scholarly journals Systematic proteomic analysis of LRRK2-mediated Rab GTPase phosphorylation establishes a connection to ciliogenesis

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Martin Steger ◽  
Federico Diez ◽  
Herschel S Dhekne ◽  
Pawel Lis ◽  
Raja S Nirujogi ◽  
...  
Keyword(s):  
Primary Cilia ◽  
Protein Transport ◽  
Serum Starvation ◽  
Rab Gtpase ◽  
Rab Gtpases ◽  
Rab Protein ◽  
Affinity Enrichment ◽  
Fold Reduction ◽  
Cilia Formation ◽  
Conserved Residue

We previously reported that Parkinson’s disease (PD) kinase LRRK2 phosphorylates a subset of Rab GTPases on a conserved residue in their switch-II domains (Steger et al., 2016) (PMID: 26824392). Here, we systematically analyzed the Rab protein family and found 14 of them (Rab3A/B/C/D, Rab5A/B/C, Rab8A/B, Rab10, Rab12, Rab29, Rab35 and Rab43) to be specifically phosphorylated by LRRK2, with evidence for endogenous phosphorylation for ten of them (Rab3A/B/C/D, Rab8A/B, Rab10, Rab12, Rab35 and Rab43). Affinity enrichment mass spectrometry revealed that the primary ciliogenesis regulator, RILPL1 specifically interacts with the LRRK2-phosphorylated forms of Rab8A and Rab10, whereas RILPL2 binds to phosphorylated Rab8A, Rab10, and Rab12. Induction of primary cilia formation by serum starvation led to a two-fold reduction in ciliogenesis in fibroblasts derived from pathogenic LRRK2-R1441G knock-in mice. These results implicate LRRK2 in primary ciliogenesis and suggest that Rab-mediated protein transport and/or signaling defects at cilia may contribute to LRRK2-dependent pathologies.

scholarly journals Functional dissection of Rab GTPases involved in primary cilium formation

2007 ◽  
Vol 178 (3) ◽  
pp. 363-369 ◽  
Author(s):  
Shin-ichiro Yoshimura ◽  
Johannes Egerer ◽  
Evelyn Fuchs ◽  
Alexander K. Haas ◽  
Francis A. Barr
Keyword(s):  
Membrane Trafficking ◽  
Primary Cilia ◽  
Rab Gtpase ◽  
Rab Gtpases ◽  
Microtubule Cytoskeleton ◽  
Gtpase Activating Proteins ◽  
Cilia Formation ◽  
Sensory Structures ◽  
And Function ◽  
Specific Membrane

Primary cilia are sensory structures involved in morphogen signalling during development, liquid flow in the kidney, mechanosensation, sight, and smell (Badano, J.L., N. Mitsuma, P.L. Beales, and N. Katsanis. 2006. Annu. Rev. Genomics Hum. Genet. 7:125–148; Singla, V., and J.F. Reiter. 2006. Science. 313:629–633.). Mutations that affect primary cilia are responsible for several diseases, including neural tube defects, polycystic kidney disease, retinal degeneration, and cancers (Badano et al., 2006; Singla and Reiter, 2006). Primary cilia formation and function requires tight integration of the microtubule cytoskeleton with membrane trafficking (Singla and Reiter, 2006), and this is poorly understood. We show that the Rab GTPase membrane trafficking regulators Rab8a, -17, and -23, and their cognate GTPase-activating proteins (GAPs), XM_037557, TBC1D7, and EVI5like, are involved in primary cilia formation. However, other human Rabs and GAPs are not. Additionally, Rab8a specifically interacts with cenexin/ODF2, a basal body and microtubule binding protein required for cilium biogenesis (Ishikawa, H., A. Kubo, S. Tsukita, and S. Tsukita. 2005. Nat. Cell Biol. 7:517–524), and is the sole Rab enriched at primary cilia. These findings provide a basis for understanding how specific membrane trafficking pathways cooperate with the microtubule cytoskeleton to give rise to the primary cilia.

LRRK2 and Rab GTPases

2018 ◽  
Vol 46 (6) ◽  
pp. 1707-1712 ◽  
Author(s):  
Suzanne R. Pfeffer
Keyword(s):  
Membrane Trafficking ◽  
Protein Function ◽  
Short Review ◽  
Rab Gtpase ◽  
Rab Gtpases ◽  
Rab Protein ◽  
Complex Interactions ◽  
Preferential Binding ◽  
Bona Fide ◽  
Mutant Lrrk2

Leucine-rich repeat kinase 2 (LRRK2) is mutated in familial Parkinson's disease, and pathogenic mutations activate the kinase activity. A tour de force screen by Mann and Alessi and co-workers identified a subset of Rab GTPases as bona fide LRRK2 substrates. Rab GTPases are master regulators of membrane trafficking and this short review will summarize what we know about the connection between LRRK2 and this family of regulatory proteins. While, in most cases, Rab GTPase phosphorylation is predicted to interfere with Rab protein function, the discovery of proteins that show preferential binding to phosphorylated Rabs suggests that more complex interactions may also contribute to mutant LRRK2-mediated pathology.

scholarly journals Induction of Ran GTP drives ciliogenesis

2011 ◽  
Vol 22 (23) ◽  
pp. 4539-4548 ◽  
Author(s):  
Shuling Fan ◽  
Eileen L. Whiteman ◽  
Toby W. Hurd ◽  
Jeremy C. McIntyre ◽  
John F. Dishinger ◽  
...  
Keyword(s):  
Primary Cilia ◽  
Protein Transport ◽  
Nucleocytoplasmic Transport ◽  
Small Gtpase ◽  
Basal Bodies ◽  
Ciliary Protein ◽  
Cilia Formation ◽  
Ran Gtp ◽  
New Evidence ◽  
Gtpase Ran

The small GTPase Ran and the importin proteins regulate nucleocytoplasmic transport. New evidence suggests that Ran GTP and the importins are also involved in conveying proteins into cilia. In this study, we find that Ran GTP accumulation at the basal bodies is coordinated with the initiation of ciliogenesis. The Ran-binding protein 1 (RanBP1), which indirectly accelerates Ran GTP → Ran GDP hydrolysis and promotes the dissociation of the Ran/importin complex, also localizes to basal bodies and cilia. To confirm the crucial link between Ran GTP and ciliogenesis, we manipulated the levels of RanBP1 and determined the effects on Ran GTP and primary cilia formation. We discovered that RanBP1 knockdown results in an increased concentration of Ran GTP at basal bodies, leading to ciliogenesis. In contrast, overexpression of RanBP1 antagonizes primary cilia formation. Furthermore, we demonstrate that RanBP1 knockdown disrupts the proper localization of KIF17, a kinesin-2 motor, at the distal tips of primary cilia in Madin–Darby canine kidney cells. Our studies illuminate a new function for Ran GTP in stimulating cilia formation and reinforce the notion that Ran GTP and the importins play key roles in ciliogenesis and ciliary protein transport.

scholarly journals PPM1H phosphatase counteracts LRRK2 signaling by selectively dephosphorylating Rab proteins

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Kerryn Berndsen ◽  
Pawel Lis ◽  
Wondwossen M Yeshaw ◽  
Paulina S Wawro ◽  
Raja S Nirujogi ◽  
...  
Keyword(s):  
Parkinson’S Disease ◽  
Parkinson's Disease ◽  
Protein Phosphatase ◽  
Primary Cilia ◽  
A549 Cells ◽  
Rab Proteins ◽  
Rab Gtpases ◽  
Biochemical Studies ◽  
Cilia Formation ◽  
Human A549

Mutations that activate LRRK2 protein kinase cause Parkinson’s disease. LRRK2 phosphorylates a subset of Rab GTPases within their Switch-II motif controlling interaction with effectors. An siRNA screen of all human protein phosphatases revealed that a poorly studied protein phosphatase, PPM1H, counteracts LRRK2 signaling by specifically dephosphorylating Rab proteins. PPM1H knockout increased endogenous Rab phosphorylation and inhibited Rab dephosphorylation in human A549 cells. Overexpression of PPM1H suppressed LRRK2-mediated Rab phosphorylation. PPM1H also efficiently and directly dephosphorylated Rab8A in biochemical studies. A “substrate-trapping” PPM1H mutant (Asp288Ala) binds with high affinity to endogenous, LRRK2-phosphorylated Rab proteins, thereby blocking dephosphorylation seen upon addition of LRRK2 inhibitors. PPM1H is localized to the Golgi and its knockdown suppresses primary cilia formation, similar to pathogenic LRRK2. Thus, PPM1H acts as a key modulator of LRRK2 signaling by controlling dephosphorylation of Rab proteins. PPM1H activity enhancers could offer a new therapeutic approach to prevent or treat Parkinson’s disease.

scholarly journals Ndel1 suppresses ciliogenesis in proliferating cells by regulating the trichoplein–Aurora A pathway

2016 ◽  
Vol 212 (4) ◽  
pp. 409-423 ◽  
Author(s):  
Hironori Inaba ◽  
Hidemasa Goto ◽  
Kousuke Kasahara ◽  
Kanako Kumamoto ◽  
Shigenobu Yonemura ◽  
...  
Keyword(s):  
Primary Cilia ◽  
Serum Starvation ◽  
Aurora A ◽  
Proliferating Cells ◽  
Quiescent Cells ◽  
Cell Cycle Reentry ◽  
Upstream Regulator ◽  
Cilia Formation ◽  
Mother Centriole ◽  
Component Protein

Primary cilia protrude from the surface of quiescent cells and disassemble at cell cycle reentry. We previously showed that ciliary reassembly is suppressed by trichoplein-mediated Aurora A activation pathway in growing cells. Here, we report that Ndel1, a well-known modulator of dynein activity, localizes at the subdistal appendage of the mother centriole, which nucleates a primary cilium. In the presence of serum, Ndel1 depletion reduces trichoplein at the mother centriole and induces unscheduled primary cilia formation, which is reverted by forced trichoplein expression or coknockdown of KCTD17 (an E3 ligase component protein for trichoplein). Serum starvation induced transient Ndel1 degradation, subsequent to the disappearance of trichoplein at the mother centriole. Forced expression of Ndel1 suppressed trichoplein degradation and axonemal microtubule extension during ciliogenesis, similar to trichoplein induction or KCTD17 knockdown. Most importantly, the proportion of ciliated and quiescent cells was increased in the kidney tubular epithelia of newborn Ndel1-hypomorphic mice. Thus, Ndel1 acts as a novel upstream regulator of the trichoplein–Aurora A pathway to inhibit primary cilia assembly.

scholarly journals Pathogenic LRRK2 control of primary cilia and Hedgehog signaling in neurons and astrocytes of mouse brain

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Shahzad S Khan ◽  
Yuriko Sobu ◽  
Herschel S Dhekne ◽  
Francesca Tonelli ◽  
Kerryn Berndsen ◽  
...  
Keyword(s):  
Hedgehog Signaling ◽  
Primary Cilia ◽  
Dorsal Striatum ◽  
Mutant Mice ◽  
Mouse Strains ◽  
Rab Gtpase ◽  
Rab Gtpases ◽  
Nigrostriatal Pathway ◽  
Cholinergic Interneurons ◽  
Lrrk2 Kinase

Activating LRRK2 mutations cause Parkinson's disease, and pathogenic LRRK2 kinase interferes with ciliogenesis. Previously, we showed that cholinergic interneurons of the dorsal striatum lose their cilia in R1441C LRRK2 mutant mice (Dhekne et al., 2018). Here, we show that cilia loss is seen as early as 10 weeks of age in these mice and also in two other mouse strains carrying the most common human G2019S LRRK2 mutation. Loss of the PPM1H phosphatase that is specific for LRRK2-phosphorylated Rab GTPases yields the same cilia loss phenotype seen in mice expressing pathogenic LRRK2 kinase, strongly supporting a connection between Rab GTPase phosphorylation and cilia loss. Moreover, astrocytes throughout the striatum show a ciliation defect in all LRRK2 and PPM1H mutant models examined. Hedgehog signaling requires cilia, and loss of cilia in LRRK2 mutant rodents correlates with dysregulation of Hedgehog signaling as monitored by in situ hybridization of Gli1 and Gdnf transcripts. Dopaminergic neurons of the substantia nigra secrete a Hedgehog signal that is sensed in the striatum to trigger neuroprotection; our data support a model in which LRRK2 and PPM1H mutant mice show altered responses to critical Hedgehog signals in the nigrostriatal pathway.

scholarly journals Evidence for Enhanced Exosome Production in Aromatase Inhibitor-Resistant Breast Cancer Cells

2020 ◽  
Vol 21 (16) ◽  
pp. 5841
Author(s):  
Giuseppina Augimeri ◽  
Giusi La Camera ◽  
Luca Gelsomino ◽  
Cinzia Giordano ◽  
Salvatore Panza ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cancer Cells ◽  
Cancer Progression ◽  
Breast Cancer Cells ◽  
Protein Transport ◽  
Small Gtpase ◽  
Rab Gtpase ◽  
Rab Gtpases ◽  
Breast Cancers ◽  
Mcf 7

Aromatase inhibitors (AIs) represent the standard anti-hormonal therapy for post-menopausal estrogen receptor-positive breast cancer, but their efficacy is limited by the emergence of AI resistance (AIR). Exosomes act as vehicles to engender cancer progression and drug resistance. The goal of this work was to study exosome contribution in AIR mechanisms, using estrogen-dependent MCF-7 breast cancer cells as models and MCF-7 LTED (Long-Term Estrogen Deprived) subline, modeling AIR. We found that exosome secretion was significantly increased in MCF-7 LTED cells compared to MCF-7 cells. MCF-7 LTED cells also exhibited a higher amount of exosomal RNA and proteins than MCF-7 cells. Proteomic analysis revealed significant alterations in the cellular proteome. Indeed, we showed an enrichment of proteins frequently identified in exosomes in MCF-7 LTED cells. The most up-regulated proteins in MCF-7 LTED cells were represented by Rab GTPases, important vesicle transport-regulators in cancer, that are significantly mapped in “small GTPase-mediated signal transduction”, “protein transport” and “vesicle-mediated transport” Gene Ontology categories. Expression of selected Rab GTPases was validated by immunoblotting. Collectively, we evidence, for the first time, that AIR breast cancer cells display an increased capability to release exosomes, which may be associated with an enhanced Rab GTPase expression. These data provide the rationale for further studies directed at clarifying exosome’s role on endocrine therapy, with the aim to offer relevant markers and druggable therapeutic targets for the management of hormone-resistant breast cancers.

scholarly journals PPM1H phosphatase counteracts LRRK2 signaling by selectively dephosphorylating Rab proteins

2019 ◽  
Author(s):  
Kerryn Berndsen ◽  
Pawel Lis ◽  
Wondwossen Yeshaw ◽  
Paulina S. Wawro ◽  
Raja S. Nirujogi ◽  
...  
Keyword(s):  
Parkinson’S Disease ◽  
Parkinson's Disease ◽  
Protein Phosphatase ◽  
Primary Cilia ◽  
Rab Proteins ◽  
Rab Gtpases ◽  
Knock Out ◽  
High Affinity ◽  
Biochemical Studies ◽  
Cilia Formation

AbstractMutations that activate LRRK2 protein kinase cause Parkinson’s disease. LRRK2 phosphorylates a subset of Rab GTPases within their Switch-II motif controlling interaction with effectors. An siRNA screen of all protein phosphatases revealed that a poorly studied protein phosphatase, PPM1H, counteracts LRRK2 signaling by specifically dephosphorylating Rab proteins. PPM1H knock out increased endogenous Rab phosphorylation and inhibited Rab dephosphorylation. Overexpression of PPM1H suppressed LRRK2-mediated Rab phosphorylation. PPM1H also efficiently and directly dephosphorylated Rab8A in biochemical studies. A “substrate-trapping” PPM1H mutant (Asp288Ala) binds with high affinity to endogenous, LRRK2-phosphorylated Rab proteins, thereby blocking dephosphorylation seen upon addition of LRRK2 inhibitors. PPM1H is localized to the Golgi and its knockdown suppresses primary cilia formation, similar to pathogenic LRRK2. Thus, PPM1H acts as a key modulator of LRRK2 signaling by controlling dephosphorylation of Rab proteins. PPM1H activity enhancers could offer a new therapeutic approach to prevent or treat Parkinson’s disease.

scholarly journals Pathogenic LRRK2 control of primary cilia and Hedgehog signaling in neurons and astrocytes of mouse brain

2021 ◽  
Author(s):  
Shahzad S. Khan ◽  
Yuriko Sobu ◽  
Herschel S. Dhekne ◽  
Francesca Tonelli ◽  
Kerryn Berndsen ◽  
...  
Keyword(s):  
Hedgehog Signaling ◽  
Primary Cilia ◽  
Dorsal Striatum ◽  
Mouse Strains ◽  
Rab Gtpase ◽  
Rab Gtpases ◽  
Lrrk2 Mutation ◽  
Cholinergic Interneurons ◽  
To Receive

AbstractPreviously, we showed that cholinergic interneurons of the dorsal striatum lose cilia in mice harboring the Parkinson’s disease associated, kinase activating, R1441C LRRK2 mutation (Dhekne et al., 2018). Here we show that this phenotype is also seen in two mouse strains carrying the most common human G2019S LRRK2 mutation. Heterozygous loss of the PPM1H phosphatase that is specific for LRRK2-phosphorylated Rab GTPases (Berndsen et al., 2019) yields the same cilia loss phenotype, strongly supporting a connection between Rab GTPase phosphorylation and cilia loss. In addition, astrocytes throughout the striatum show a ciliation defect in LRRK2 and PPM1H-/+ mutant models. Hedgehog signaling requires cilia, and loss of cilia correlates here with a loss in induction of Hedgehog signaling as monitored by in situ hybridization of Gli1 transcripts. These data support a model in which LRRK2 and PPM1H mutant mice struggle to receive and respond to critical Hedgehog signals in the nigral-striatal pathway.

scholarly journals A comprehensive analysis of Rab GTPases reveals a role for Rab34 in serum starvation-induced primary ciliogenesis

2020 ◽  
Vol 295 (36) ◽  
pp. 12674-12685 ◽  
Author(s):  
Mai E. Oguchi ◽  
Koki Okuyama ◽  
Yuta Homma ◽  
Mitsunori Fukuda
Keyword(s):  
Membrane Trafficking ◽  
Primary Cilia ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Small Gtpases ◽  
Terminal Region ◽  
Serum Starvation ◽  
Rab Gtpases ◽  
Human Telomerase Reverse Transcriptase ◽  
Specific Sequence

Primary cilia are sensors of chemical and mechanical signals in the extracellular environment. The formation of primary cilia (i.e. ciliogenesis) requires dynamic membrane trafficking events, and several Rab small GTPases, key regulators of membrane trafficking, have recently been reported to participate in ciliogenesis. However, the precise mechanisms of Rab-mediated membrane trafficking during ciliogenesis remain largely unknown. In the present study, we used a collection of siRNAs against 62 human Rabs to perform a comprehensive knockdown screening for Rabs that regulate serum starvation-induced ciliogenesis in human telomerase reverse transcriptase retinal pigment epithelium 1 (hTERT-RPE1) cells and succeeded in identifying Rab34 as an essential Rab. Knockout (KO) of Rab34, but not of Rabs previously reported to regulate ciliogenesis (e.g. Rab8 and Rab10) in hTERT-RPE1 cells, drastically impaired serum starvation-induced ciliogenesis. Rab34 was also required for serum starvation-induced ciliogenesis in NIH/3T3 cells and MCF10A cells but not for ciliogenesis in Madin-Darby canine kidney (MDCK)-II cysts. We then attempted to identify a specific region(s) of Rab34 that is essential for ciliogenesis by performing deletion and mutation analyses of Rab34. Unexpectedly, instead of a specific sequence in the switch II region, which is generally important for recognizing effector proteins (e.g. Rab interacting lysosomal protein [RILP]), a unique long N-terminal region of Rab34 before the conserved GTPase domain was found to be essential. These findings suggest that Rab34 is an atypical Rab that regulates serum starvation-induced ciliogenesis through its unique N-terminal region.

Sign in / Sign up

Export Citation Format

Share Document