Characterization of the Roco Protein Family in Dictyostelium discoideum

Abstract

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...

Roco kinase structures give insights into the mechanism of Parkinson disease-related leucine-rich-repeat kinase 2 mutations

“…Dictyostelium cells with a disruption in the roco4 gene cannot synthesize cellulose, resulting in instable stalks that are unable to lift the spore head ( Fig. 1C) (20). This strong developmental defect of roco4-nulls cells can be rescued completely by the expression of wild-type Roco4 and also by a chimeric protein of Roco4 in which the kinase domain is replaced by that of LRRK2 (Fig.…”

“…Remarkably, the social amoeba Dictyostelium contains 11 Roco family members that contain a large variety of domains and have been studied in detail (19)(20)(21). In this study we used Dictyostelium Roco4, which has the same domain topology as LRRK2 (Fig.…”

“…Development of Dictyostelium mutants was tested as previously described (20). Briefly, cells were harvested in phosphate buffer (11 mM KH 2 PO 4 , 2.8 mM Na 2 HPO 4 ), plated at a density of 1 × 10 6 cells/cm 2 , and allowed to develop for 48 h on nutrient-free plates containing phosphate buffer supplemented with 15 g/L agar.…”

Structural model of the dimeric Parkinson’s protein LRRK2 reveals a compact architecture involving distant interdomain contacts

“…In addition, based on the LRRK2 primary protein sequence, the LRR domain is directly coupled to both the C terminus of the ankyrin domain and the N terminus of the Roc G-domain. Although mechanistically not well understood, recent studies did show that the N terminus of human LRRK2 and related Roco proteins is essential for function in vivo (45). Our data suggest that the LRRK2 ankyrin-LRR domains may play a direct role in the regulation of the LRRK2 kinase activity, guiding future in-depth functional characterization of LRRK2 at a molecular level.…”

Intracellular photoactivation of caged cGMP induces myosin II and actin responses in motile cells

“…12,22 Subsequent studies revealed a rich topology of domains that led to the classification of GbpC as a member of the Roco family of proteins. 23 A multi-step intramolecular activation mechanism has been identified for this protein 24,25 and its subcellular localization in response to cAMP-stimuli and osmotic shock was studied. 26 Despite our detailed mechanistic knowledge of GbpC activation, many open questions remain.…”