Homo- and heterodimerization of ROCO kinases: LRRK2 kinase inhibition by the LRRK2 ROCO fragment

Mutations in the gene encoding leucine-rich repeat kinase 2 (LRRK2) are the most common cause of autosomal-dominant familial and late-onset sporadic Parkinson's disease (PD). LRRK2 is a large multi-domain protein featuring a GTP-binding C-terminal of Ras of complex proteins (ROC) (ROCO) domain combination unique for the ROCO protein family, directly followed by a kinase domain. Dimerization is a well-established phenomenon among protein kinases. Here, we confirm LRRK2 self-interaction, and provide evidence for general homo- and heterodimerization potential among the ROCO kinase family (LRRK2, LRRK1, and death-associated protein kinase 1). The ROCO domain was critically, though not exclusively involved in dimerization, as a LRRK2 deletion mutant lacking the ROCO domain retained dimeric properties. GTP binding did not appear to influence ROCO^LRRK2 self-interaction. Interestingly, ROCO^LRRK2 fragments exerted an inhibitory effect on both wild-type and the elevated G2019S LRRK2 autophosphorylation activity. Insertion of PD mutations into ROCO^LRRK2 reduced self-interaction and led to a reduction of LRRK2 kinase inhibition. Collectively, these results suggest a functional link between ROCO interactions and kinase activity of wild-type and mutant LRRK2. Importantly, our finding of ROCO^LRRK2 fragment-mediated LRRK2 kinase inhibition offers a novel lead for drug design and thus might have important implications for new therapeutic avenues in PD.     

The emerging interrelation between ROCO and related kinases,intracellular Ca2+ signaling,and autophagy

ROCO kinases form a family of proteins characterized by kinase activity in addition to the presence of the so-called ROC (Ras of complex proteins)/COR (C-terminal of ROC) domains having a role in their GTPase activity. These are the death-associated protein kinase (DAPK) 1 and the leucine-rich repeat kinases (LRRK) 1 and 2. These kinases all play roles in cellular life and death decisions and in autophagy in particular. Related to the ROCO kinases is DAPK 2 that however cannot be classified as a ROCO protein due to the absence of the ROC/COR domains. This review aims to bring together what is known about the relation between these proteins and intracellular Ca²⁺ signals in the induction and regulation of autophagy. Interestingly, DAPK 1 and 2 and LRRK2 are all linked to Ca²⁺ signaling in their effects on autophagy, though in various ways. Present evidence supports an upstream role for LRRK2 that via lysosomal and endoplasmic reticulum Ca²⁺ release can trigger autophagy induction. In contrast herewith, DAPK1 and 2 react on existing Ca²⁺ signals to stimulate the autophagic pathway. Further research will be needed for obtaining a full understanding of the role of these various kinases in autophagy and to assess their exact relation with intracellular Ca²⁺ signaling as this would be helpful in the development of novel therapeutic strategies against neurodegenerative disorders, cancer and auto-immune diseases.This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.     

GTP binding to the ROC domain of DAP-kinase regulates its function through intramolecular signalling

Death-associated protein kinase (DAPk) was recently suggested by sequence homology to be a member of the ROCO family of proteins. Here, we show that DAPk has a functional ROC (Ras of complex proteins) domain that mediates homo-oligomerization and GTP binding through a defined P-loop motif. Upon binding to GTP, the ROC domain negatively regulates the catalytic activity of DAPk and its cellular effects. Mechanistically, GTP binding enhances an inhibitory autophosphorylation at a distal site that suppresses kinase activity. This study presents a new mechanism of intramolecular signal transduction, by which GTP binding operates in cis to affect the catalytic activity of a distal domain in the protein.     

Highly dynamic exon shuffling in candidate pathogen receptors ... what if brown algae were capable of adaptive immunity?

Pathogen recognition is the first step of immune reactions. In animals and plants, direct or indirect pathogen recognition is often mediated by a wealth of fast-evolving receptors, many of which contain ligand-binding and signal transduction domains, such as leucine-rich or tetratricopeptide repeat (LRR/TPR) and NB-ARC domains, respectively. In order to identify candidates potentially involved in algal defense, we mined the genome of the brown alga Ectocarpus siliculosus for homologues of these genes and assessed the evolutionary pressures acting upon them. We thus annotated all Ectocarpus LRR-containing genes, in particular an original group of LRR-containing GTPases of the ROCO family, and 24 NB-ARC-TPR proteins. They exhibit high birth and death rates, while a diversifying selection is acting on their LRR (respectively TPR) domain, probably affecting the ligand-binding specificities. Remarkably, each repeat is encoded by an exon, and the intense exon shuffling underpins the variability of LRR and TPR domains. We conclude that the Ectocarpus ROCO and NB-ARC-TPR families are excellent candidates for being involved in recognition/transduction events linked to immunity. We further hypothesize that brown algae may generate their immune repertoire via controlled somatic recombination, so far only known from the vertebrate adaptive immune systems.     

There is more to life and death than mitochondria: Bcl-2 proteins at the endoplasmic reticulum

Proteins of the Bcl-2 family are important regulators of cell fate. The role of these proteins in controlling mitochondrial apoptotic processes has been extensively investigated, although exact molecular mechanisms are incompletely understood. However, mounting evidence indicates that these proteins also function at the endoplasmic reticulum and other locations within the cell. Both pro- and anti-apoptotic Bcl-2 family members can regulate endoplasmic reticulum calcium, cellular pH and endoplasmic reticulum resident proteins. In this review, we discuss the activities and potential targets of Bcl-2 family members at the endoplasmic reticulum and other cellular locations.     

A new class of membrane-associated calcium-binding proteins

Calcium ions act as modulators of many fundamental processes in eukaryotic cells. Although these processes apparently involve initial interactions between calcium ions and cell membranes, the identity of the putative membrane Ca²⁺-binding proteins has until recently been obscure. This article describes a recently discovered family of mammalian membrane proteins, of perhaps ancient origin, that may fulfil this function.     

LRRK1 protein kinase activity is stimulated upon binding of GTP to its Roc domain

Human leucine-rich repeat kinase 1 (LRRK1) is a multi-domain protein of unknown function belonging to the ROCO family of complex proteins. Here, we report the molecular characterization of human LRRK1 and show, for the first time, that LRRK1 is both a functional protein kinase and a GDP/GTP-binding protein. Binding of GTP to LRRK1 is specific, requires the GTPase-like Roc domain, and leads to a stimulation of LRRK1 kinase activity. LRRK1 is the first example of a GTP-regulated protein kinase harboring both the kinase effector domain and the GTP-binding regulatory domain. Hence, we propose a model in which LRRK1 cycles between a GTP-bound active and a GDP-bound inactive state. Moreover, we mutated LRRK1 to mimic mutations previously identified in LRRK2/dardarin, the only human paralogue of LRRK1, that have been linked to autosomal-dominant parkinsonism. We demonstrate that three of four mutations analyzed significantly downregulate LRRK1 kinase activity. Ultimately, the results presented for LRRK1 may contribute to the elucidation of LRRK2's role in the pathogenesis of Parkinson's disease.     

The Parkinson's disease-associated protein, leucine-rich repeat kinase 2 (LRRK2), is an authentic GTPase that stimulates kinase activity

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the leading cause of autosomal dominant Parkinson's disease (PD). LRRK2, a member of the ROCO protein family, contains both Ras GTPase-like (Roc) and kinase (MAPKKK) domains, as well as other functional motifs. Here, we have identified LRRK2 as the first mammalian ROCO protein that is an authentic and functional GTPase, defined by the ability to bind GTP and undergo intrinsic GTP hydrolysis. Furthermore, the Roc domain is sufficient for this native GTPase activity and binds and hydrolyzes GTP indistinguishably from the Ras-related small GTPase, Rac1. The PD-associated mutation, R1441C, located within the Roc domain, leads to an increase in LRRK2 kinase activity and a decrease in the rate of GTP hydrolysis, compared to the wild-type protein, in an in vitro assay. This finding suggests that the R1441C mutation may help stabilize an activated state of LRRK2. Additionally, LRRK2-mediated phosphorylation is stimulated upon binding of non-hydrolyzable GTP analogs, suggesting that LRRK2 is an MAPKKK-activated intramolecularly by its own GTPase. Since GTPases and MAPKKKs are upstream regulators of multiple signal transduction cascades, LRRK2 may play a central role in integrating pathways involved in neuronal cell signaling and the pathogenesis of PD.     

The Drosophila CD2AP/CIN85 orthologue Cindr regulates junctions and cytoskeleton dynamics during tissue patterning

Developing tissues require cells to undergo intricate processes to shift into appropriate niches. This requires a functional connection between adhesion-mediating events at the cell surface and a cytoskeletal reorganization to permit directed movement. A small number of proteins are proposed to link these processes. Here, we identify one candidate, Cindr, the sole Drosophila melanogaster member of the CD2AP/CIN85 family (this family has been previously implicated in a variety of processes). Using D. melanogaster retina, we demonstrate that Cindr links cell surface junctions (E-cadherin) and adhesion (Roughest) with multiple components of the actin cytoskeleton. Reducing cindr activity leads to defects in local cell movement and, consequently, tissue patterning and cell death. Cindr activity is required for normal localization of Drosophila E-cadherin and Roughest, and we show additional physical and functional links to multiple components of the actin cytoskeleton, including the actin-capping proteins capping protein alpha and capping protein beta. Together, these data demonstrate that Cindr is involved in dynamic cell rearrangement in an emerging epithelium.     

Leucine-rich repeat kinase 2: relevance to Parkinson's disease

Human leucine-rich repeat kinase 2 (LRRK2) is a novel kinase belonging to the ROCO protein superfamily (Ras of complex proteins (Roc) with a C-terminal of Roc domain). This large complex protein of 280kDa contains several functional domains including leucine-rich repeats, Ras-related GTPase, mitogen-activated protein kinase kinase kinase (MAPKKK), and WD40 repeats. While definitive functions of LRRK2 have yet to be described, the domain structure of LRRK2 suggests that it plays an important role in the regulation of signal transduction cascades through its dual enzymatic activities of GTPase and MAPKKK. Moreover, mutations in LRRK2 have been found to be thus far the most frequent cause of late-onset familial and idiopathic Parkinson's disease. Further investigations should allow for the elucidation of how pathogenic mutations trigger changes in the structure and function of LRRK2 that lead to aberrant signal transduction and neurodegeneration in Parkinson's disease.     

Fasciculation and elongation zeta proteins 1 and 2: From structural flexibility to functional diversity

Fasciculation and elongation zeta/zygin(FEZ) proteins are a family of hub proteins and share many characteristics like high connectivity in interaction networks, they are involved in several cellular processes, evolve slowly and in general have intrinsically disordered regions. In 1985, unc-76 gene was firstly described and involved in axonal growth in C. elegans, and in 1997 Bloom and Horvitz enrolled also the human homologues genes, FEZ1 and FEZ2, in this process. While nematodes possess one gene(unc-76), mammalians have one more copy(FEZ1 and FEZ2). Several animal models have been used to study FEZ family functions like: C. elegans, D. melanogaster, R. novergicus and human cells.Complementation assays were performed and demonstrated the function conservation between paralogues. Human FEZ1 protein is more studied followed by UNC-76 and FEZ2 proteins, respectively. While FEZ1 and UNC-76 shared interaction partners, FEZ2 evolved and increased the number of protein-protein interactions(PPI) with cytoplasmatic partners. FEZ proteins are implicated in intracellular transport, acting as bivalent cargo transport adaptors in kinesinmediated movement. Especially in light of this cellular function, this family of proteins has been involved in several processes like neuronal development,neurological disorders, viral infection and autophagy. However, nuclear functions of FEZ proteins have been explored as well, due to high content of PPI with nuclear proteins, correlating FEZ1 expression to Sox2 and Hoxb4 gene regulation and retinoic acid signaling. These recent findings open new avenue to study FEZ proteins functions and its involvement in already described processes.This review intends to reunite aspects of evolution, structure, interaction partners and function of FEZ proteins and correlate them to physiological and pathological processes.     

Pombe Cdc15 homology (PCH) proteins: coordinators of membrane-cytoskeletal interactions

Cellular adhesion, motility, endocytosis, exocytosis and cytokinesis involve the coordinated reorganization of the cytoskeleton and of the plasma membrane. The 'Pombe Cdc15 homology' (PCH) family of adaptor proteins has recently been shown to coordinate the membrane and cytoskeletal dynamics involved in these processes by curving membranes, recruiting dynamin and controlling the architecture of the actin cytoskeleton. Mutations in PCH family members or proteins that interact with them are associated with autoinflammatory, neurological or neoplastic diseases. Here, we review the nature, actions and disease associations of the vertebrate PCH family members, highlighting their fundamental roles in the regulation of processes involving membrane-cytoskeletal interactions.     

The PsbP family of proteins

The PsbP family of proteins consists of 11 evolutionarily related thylakoid lumenal components. These include the archetypal PsbP protein, which is an extrinsic subunit of eukaryotic photosystem II, three PsbP-like proteins (CyanoP of the prokaryotic cyanobacteria and green oxyphotobacteria, and the PPL1 and PPL2 proteins found in many eukaryotes), and seven PsbP-domain (PPD) proteins (PPD1–PPD7, most of which are found in the green plant lineage). All of these possess significant sequence and structural homologies while having very diverse functions. While the PsbP protein has been extensively studied and plays a functional role in the optimization of photosynthetic oxygen evolution at physiological calcium and chloride concentrations, the molecular functions of the other family members are poorly understood. Recent investigations have begun to illuminate the roles that these proteins play in membrane protein complex assembly/stability, hormone biosynthesis, and other metabolic processes. In this review we have examined this functional information within the context of recent advances examining the structure of these components.     

Dynamin at the actin-membrane interface

Many important cellular processes such as phagocytosis, cell motility and endocytosis require the participation of a dynamic and interactive actin cytoskeleton that acts to deform cellular membranes. The extensive family of non-traditional myosins has been implicated in linking the cortical actin gel with the plasma membrane. Recently, however, the dynamins have also been included in these cell processes as a second family of mechanochemical enzymes that self-associate and hydrolyze nucleotides to perform 'work' while linking cellular membranes to the actin cytoskeleton. The dynamins are believed to form large helical polymers from which extend many interactive proline-rich tail domains, and these domains bind to a variety of SH3-domain-containing proteins, many of which appear to be actin-binding proteins. Recent data support the concept that the dynamin family might act as a 'polymeric contractile scaffold' at the interface between biological membranes and filamentous actin.     

Are the molecular strategies that control apoptosis conserved in bacteria?

The Staphylococcus aureus cid and lrg operons have been shown to encode putative membrane proteins that are involved in the regulation of murein hydrolase activity and penicillin tolerance. Cid proteins enhance murein hydrolase activity and penicillin sensitivity, whereas Lrg proteins have an inhibitory effect on these processes. It has been proposed that the Cid and Lrg proteins function in a way analogous to bacteriophage-encoded holins and antiholins, respectively, which control the timing of bacteriophage-induced lysis. This article explores the possibility that the Cid-Lrg regulatory system controls bacterial programmed cell death using a molecular strategy that it is functionally analogous to that mediated by the eukaryotic Bcl-2 family of apoptosis regulatory proteins.     

Assaying the Kinase Activity of LRRK2 in vitro

Leucine Rich Repeat Kinase 2 (LRRK2) is a 2527 amino acid member of the ROCO family of proteins, possessing a complex, multidomain structure including a GTPase domain (termed ROC, for Ras of Complex proteins) and a kinase domain¹. The discovery in 2004 of mutations in LRRK2 that cause Parkinson''s disease (PD) resulted in LRRK2 being the focus of a huge volume of research into its normal function and how the protein goes awry in the disease state^2,3. Initial investigations into the function of LRRK2 focused on its enzymatic activities^4-6. Although a clear picture has yet to emerge of a consistent alteration in these due to mutations, data from a number of groups has highlighted the importance of the kinase activity of LRRK2 in cell death linked to mutations^7,8. Recent publications have reported inhibitors targeting the kinase activity of LRRK2, providing a key experimental tool^9-11. In light of these data, it is likely that the enzymatic properties of LRRK2 afford us an important window into the biology of this protein, although whether they are potential drug targets for Parkinson''s is open to debate.A number of different approaches have been used to assay the kinase activity of LRRK2. Initially, assays were carried out using epitope tagged protein overexpressed in mammalian cell lines and immunoprecipitated, with the assays carried out using this protein immobilised on agarose beads^4,5,7. Subsequently, purified recombinant fragments of LRRK2 in solution have also been used, for example a GST tagged fragment purified from insect cells containing residues 970 to 2527 of LRRK2¹². Recently, Daniëls et al. reported the isolation of full length LRRK2 in solution from human embryonic kidney cells, however this protein is not widely available¹³. In contrast, the GST fusion truncated form of LRRK2 is commercially available (from Invitrogen, see table 1 for details), and provides a convenient tool for demonstrating an assay for LRRK2 kinase activity. Several different outputs for LRRK2 kinase activity have been reported. Autophosphorylation of LRRK2 itself, phosphorylation of Myelin Basic Protein (MBP) as a generic kinase substrate and phosphorylation of an artificial substrate - dubbed LRRKtide, based upon phosphorylation of threonine 558 in Moesin - have all been used, as have a series of putative physiological substrates including α-synuclein, Moesin and 4-EBP^14-17. The status of these proteins as substrates for LRRK2 remains unclear, and as such the protocol described below will focus on using MBP as a generic substrate, noting the utility of this system to assay LRRK2 kinase activity directed against a range of potential substrates.     

Rhomboid proteins: a role in keratinocyte proliferation and cancer

The Rhomboids represent a relatively recently discovered family of proteins, consisting in a variety of intramembrane serine proteases and their inactive homologues, the iRhoms. Rhomboids typically contain six or seven transmembrane domains (TMD) and have been classified into four subgroups: Secretase A and B, Presenilin-Associated-Rhomboid-Like (PARL) and iRhoms. Although the iRhoms, iRhom1 and iRhom2, have lost their protease activity during evolution, they retain key non-protease functions and have been implicated in the regulation of epidermal growth factor (EGF) signalling. EGF is moreover a substrate of RHBDL2, their active Rhomboid relative. Other substrates of RHBDL2 include members of the EphrinB family and thrombomodulin. RHBDL2 has also previously been demonstrated to be important in wound healing in cutaneous keratinocytes through the cleavage of thrombomodulin. Additional roles for these intriguing proteins seem likely to be revealed in the future. This review focuses on our current understanding of Rhomboids and, in particular, on RHBDL2 and iRhom2 and their roles in cellular processes and human disease.