Compared with their widely known function in cell growth, differentiation, and tumorigenesis, the role of Ras proteins in cell motility has been relatively unexplored. In the current work, we performed a series of experiments to investigate how RasC regulates chemotaxis via TORC2. Based on our results, we propose a network of signaling events centered around Ras-mediated activation of TORC2 that controls cytoskeletal activity and chemotaxis. As illustrated in , chemoattractants activate RasC through the G protein–coupled receptor cAR1, leading to TORC2-mediated activation of PKBR1 and PKBA, which phosphorylate PKB substrates and regulate chemotactic responses of the cell. This model is supported by several findings. First, we show that gain of function in RasC results in prolonged phosphorylation of PKBR1 and PKB substrates (), which in turn leads to extended ACA activation, elevated actin polymerization, and impaired chemotaxis (). Second, disruption of TORC2 activity specifically suppresses the aberrant chemotactic responses caused by persistently activated RasC ( and ). Third, RasC is required for activation of the PKBs in vivo () and stimulates TORC2-dependent phosphorylation of the PKBs in vitro ( and ). Fourth, we show that RasC does not activate PI3Ks and that the activation of the TORC2 pathway by RasC does not require PIP3 (). We conclude that there is a RasC–TORC2–PKB pathway in D. discoideum that plays a major role in temporal and spatial regulation of chemotactic responses. This finding has several implications in our understanding of chemotaxis and TORC2 signaling.
Figure 9. Schematic diagram of RasC-mediated signaling pathways that control chemotaxis. The chemoattractant cAMP signals through the G protein–coupled receptor cAR1 to RasC, leading to TORC2-mediated activation of PKBR1 and PKBA. The activation of PKBA (more ...)
Our results provide new insights about the negative regulators in the chemotactic signaling pathways. Many G protein–coupled signaling events rapidly subside when cells are exposed to a step increase in chemoattractant. The time courses of RasC activation, PKB phosphorylation, and downstream chemotactic responses all reflect this rapid shutoff ( and ). When the activation of RasC is prolonged, as in cells expressing the Q62L or G13V mutation, the kinetics of all of the downstream responses are similarly extended, suggesting that RasC inactivation is a rate-limiting step for the shutoff. Previous experiments of G protein signaling showed that α and βγ subunits of G protein remain dissociated (i.e., activated) as long as chemoattractant receptors are occupied (Janetopoulos et al., 2001
). Together, these results argue that the site of shutoff lies between G proteins and the Ras regulators ().
Why does prolonged activation of RasC impair chemotaxis? Chemotaxis involves the interaction between spontaneous motility and directional sensing. In the absence of a chemoattractant gradient, actin-based membrane protrusions are formed along the cell perimeter, propelling the cell in random directions. In a gradient, directional sensing generates intracellular asymmetries that bias motility. Activation of Ras proteins and TORC2-mediated PKB phosphorylation have been shown to occur selectively at the leading edge of chemotaxing cells as well as at the tips of pseudopodia and are thus in a position to bias motility (Sasaki et al., 2004
; Kamimura et al., 2008
). We speculate that extending RasC activation interferes with chemotaxis in multiple ways. First, pseudopodia extension and retraction that underlie random migration as well as chemotaxis likely depend on the timely activation and inactivation of Ras proteins. Indeed, we have observed that both basal motility and responses to chemotactic stimuli are altered in cells expressing RasCQ62L
. The character and distribution of pseudopods in cells expressing RasCQ62L
appear to be different from those in wild-type cells (). Second, because the dispersion length of a protein is proportional to its half-life, the constitutively activated form of RasCQ62L
is expected to have a broader spatial distribution than that of wild-type RasC. As RasCQ62L
diffuses further before being inactivated, the downstream responses it controls will be delocalized as well, impairing cells’ ability to interpret the directional signal. Therefore, the rapid inactivation of RasC is critical for localizing downstream responses and directional sensing.
Our results provide the first substantial evidence that TORC2 is under the control of a Ras protein and open the possibility for further study of the regulatory mechanism of TORC2. Loss of function in RasC greatly reduces PKB phosphorylation (), and the effects of RasCQ62L on PKBR1 phosphorylation are suppressed by deleting PiaA (). These in vivo results indicate that the major effects of RasC on chemotaxis are mediated through TORC2 and PKBR1. The active form of RasC promotes TORC2-dependent phosphorylation of the two PKBs in vitro in the absence of any stimulus ( and ). This reaction exhibits several characteristics that are consistent with a RasC–TORC2–PKB pathway. It is blocked by exogenous RBD and does not occur in lysates lacking the TORC2 component PiaA or Rip3 but can be rapidly initiated by adding Flag immunoprecipitate from piaA− cells expressing Flag-PiaA. Further experiments indicate that the reconstituting activity associated with the Flag immunoprecipitate likely contains a functional TOR complex (). Importantly, TORC2 binds specifically to RasCQ62L but not to inactivated wild-type RasC (), suggesting that a regulated interaction may contribute to the stimulation of TORC2 activity by RasCQ62L. Interestingly, we found that RasCQ62L lacking the C-terminal CAAX motif is not able to extend the kinetics of PKB phosphorylation (unpublished data), indicating that the membrane localization of RasC is critical for its in vivo function in activating TORC2.
A role for a Ras protein in PKB activation and cell migration may not seem surprising, as it has been shown that Ras proteins bind to both D. discoideum
and mammalian PI3Ks (Rodriguez-Viciana et al., 1994
; Pacold et al., 2000
; Funamoto et al., 2002
; Kae et al., 2004
). However, our data show that the effects of RasCQ62L
on chemotactic responses and the cytoskeletal activity are primarily mediated through TORC2. Furthermore, we found that disruption of PKBA but not PKBR1 in pten−
cells suppresses its chemotaxis defects (unpublished data). Therefore, although expressing RasCQ62L
and deleting PTEN both result in hyperstimulation of the PKB signaling, their effects appear to be mediated through different PKB isoforms. Our finding that TORC2 is a critical effector in Ras-mediated cell migration may serve as a stepping stone to understand cell motility in other organisms. PIP3-independent chemotaxis and chemoattractant-stimulated Ras activation have also been observed in human neutrophils (Worthen et al., 1994
; Zheng et al., 1997
). In many cancer cells that display altered cell migration, Ras proteins are found to be persistently activated (Oxford and Theodorescu, 2003
). In addition, TORC2 has been suggested to regulate cytoskeletal-based events in various systems (Jacinto et al., 2004
; Sarbassov et al., 2004
; Kamada et al., 2005
). It will be of great interest to learn whether the Ras–TORC2 pathway plays a similar role in the cytoskeleton organization and migration of these cells.