Karsten Boldt

The Parkinson disease-associated protein kinase LRRK2 exhibits MAPKKK activity and phosphorylates MKK3/6 and MKK4/7, in vitro

Autosomal dominant mutations in the human Leucine‐Rich Repeat Kinase 2 (LRRK2) gene represent the most common monogenetic cause of Parkinson disease (PD) and increased kinase activity observed in pathogenic mutants of LRRK2 is most likely causative for PD‐associated neurotoxicity. The sequence of the LRRK2 kinase domain shows similarity to MAP kinase kinase kinases. Furthermore, LRRK2 shares highest sequence homology with mixed linage kinases which act upstream of canonical MAPKK and are involved in cellular stress responses. Therefore, we addressed the question if LRRK2 exhibits MAPKKK activity by systematically testing MAPKKs as candidate substrates, in vitro. We demonstrate that LRRK2 variants phosphorylate mitogen‐activated protein kinase kinases (MAPKK), including MKK3 ‐4, ‐6 and ‐7. MKKs act upstream of the MAPK p38 and JNK mediating oxidative cell stress, neurotoxicity and apoptosis. The disease‐associated LRRK2 G2019S and I2020T mutations show an increased phosphotransferase activity towards MKKs correlating with the activity shown for its autophosphorylation. Our findings present evidence of a new class of molecular targets for mutant LRRK2 that link to neurotoxicity, cellular stress, cytoskeletal dynamics and vesicular transport.

Mutations in LCA5, encoding the ciliary protein lebercilin, cause Leber congenital amaurosis.

Leber congenital amaurosis (LCA) causes blindness or severe visual impairment at or within a few months of birth. Here we show, using homozygosity mapping, that the LCA5 gene on chromosome 6q14, which encodes the previously unknown ciliary protein lebercilin, is associated with this disease. We detected homozygous nonsense and frameshift mutations in LCA5 in five families affected with LCA. In a sixth family, the LCA5 transcript was completely absent. LCA5 is expressed widely throughout development, although the phenotype in affected individuals is limited to the eye. Lebercilin localizes to the connecting cilia of photoreceptors and to the microtubules, centrioles and primary cilia of cultured mammalian cells. Using tandem affinity purification, we identified 24 proteins that link lebercilin to centrosomal and ciliary functions. Members of this interactome represent candidate genes for LCA and other ciliopathies. Our findings emphasize the emerging role of disrupted ciliary processes in the molecular pathogenesis of LCA.

DYX1C1 is required for axonemal dynein assembly and ciliary motility

DYX1C1 has been associated with dyslexia and neuronal migration in the developing neocortex. Unexpectedly, we found that deleting exons 2–4 of Dyx1c1 in mice caused a phenotype resembling primary ciliary dyskinesia (PCD), a disorder characterized by chronic airway disease, laterality defects and male infertility. This phenotype was confirmed independently in mice with a Dyx1c1 c.T2A start-codon mutation recovered from an N-ethyl-N-nitrosourea (ENU) mutagenesis screen. Morpholinos targeting dyx1c1 in zebrafish also caused laterality and ciliary motility defects. In humans, we identified recessive loss-of-function DYX1C1 mutations in 12 individuals with PCD. Ultrastructural and immunofluorescence analyses of DYX1C1-mutant motile cilia in mice and humans showed disruptions of outer and inner dynein arms (ODAs and IDAs, respectively). DYX1C1 localizes to the cytoplasm of respiratory epithelial cells, its interactome is enriched for molecular chaperones, and it interacts with the cytoplasmic ODA and IDA assembly factor DNAAF2 (KTU). Thus, we propose that DYX1C1 is a newly identified dynein axonemal assembly factor (DNAAF4).

Phosphopeptide Analysis Reveals Two Discrete Clusters of Phosphorylation in the N-Terminus and the Roc Domain of the Parkinson-Disease Associated Protein Kinase LRRK2

Mutations in leucine-rich repeat kinase 2 (LRRK2) that increase its kinase activity associate with familial forms of Parkinson disease (PD). As phosphorylation determines the functional state of most protein kinases, we systematically mapped LRRK2 phosphorylation sites by mass spectrometry. Our analysis revealed a high degree of constitutive phosphorylation in a narrow serine-rich region preceding the LRR-domain. Allowing de novo autophosphorylation of purified LRRK2 in an in vitro autokinase assay prior to mass spectrometric analysis, we discovered multiple sites of autophosphorylation. Solely serine and threonine residues were found phosphorylated suggesting LRRK2 as a true serine threonine kinase. Autophosphorylation mainly targets the ROC GTPase domain and its clustering around the GTP binding pocket of ROC suggests cross-regulatory activity between kinase and Roc domain. In conclusion, the phosphoprotein LRRK2 functions as an autocatalytically active serine threonine kinase. Clustering of phosphosites within two discrete domains suggest that phosphorylation may regulate its biological functions in a yet unknown fashion.

ANKS6 is a central component of a nephronophthisis module linking NEK8 to INVS and NPHP3

Nephronophthisis is an autosomal recessive cystic kidney disease that leads to renal failure in childhood or adolescence. Most NPHP gene products form molecular networks. Here we identify ANKS6 as a new NPHP family member that connects NEK8 (NPHP9) to INVS (NPHP2) and NPHP3. We show that ANKS6 localizes to the proximal cilium and confirm its role in renal development through knockdown experiments in zebrafish and Xenopus laevis. We also identify six families with ANKS6 mutations affected by nephronophthisis, including severe cardiovascular abnormalities, liver fibrosis and situs inversus. The oxygen sensor HIF1AN hydroxylates ANKS6 and INVS and alters the composition of the ANKS6-INVS-NPHP3 module. Knockdown of Hif1an in Xenopus results in a phenotype that resembles loss of other NPHP proteins. Network analyses uncovered additional putative NPHP proteins and placed ANKS6 at the center of this NPHP module, explaining the overlapping disease manifestation caused by mutation in ANKS6, NEK8, INVS or NPHP3.

Pitchfork Regulates Primary Cilia Disassembly and Left-Right Asymmetry

A variety of developmental disorders have been associated with ciliary defects, yet the controls that govern cilia disassembly are largely unknown. Here we report a mouse embryonic node gene, which we named Pitchfork (Pifo). Pifo associates with ciliary targeting complexes and accumulates at the basal body during cilia disassembly. Haploinsufficiency causes a unique node cilia duplication phenotype, left-right asymmetry defects, and heart failure. This phenotype is likely relevant in humans, because we identified a heterozygous R80K PIFO mutation in a fetus with situs inversus and cystic liver and kidneys, and in patient with double-outflow right ventricle. We show that PIFO, but not R80K PIFO, is sufficient to activate Aurora A, a protooncogenic kinase that induces cilia retraction, and that Pifo/PIFO mutation causes cilia retraction, basal body liberation, and overreplication defects. Thus, the observation of a disassembly phenotype in vivo provides an entry point to understand and categorize ciliary disease. AUTHOR AUDIO:

A QUICK screen for Lrrk2 interaction partners - leucine-rich repeat kinase 2 is involved in actin cytoskeleton dynamics

Mutations in human leucine-rich repeat kinase 2 (Lrrk2), a protein of yet unknown function, are linked to Parkinsons disease caused by degeneration of midbrain dopaminergic neurons. The protein comprises several domains including a GTPase and a kinase domain both affected by several pathogenic mutations. To elucidate the molecular interaction network of endogenous Lrrk2 under stoichiometric constraints, we applied QUICK (quantitative immunoprecipitation combined with knockdown) in NIH3T3 cells. The identified interactome reveals actin isoforms as well as actin-associated proteins involved in actin filament assembly, organization, rearrangement, and maintenance, suggesting that the biological function of Lrrk2 is linked to cytoskeletal dynamics. In fact, we demonstrate Lrrk2 de novo binding to F-actin and its ability to modulate its assembly in vitro. When tested in intact cells, knockdown of Lrrk2 causes morphological alterations in NIH3T3 cells. In developing dopaminergic midbrain primary neurons, Lrrk2 knockdown results in shortened neurite processes, indicating a physiological role of Lrrk2 in cytoskeletal organization and dynamics of dopaminergic neurons. Hence, our results demonstrate that molecular interactions as well as the physiological function of Lrrk2 are closely related to the organization of the actin-based cytoskeleton, a crucial feature of neuronal development and neuron function.

An siRNA-based functional genomics screen for the identification of regulators of ciliogenesis and ciliopathy genes

Defects in primary cilium biogenesis underlie the ciliopathies, a growing group of genetic disorders. We describe a whole-genome siRNA-based reverse genetics screen for defects in biogenesis and/or maintenance of the primary cilium, obtaining a global resource. We identify 112 candidate ciliogenesis and ciliopathy genes, including 44 components of the ubiquitin–proteasome system, 12 G-protein-coupled receptors, and 3 pre-mRNA processing factors (PRPF6, PRPF8 and PRPF31) mutated in autosomal dominant retinitis pigmentosa. The PRPFs localize to the connecting cilium, and PRPF8- and PRPF31-mutated cells have ciliary defects. Combining the screen with exome sequencing data identified recessive mutations in PIBF1, also known as CEP90, and C21orf2, also known as LRRC76, as causes of the ciliopathies Joubert and Jeune syndromes. Biochemical approaches place C21orf2 within key ciliopathy-associated protein modules, offering an explanation for the skeletal and retinal involvement observed in individuals with C21orf2 variants. Our global, unbiased approaches provide insights into ciliogenesis complexity and identify roles for unanticipated pathways in human genetic disease.

Active Transport and Diffusion Barriers Restrict Joubert Syndrome-Associated ARL13B/ARL-13 to an Inv-like Ciliary Membrane Subdomain

Cilia are microtubule-based cell appendages, serving motility, chemo-/mechano-/photo- sensation, and developmental signaling functions. Cilia are comprised of distinct structural and functional subregions including the basal body, transition zone (TZ) and inversin (Inv) compartments, and defects in this organelle are associated with an expanding spectrum of inherited disorders including Bardet-Biedl syndrome (BBS), Meckel-Gruber Syndrome (MKS), Joubert Syndrome (JS) and Nephronophthisis (NPHP). Despite major advances in understanding ciliary trafficking pathways such as intraflagellar transport (IFT), how proteins are transported to subciliary membranes remains poorly understood. Using Caenorhabditis elegans and mammalian cells, we investigated the transport mechanisms underlying compartmentalization of JS-associated ARL13B/ARL-13, which we previously found is restricted at proximal ciliary membranes. We now show evolutionary conservation of ARL13B/ARL-13 localisation to an Inv-like subciliary membrane compartment, excluding the TZ, in many C. elegans ciliated neurons and in a subset of mammalian ciliary subtypes. Compartmentalisation of C. elegans ARL-13 requires a C-terminal RVVP motif and membrane anchoring to prevent distal cilium and nuclear targeting, respectively. Quantitative imaging in more than 20 mutants revealed differential contributions for IFT and ciliopathy modules in defining the ARL-13 compartment; IFT-A/B, IFT-dynein and BBS genes prevent ARL-13 accumulation at periciliary membranes, whereas MKS/NPHP modules additionally inhibit ARL-13 association with TZ membranes. Furthermore, in vivo FRAP analyses revealed distinct roles for IFT and MKS/NPHP genes in regulating a TZ barrier to ARL-13 diffusion, and intraciliary ARL-13 diffusion. Finally, C. elegans ARL-13 undergoes IFT-like motility and quantitative protein complex analysis of human ARL13B identified functional associations with IFT-B complexes, mapped to IFT46 and IFT74 interactions. Together, these findings reveal distinct requirements for sequence motifs, IFT and ciliopathy modules in defining an ARL-13 subciliary membrane compartment. We conclude that MKS/NPHP modules comprise a TZ barrier to ARL-13 diffusion, whereas IFT genes predominantly facilitate ARL-13 ciliary entry and/or retention via active transport mechanisms.

Tandem affinity purification of protein complexes from mammalian cells by the Strep/FLAG (SF)-TAP tag.

Isolation and dissection of native multiprotein complexes is a central theme in functional genomics. The development of the tandem affinity purification (TAP) tag has enabled efficient and large-scale purification of native protein complexes. The SF-TAP tag, a modified version of the TAP tag, allows a fast and straightforward purification of protein complexes from mammalian cells. It consists of a tandem Strep-tag II and a FLAG epitope (SF-TAP). The SF-TAP tag allows a native elution of protein complexes without proteolytic cleavage needed in the original TAP procedure. Besides the SF-TAP protocol, the principal idea of a pathway mapping by subsequent tagging of copurified proteins is demonstrated for the interactome of the MAPKKK Raf.