Regulation of TORC2 complex in Dictyostelium discoideum

Dictyostelium is an amoeba that lives in the soil where it feeds on bacteria. During scarcity of food, Dictyostelium cells undergo a highly regulated developmental process in which the cells aggregate by chemotaxing towards pulsatile emission of extracellular cAMP from a signaling center; the cells move up the cAMP gradient and also relay the cAMP signal. In our laboratory we use Dictyostelium to unravel the molecular mechanisms of chemotaxis. Binding of the chemoattractant cAMP to receptor proteins on the surface of Dictyostelium cells lead to the intracellular activation of heterotrimeric G proteins (Gα’s and Gβγ), which in turn activate monomeric G proteins (Ras) by exchange of GDP for GTP. The activation of Ras leads to F-actin formation at the front of the cell, thus, enabling a cell to extend a protrusion in the direction of the highest cAMP concentration. Four Ras-mediated chemotaxis pathways have been described namely: sGC (soluble Guanylyl Cyclase), PLA2 (Phospholipase A2), PI3K (Phosphoinositide 3-kinase) and TORC2 (Target Of Rapamycin Complex 2). We have shown that none of these four pathways is essential for Ras activation or chemotaxis in steep gradients of cAMP in the lab; however they play a very important role in amplification of the weak chemoattractant gradients as it occurs in nature. This study concentrates on the regulation and function of TORC2 in Dictyostelium chemotaxis and development. TORC2 is a multiprotein complex consisting of four proteins: Lst8, Rip3, Pia and Tor. Previous genetic studies have implicated TORC2 in regulating chemotaxis, activation of adenylyl cyclase and development in Dictyostelium. All the four protein components of TORC2 are required to mediate activation of adenylyl cyclase, but the exact mechanism by which these protein components regulate the activity of TORC2 remains largely unknown. The Tor gene encodes a kinase that phosphorylates down-stream proteins. The Pia gene product is a cytosolic regulator of Adenylyl Cyclase (ACA) which in turn plays a role in signal relay by producing cAMP. This suggests that the TORC2 complex may have two outputs, Tor and Pia. It has been demonstrated that the TORC2 complex can be activated by multiple signals, including the heterotrimeric G-protein G2, and the monomeric RasC and Rap1. RasC is a major regulator of TORC2 signaling. In this thesis, we have shown that RasC activates the TORC2 complex via direct binding to the TOR component of the complex. Additionally, we have also confirmed that RIP3 is not binding RasC, but the small G protein Rap1. Since G2 activates RasC and Rap1, it has always been proposed that activation of TORC2 by G2 is mediated through RasC and Rap1. However, our study provides strong evidence that G2 can also regulate TORC2 activity directly, because G2 strongly binds to Lst8 and Gbinds to Pia. Thus, each protein of the TORC2 complex interacts with a different upstream regulator. While some individual TORC2 regulators (Gα2, RasC and Rap1) were shown to activate the TORC2 complex others (Gβγ) does not activate TORC2. We have not yet investigated the activity of different combinations of these four regulators on TORC2 activity. Do they form AND gates (both must be present) or OR gates (one of two is sufficient)? Is one regulator recruiting TORC2 while another regulator changes the local activity? Based on our studies we have postulated a new model of regulation of Dictyostelium chemotaxis pathway. According to this model, TORC2 is regulated by both monomeric & heterotrimeric G-proteins which bind to distinct TORC2 components. These regulators of TORC2 are itself involved in several interconnected pathways and feedback loops which regulate distinct chemotactic processes. This could explains a role of the TORC2 complex as an integrator and processor of chemotaxis signaling pathways. Owing to its homology to mammalian counterparts, research about TORC2 regulation is of prime importance as it may open new portals to understand and design drugs to treat several human diseases and disorders. mTOR pathway has been found to be dysregulated in many diseases such as diabetes, obesity, depression and certain cancers. Recently, mTOR signaling has also been linked to Alzheimer’s disease pathology. Hence, our study about the regulation of TORC2 complex throws light in this area since we have shown that some regulators of TORC2 activate its kinase activity whereas others inhibit it. To better understand the role(s) played by heterotrimeric G proteins in TORC2 regulation, future research should focus on studying TORC2 activation in response to all permutations and combinations possible among heterotrimeric G proteins