In this review, we discuss the evolutionary, biochemical, and functional data available for members of the Roco protein family. They are characterized by having a conserved supradomain that contains a Ras-like GTPase domain, called Roc, and a characteristic COR (C-terminal of Roc) domain. A kinase domain and diverse regulatory and protein-protein interaction domains are also often found in Roco proteins. First detected in the slime mold Dictyostelium discoideum, they have a broad phylogenetic range, being present in both prokaryotes and eukaryotes. The functions of these proteins are diverse. The best understood are Dictyostelium Rocos, which are involved in cell division, chemotaxis, and development. However, this family has received extensive attention because mutations in one of the human Roco genes (LRRK2) cause familial Parkinson disease. Other human Rocos are involved in epilepsy and cancer. Biochemical data suggest that Roc domains are capable of activating kinase domains intramolecularly. Interestingly, some of the dominant, disease-causing mutations in both the GTPase and kinase domains of LRRK2 increase kinase activity. Thus, Roco proteins may act as stand-alone transduction units, performing roles that were thought so far to require multiple proteins, as occur in the Ras transduction pathway.

Central to chemotaxis is the molecular mechanism by which cells exhibit directed movement in shallow gradients of a chemoattractant. We used Dictyostelium mutants to investigate the minimal requirements for chemotaxis, and identified a basal signalling module providing activation of Ras at the leading edge, which is sufficient for chemotaxis. The signalling enzymes PI3K, TorC2, PLA2 and sGC are not required for Ras activation and chemotaxis to folate or to steep gradients of cAMP, but they provide a memory of direction and improved orientation of the cell, which together increase the sensitivity about 150-fold for chemotaxis in shallow cAMP gradients.

Switching between attractive and repulsive migration in cell movement in response to extracellular guidance cues has been found in various cell types and is an important cellular function for translocation during cellular and developmental processes. Here we show that the preferential direction of migration during electrotaxis in Dictyostelium cells can be reversed by genetically modulating both guanylyl cyclases (GCases) and the cyclic guanosine monophosphate (cGMP)-binding protein C (GbpC) in combination with the inhibition of phosphatidylinositol-3-OH kinases (PI3Ks). The PI3K-dependent pathway is involved in cathode-directed migration under a direct-current electric field. The catalytic domains of soluble GCase (sGC) and GbpC also mediate cathode-directed signaling via cGMP, whereas the N-terminal domain of sGC mediates anode-directed signaling in conjunction with both the inhibition of PI3Ks and cGMP production. These observations provide an identification of the genes required for directional switching in electrotaxis and suggest that a parallel processing of electric signals, in which multiple-signaling pathways act to bias cell movement toward the cathode or anode, is used to determine the direction of migration.cGMP ͉ chemotaxis ͉ electrotaxis ͉ PI3K

The Roco family consists of multidomain Ras-GTPases that include LRRK2, a protein mutated in familial Parkinson's disease. The genome of the cellular slime mold Dictyostelium discoideum encodes 11 Roco proteins. To study the functions of these proteins, we systematically knocked out the roco genes. Previously described functions for GbpC, Pats1, and QkgA (Roco1 to Roco3) were confirmed, while novel developmental defects were identified in roco4-and roco11-null cells. Cells lacking Roco11 form larger fruiting bodies than wild-type cells, while roco4-null cells show strong developmental defects during the transition from mound to fruiting body; prestalk cells produce reduced levels of cellulose, leading to unstable stalks that are unable to properly lift the spore head. Detailed phylogenetic analysis of four slime mold species reveals that QkgA and Roco11 evolved relatively late by duplication of an ancestor roco4 gene (later than ϳ300 million years ago), contrary to the situation with other roco genes, which were already present before the split of the common ancestor of D. discoideum and Polysphondylium pallidum (before ϳ600 million years ago). Together, our data show that the Dictyostelium Roco proteins serve a surprisingly diverse set of functions and highlight Roco4 as a key protein for proper stalk cell formation.The Roco protein family is characterized by sharing a conserved core, consisting of a Ras-like GTPase called Roc (Ras of complex proteins) and a COR (C-terminal Of Roc) domain, often with a C-terminal kinase domain and several N-terminal leucine-rich repeats (LRR) (5, 14). The Dictyostelium cyclic GMP (cGMP)-binding protein GbpC was the seed of the family, in combination with 10 other genes encoding Roco proteins in Dictyostelium. Although the family did not draw much attention in the first years after its discovery, this rapidly changed when mutations in the human Roco protein LRRK2 were linked to the development of Parkinson's disease (PD) (3,15,17,22,28). Since then, most work on Roco proteins has focused on the biological and biochemical characterization of LRRK2 and GbpC. Phosphorylation studies have revealed that pathogenic mutations in LRRK2 lead to an increase in kinase activity and neuronal toxicity (26,27). Currently, it is not well understood how mutations in LRRK2 exactly lead to the loss of dopaminergic neurons and formation of so-called Lewis bodies, which are characteristic for the development of PD, but recent evidence hints at a role for LRRK2 in the activation of programmed cell death, through activation of caspase-8 (10).Dictyostelium cells that lack the cGMP-binding protein GbpC show abnormal phosphorylation and assembly of myosin II, which is needed to control the back of the cell during chemotaxis (7). The role of cGMP, and thus GbpC, in chemotaxis becomes even more evident when two other signaling pathways for chemotaxis (PLA2 and PI3K) are inhibited: under these circumstances, cells become solely dependent on the cGMP pathway for chemotaxis toward the chemoattractant cAMP (25...

GbpC is a large multidomain protein involved in cGMP-mediated chemotaxis in the cellular slime mold Dictyostelium discoideum. GbpC belongs to the Roco family of proteins that often share a central core region, consisting of leucine-rich repeats, a Ras domain (Roc), a Cor domain, and a MAPKKKinase domain. In addition to this core, GbpC contains a RasGEF domain and two cGMP-binding domains. Here, we report on an intramolecular signaling cascade of GbpC. In vitro, the RasGEF domain of GbpC specifically accelerates the GDP/GTP exchange of the Roc domain. Moreover, cGMP binding to GbpC strongly stimulates the binding of GbpC to GTP-agarose, suggesting cGMP-stimulated GDP/GTP exchange at the Roc domain. The function of the protein in vivo was investigated by rescue analysis of the chemotactic defect of gbpC null cells. Mutants that lack a functional guanine exchange factor (GEF), Roc, or kinase domain are inactive in vivo. Together, the results suggest a four-step intramolecular activation mechanism of the Roco protein GbpC: cGMP binding to the cyclic nucleotide-binding domains, activation of the GEF domain, GDP/GTP exchange of Roc, and activation of the MAPKKK domain.Extracellular cAMP is a chemoattractant for Dictyostelium cells. Upon binding of cAMP to surface receptors, several signaling cascades are activated that cause cells to crawl toward the source of cAMP (1, 2). Some of the signaling molecules involved in chemotaxis are the second messengers phosphatidylinositol 1,4,5-trisphosphate, inositol 1,4,5-trisphosphate, Ca 2ϩ , and the product(s) of PLA 2 , 2 as well as the cyclic nucleotides cAMP and cGMP (2-4). These second messengers have important roles in transducing signals that lead to actin polymerization at the front of the cell (5) and phosphorylation of myosin in the back of the cell, which are vital for cells to move (6, 7). One of the second messengers that have been implicated in myosin regulation is cGMP (8, 9). Recently, the proteins that are involved in the formation and degradation of cGMP have been identified and characterized (4, 10). Binding of extracellular cAMP to the surface cAMP receptor cAR1 causes a G-proteindependent activation of two guanylyl cyclases, soluble guanylyl cyclase (sGC) and membrane-bound guanylyl cyclase (GCA). These two enzymes are responsible for the rapid synthesis of cGMP in cells, whereas three cGMP-degrading enzymes, PDE3, PDE5, and PDE6, cause a subsequent decrease of cGMP in the cells, back to basal levels.In Dictyostelium, the function of cGMP is transduced by the target protein GbpC. Disruption of the gene coding for GbpC yields a cell line that has impaired regulation of myosin II as well as defects in chemotaxis, comparable with cells that lack both guanylyl cyclases (9). GbpC was shown to contain two cyclic nucleotidebinding domains that are able to bind cGMP with high affinity. Furthermore, this protein has many other domains: leucine-rich repeats and domains that have homology to Ras-GTPases (Roc for Ras of complex proteins), Cor (for C terminus of Roc), MAP...