Gα13 Stimulates the Tyrosine Phosphorylation of Ric-8A.
The G12 family of heterotrimeric G proteins is defined by their α-subunits, Gα12 and Gα13. 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 Gα13, we subjected Gα13 to tandem affinity purification-coupled mass spectrometric analysis. Our results from such analysis indicate that Gα13 potently interacts with mammalian Ric-8A. Our mass spectrometric analysis data also indicates that Ric-8A, which was tandem affinity purified along with Gα13, 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 Gα13 containing the guanine-ring interaction site is essential and sufficient for its interaction with Ric-8A. Evaluation of Gα13-specific signaling pathways in SKOV3 or HeyA8 ovarian cancer cell lines indicate that Ric-8A potentiates Gα13-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 Gα13. Our results also indicate that the stimulation of tyrosine-phosphorylation of Ric-8A by Gα13 is partially sensitive to inhibitors of Src-family of kinases, namely PP2 and SI. Furthermore, we demonstrate that Gα13 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 Gα13 stimulates the tyrosine phosphorylation of Ric-8A and Gα13-mediated tyrosine-phosphorylation plays a critical role in the translocation of Ric-8A to plasma membrane.
Stephenson Cancer Center and Department of Cell Biology, 975 NE 10th Street, The University of Oklahoma Health Sciences Center, OK 73104, United States.
Mingda
M
Yan
Stephenson Cancer Center and Department of Cell Biology, 975 NE 10th Street, The University of Oklahoma Health Sciences Center, OK 73104, United States.
Ji Hee
JH
Ha
Stephenson Cancer Center and Department of Cell Biology, 975 NE 10th Street, The University of Oklahoma Health Sciences Center, OK 73104, United States.
Danny N
DN
Dhanasekaran
Cdc42; Gα13; Ric-8A; Src; membrane-translocation; p38MAPK