα(13), we subjected Gα(13) to tandem affinity purification-coupled mass spectrometric analysis. Our results from such analysis indicate that Gα(13) potently interacts with mammalian Ric-8A. Our mass spectrometric analysis data also indicates that Ric-8A, which was tandem affinity purified along with Gα(13), is phosphorylated at Ser-436, Thr-441, Thr-443 and Tyr-435. Using a serial deletion approach, we have defined that the C-terminus of Gα(13) containing the guanine-ring interaction site is essential and sufficient for its interaction with Ric-8A. Evaluation of Gα(13)-specific signaling pathways in SKOV3 or HeyA8 ovarian cancer cell lines indicate that Ric-8A potentiates Gα(13)-mediated activation of RhoA, Cdc42, and the downstream p38MAPK. We also establish that the tyrosine phosphorylation of Ric-8A, thus far unidentified, is potently stimulated by Gα(13). Our results also indicate that the stimulation of tyrosine-phosphorylation of Ric-8A by Gα(13) is partially sensitive to inhibitors of Src-family of kinases, namely PP2 and SI. Furthermore, we demonstrate that Gα(13) promotes the translocation of Ric-8A to plasma membrane and this translocation is attenuated by the Src-inhibitors, SI1 and PP2. Thus, our results demonstrate for the first time that Gα(13) stimulates the tyrosine phosphorylation of Ric-8A and Gα(13)-mediated tyrosine-phosphorylation plays a critical role in the translocation of Ric-8A to plasma membrane.