Mutations in LRRK2 are a common cause of genetic Parkinson’s disease (PD). LRRK2 is a multi-domain Roco protein, harbouring kinase and GTPase activity. In analogy with a bacterial homologue, LRRK2 was proposed to act as a GTPase activated by dimerization (GAD), while recent reports suggest LRRK2 to exist under a monomeric and dimeric form in vivo. It is however unknown how LRRK2 oligomerization is regulated. Here, we show that oligomerization of a homologous bacterial Roco protein depends on the nucleotide load. The protein is mainly dimeric in the nucleotide-free and GDP-bound states, while it forms monomers upon GTP binding, leading to a monomer-dimer cycle during GTP hydrolysis. An analogue of a PD-associated mutation stabilizes the dimer and decreases the GTPase activity. This work thus provides insights into the conformational cycle of Roco proteins and suggests a link between oligomerization and disease-associated mutations in LRRK2.

Eukaryotic cells chemotax in a wide range of chemoattractant concentration gradients, and thus need inhibitory processes that terminate cell responses to reach adaptation while maintaining sensitivity to higher-concentration stimuli. However, the molecular mechanisms underlying inhibitory processes are still poorly understood. Here, we reveal a locally controlled inhibitory process in a GPCR-mediated signaling network for chemotaxis in We identified a negative regulator of Ras signaling, C2GAP1, which localizes at the leading edge of chemotaxing cells and is activated by and essential for GPCR-mediated Ras signaling. We show that both C2 and GAP domains are required for the membrane targeting of C2GAP1, and that GPCR-triggered Ras activation is necessary to recruit C2GAP1 from the cytosol and retains it on the membrane to locally inhibit Ras signaling. C2GAP1-deficient cells have altered Ras activation that results in impaired gradient sensing, excessive polymerization of F actin, and subsequent defective chemotaxis. Remarkably, these cellular defects of cells are chemoattractant concentration dependent. Thus, we have uncovered an inhibitory mechanism required for adaptation and long-range chemotaxis.

Target of Rapamycin Complex 2 (TORC2) has conserved roles in regulating cytoskeleton dynamics and cell migration and has been linked to cancer metastasis. However, little is known about the mechanisms regulating TORC2 activity and function in any system. In Dictyostelium, TORC2 functions at the front of migrating cells downstream of the Ras protein RasC, controlling F-actin dynamics and cAMP production. Here, we report the identification of the small GTPase Rap1 as a conserved binding partner of the TORC2 component RIP3/SIN1, and that Rap1 positively regulates the RasC-mediated activation of TORC2 in Dictyostelium. Moreover, we show that active RasC binds to the catalytic domain of TOR, suggesting a mechanism of TORC2 activation that is similar to Rheb activation of TOR complex 1. Dual Ras/Rap1 regulation of TORC2 may allow for integration of Ras and Rap1 signaling pathways in directed cell migration.

BackgroundThe small G-protein Rap1 is an important regulator of cellular adhesion in Dictyostelium, however so far the downstream signalling pathways for cell adhesion are not completely characterized. In mammalian cells talin is crucial for adhesion and Rap1 was shown to be a key regulator of talin signalling.ResultsIn a proteomic screen we identified TalinB as a potential Rap1 effector in Dictyostelium. In subsequent pull-down experiments we demonstrate that the Ras association (RA) domain of TalinB interacts specifically with active Rap1. Studies with a mutated RA domain revealed that the RA domain is essential for TalinB-Rap1 interaction, and that this interaction contributes to cell-substrate adhesion during single-celled growth and is crucial for cell-cell adhesion during multicellular development.ConclusionsDictyostelium Rap1 directly binds to TalinB via the conserved RA domain. This interaction is critical for adhesion, which becomes essential for high adhesive force demanding processes, like morphogenesis during multicellular development of Dictyostelium. In mammalian cells the established Rap1-talin interaction is indirect and acts through the scaffold protein - RIAM. Interestingly, direct binding of mouse Rap1 to the RA domain of Talin1 has recently been demonstrated.Electronic supplementary materialThe online version of this article (doi:10.1186/s12860-015-0078-0) contains supplementary material, which is available to authorized users.

Highlights d We identify a new regulator that shapes macropinocytic and phagocytic cups d Shaping protrusions into cups requires differential regulation of Ras and Rac d Cups are organized by integrating interactions with phospholipids and multiple GTPases d Defective cup formation causes a target shape-specific defect in phagocytosis