The Experts below are selected from a list of 7557 Experts worldwide ranked by ideXlab platform
Kunihiro Matsumoto - One of the best experts on this subject based on the ideXlab platform.
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LRRK1 regulates spindle orientation by phosphorylating cdk5rap2
Cell Cycle, 2015Co-Authors: Hiroshi Hanafusa, Kunihiro MatsumotoAbstract:Precise orientation of the mitotic spindle determines the correct cell division axis and is essential for tissue development and homeostasis. It is known that spindle misorientation underlies some mammalian diseases, such as tumourigenesis and polycystic kidney disease. Two kinases, Polo-like kinase 1 (PLK1) and leucine-rich repeat kinase 1 (LRRK1), were recently identified as candidate kinases required for spindle orientation by an RNAi-based screen.1 PLK1 is a mitotic kinase that regulates diverse mitotic events, including centrosome maturation. However, its precise role in spindle orientation has not been well understood. LRRK1 is related to the familial Parkinsonism gene product Park8/LRRK2, and contains a Ras of complex proteins (ROC) GTPase domain and a MAPKKK-like kinase domain. We have previously reported that LRRK1 participates in the intercellular trafficking of the epidermal growth factor (EGF) receptor.2 However, the role of LRRK1 in mitosis has remained unknown.
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LRRK1 regulates spindle orientation by phosphorylating cdk5rap2
Cell Cycle, 2015Co-Authors: Hiroshi Hanafusa, Kunihiro MatsumotoAbstract:Precise orientation of the mitotic spindle determines the correct cell division axis and is essential for tissue development and homeostasis. It is known that spindle misorientation underlies some mammalian diseases, such as tumourigenesis and polycystic kidney disease. Two kinases, Polo-like kinase 1 (PLK1) and leucine-rich repeat kinase 1 (LRRK1), were recently identified as candidate kinases required for spindle orientation by an RNAi-based screen.1 PLK1 is a mitotic kinase that regulates diverse mitotic events, including centrosome maturation. However, its precise role in spindle orientation has not been well understood. LRRK1 is related to the familial Parkinsonism gene product Park8/LRRK2, and contains a Ras of complex proteins (ROC) GTPase domain and a MAPKKK-like kinase domain. We have previously reported that LRRK1 participates in the intercellular trafficking of the epidermal growth factor (EGF) receptor.2 However, the role of LRRK1 in mitosis has remained unknown.
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plk1 dependent activation of LRRK1 regulates spindle orientation by phosphorylating cdk5rap2
Nature Cell Biology, 2015Co-Authors: Hiroshi Hanafusa, Shin Kedashiro, Motohiro Tezuka, Motoki Funatsu, Fumiko Toyoshima, Satoshi Usami, Kunihiro MatsumotoAbstract:Hanafusa and Matsumato and colleagues find that LRRK1 is a substrate for PLK1 in mitosis. Phosphorylated LRRK1 in turn phosphorylates CDK5RAP2 to promote centrosomal microtubule nucleation and correct spindle orientation.
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PLK1-dependent activation of LRRK1 regulates spindle orientation by phosphorylating CDK5RAP2
Nature Cell Biology, 2015Co-Authors: Hiroshi Hanafusa, Shin Kedashiro, Motohiro Tezuka, Motoki Funatsu, Fumiko Toyoshima, Satoshi Usami, Kunihiro MatsumotoAbstract:Hanafusa and Matsumato and colleagues find that LRRK1 is a substrate for PLK1 in mitosis. Phosphorylated LRRK1 in turn phosphorylates CDK5RAP2 to promote centrosomal microtubule nucleation and correct spindle orientation. Correct formation of the cell division axis requires the initial precise orientation of the mitotic spindle. Proper spindle orientation depends on centrosome maturation, and Polo-like kinase 1 (PLK1) is known to play a crucial role in this process. However, the molecular mechanisms that function downstream of PLK1 are not well understood. Here we show that LRRK1 is a PLK1 substrate that is phosphorylated on Ser 1790. PLK1 phosphorylation is required for CDK1-mediated activation of LRRK1 at the centrosomes, and this in turn regulates mitotic spindle orientation by nucleating the growth of astral microtubules from the centrosomes. Interestingly, LRRK1 in turn phosphorylates CDK5RAP2(Cep215), a human homologue of Drosophila Centrosomin (Cnn), in its γ-tubulin-binding motif, thus promoting the interaction of CDK5RAP2 with γ-tubulin. LRRK1 phosphorylation of CDK5RAP2 Ser 140 is necessary for CDK5RAP2-dependent microtubule nucleation. Thus, our findings provide evidence that LRRK1 regulates mitotic spindle orientation downstream of PLK1 through CDK5RAP2-dependent centrosome maturation.
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Phosphorylation of CLIP-170 by LRRK1 regulates EGFR trafficking by promoting recruitment of p150Glued to MT plus-ends
Journal of cell science, 2015Co-Authors: Shin Kedashiro, Kunihiro Matsumoto, Strahil Iv. Pastuhov, Tomoki Nishioka, Takashi Watanabe, Kozo Kaibuchi, Hiroshi HanafusaAbstract:Ligand-induced activation of the EGF receptor (EGFR) initiates trafficking events that relocalize the receptors from the cell surface to intracellular endocytic compartments. Early endosomes containing activated EGFR migrate along microtubules as they mature into late endosomes. We have recently shown that LRRK1, which is related to the familial Parkinsonism gene product Park8/LRRK2, regulates this EGFR transport in a manner dependent on LRRK1 kinase activity. However, the downstream targets of LRRK1 that may modulate this transport function have not been identified. Here, we identify CLIP-170, a microtubule plus-end protein, as a substrate of LRRK1. LRRK1 phosphorylates CLIP-170 at Thr-1384, located in its C-terminal zinc knuckle motif, and this promotes its association with dynactin–dynein complexes. We find that LRRK1 phosphorylation of CLIP-170 causes the accumulation of p150 Glued , a subunit of dynactin, at microtubule plus-ends, thereby facilitating the migration of EGFR-containing endosomes. Thus, our findings provide new mechanistic insights into the dynein-driven transport of EGFR.
Hiroshi Hanafusa - One of the best experts on this subject based on the ideXlab platform.
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LRRK1 regulates spindle orientation by phosphorylating cdk5rap2
Cell Cycle, 2015Co-Authors: Hiroshi Hanafusa, Kunihiro MatsumotoAbstract:Precise orientation of the mitotic spindle determines the correct cell division axis and is essential for tissue development and homeostasis. It is known that spindle misorientation underlies some mammalian diseases, such as tumourigenesis and polycystic kidney disease. Two kinases, Polo-like kinase 1 (PLK1) and leucine-rich repeat kinase 1 (LRRK1), were recently identified as candidate kinases required for spindle orientation by an RNAi-based screen.1 PLK1 is a mitotic kinase that regulates diverse mitotic events, including centrosome maturation. However, its precise role in spindle orientation has not been well understood. LRRK1 is related to the familial Parkinsonism gene product Park8/LRRK2, and contains a Ras of complex proteins (ROC) GTPase domain and a MAPKKK-like kinase domain. We have previously reported that LRRK1 participates in the intercellular trafficking of the epidermal growth factor (EGF) receptor.2 However, the role of LRRK1 in mitosis has remained unknown.
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LRRK1 regulates spindle orientation by phosphorylating cdk5rap2
Cell Cycle, 2015Co-Authors: Hiroshi Hanafusa, Kunihiro MatsumotoAbstract:Precise orientation of the mitotic spindle determines the correct cell division axis and is essential for tissue development and homeostasis. It is known that spindle misorientation underlies some mammalian diseases, such as tumourigenesis and polycystic kidney disease. Two kinases, Polo-like kinase 1 (PLK1) and leucine-rich repeat kinase 1 (LRRK1), were recently identified as candidate kinases required for spindle orientation by an RNAi-based screen.1 PLK1 is a mitotic kinase that regulates diverse mitotic events, including centrosome maturation. However, its precise role in spindle orientation has not been well understood. LRRK1 is related to the familial Parkinsonism gene product Park8/LRRK2, and contains a Ras of complex proteins (ROC) GTPase domain and a MAPKKK-like kinase domain. We have previously reported that LRRK1 participates in the intercellular trafficking of the epidermal growth factor (EGF) receptor.2 However, the role of LRRK1 in mitosis has remained unknown.
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plk1 dependent activation of LRRK1 regulates spindle orientation by phosphorylating cdk5rap2
Nature Cell Biology, 2015Co-Authors: Hiroshi Hanafusa, Shin Kedashiro, Motohiro Tezuka, Motoki Funatsu, Fumiko Toyoshima, Satoshi Usami, Kunihiro MatsumotoAbstract:Hanafusa and Matsumato and colleagues find that LRRK1 is a substrate for PLK1 in mitosis. Phosphorylated LRRK1 in turn phosphorylates CDK5RAP2 to promote centrosomal microtubule nucleation and correct spindle orientation.
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PLK1-dependent activation of LRRK1 regulates spindle orientation by phosphorylating CDK5RAP2
Nature Cell Biology, 2015Co-Authors: Hiroshi Hanafusa, Shin Kedashiro, Motohiro Tezuka, Motoki Funatsu, Fumiko Toyoshima, Satoshi Usami, Kunihiro MatsumotoAbstract:Hanafusa and Matsumato and colleagues find that LRRK1 is a substrate for PLK1 in mitosis. Phosphorylated LRRK1 in turn phosphorylates CDK5RAP2 to promote centrosomal microtubule nucleation and correct spindle orientation. Correct formation of the cell division axis requires the initial precise orientation of the mitotic spindle. Proper spindle orientation depends on centrosome maturation, and Polo-like kinase 1 (PLK1) is known to play a crucial role in this process. However, the molecular mechanisms that function downstream of PLK1 are not well understood. Here we show that LRRK1 is a PLK1 substrate that is phosphorylated on Ser 1790. PLK1 phosphorylation is required for CDK1-mediated activation of LRRK1 at the centrosomes, and this in turn regulates mitotic spindle orientation by nucleating the growth of astral microtubules from the centrosomes. Interestingly, LRRK1 in turn phosphorylates CDK5RAP2(Cep215), a human homologue of Drosophila Centrosomin (Cnn), in its γ-tubulin-binding motif, thus promoting the interaction of CDK5RAP2 with γ-tubulin. LRRK1 phosphorylation of CDK5RAP2 Ser 140 is necessary for CDK5RAP2-dependent microtubule nucleation. Thus, our findings provide evidence that LRRK1 regulates mitotic spindle orientation downstream of PLK1 through CDK5RAP2-dependent centrosome maturation.
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Phosphorylation of CLIP-170 by LRRK1 regulates EGFR trafficking by promoting recruitment of p150Glued to MT plus-ends
Journal of cell science, 2015Co-Authors: Shin Kedashiro, Kunihiro Matsumoto, Strahil Iv. Pastuhov, Tomoki Nishioka, Takashi Watanabe, Kozo Kaibuchi, Hiroshi HanafusaAbstract:Ligand-induced activation of the EGF receptor (EGFR) initiates trafficking events that relocalize the receptors from the cell surface to intracellular endocytic compartments. Early endosomes containing activated EGFR migrate along microtubules as they mature into late endosomes. We have recently shown that LRRK1, which is related to the familial Parkinsonism gene product Park8/LRRK2, regulates this EGFR transport in a manner dependent on LRRK1 kinase activity. However, the downstream targets of LRRK1 that may modulate this transport function have not been identified. Here, we identify CLIP-170, a microtubule plus-end protein, as a substrate of LRRK1. LRRK1 phosphorylates CLIP-170 at Thr-1384, located in its C-terminal zinc knuckle motif, and this promotes its association with dynactin–dynein complexes. We find that LRRK1 phosphorylation of CLIP-170 causes the accumulation of p150 Glued , a subunit of dynactin, at microtubule plus-ends, thereby facilitating the migration of EGFR-containing endosomes. Thus, our findings provide new mechanistic insights into the dynein-driven transport of EGFR.
Andrew B West - One of the best experts on this subject based on the ideXlab platform.
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lrrk2 phosphorylates membrane bound rabs and is activated by gtp bound rab7l1 to promote recruitment to the trans golgi network
Human Molecular Genetics, 2018Co-Authors: Nicole Bryant, Ravindran Kumaran, Asa Abeliovich, Alexandra Beilina, Mark R. Cookson, Andrew B WestAbstract:Human genetic studies implicate LRRK2 and RAB7L1 in susceptibility to Parkinson disease (PD). These two genes function in the same pathway, as knockout of Rab7L1 results in phenotypes similar to LRRK2 knockout, and studies in cells and model organisms demonstrate LRRK2 and Rab7L1 interact in the endolysosomal system. Recently, a subset of Rab proteins have been identified as LRRK2 kinase substrates. Herein, we find that Rab8, Rab10, and Rab7L1 must be membrane and GTP-bound for LRRK2 phosphorylation. LRRK2 mutations that cause PD including R1441C, Y1699C, and G2019S all increase LRRK2 phosphorylation of Rab7L1 four-fold over wild-type LRRK2 in cells, resulting in the phosphorylation of nearly one-third the available Rab7L1 protein in cells. In contrast, the most common pathogenic LRRK2 mutation, G2019S, does not upregulate LRRK2-mediated phosphorylation of Rab8 or Rab10. LRRK2 interaction with membrane and GTP-bound Rab7L1, but not Rab8 or Rab10, results in the activation of LRRK2 autophosphorylation at the serine 1292 position, required for LRRK2 toxicity. Further, Rab7L1 controls the proportion of LRRK2 that is membrane-associated, and LRRK2 mutations enhance Rab7L1-mediated recruitment of LRRK2 to the trans-Golgi network. Interaction studies with the Rab8 and Rab10 GTPase-activating protein TBC1D4/AS160 demonstrate that LRRK2 phosphorylation may block membrane and GTP-bound Rab protein interaction with effectors. These results suggest reciprocal regulation between LRRK2 and Rab protein substrates, where Rab7L1-mediated upregulation of LRRK2 kinase activity results in the stabilization of membrane and GTP-bound Rab proteins that may be unable to interact with Rab effector proteins.
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Elevated LRRK2 autophosphorylation in brain-derived and peripheral exosomes in LRRK2 mutation carriers.
Acta neuropathologica communications, 2017Co-Authors: Shijie Wang, Omar S. Mabrouk, Tyler Maltbie, Jan O. Aasly, Tao Ye, Andrew B WestAbstract:Missense mutations in the leucine-rich repeat kinase 2 (LRRK2) gene can cause late-onset Parkinson disease (PD). LRRK2 mutations increase LRRK2 kinase activities that may increase levels of LRRK2 autophosphorylation at serine 1292 (pS1292) and neurotoxicity in model systems. pS1292-LRRK2 protein can be packaged into exosomes and measured in biobanked urine. Herein we provide evidence that pS1292-LRRK2 protein is robustly expressed in cerebral spinal fluid (CSF) exosomes. In a novel cohort of Norwegian subjects with and without the G2019S-LRRK2 mutation, with and without PD, we quantified levels of pS1292-LRRK2, total LRRK2, and other exosome proteins in urine from 132 subjects and in CSF from 82 subjects. CSF and urine were collected from the same morning clinic visit in 55 of the participants. We found that total LRRK2 protein concentration was similar in exosomes purified from either CSF or urine but the levels did not correlate. pS1292-LRRK2 levels were higher in urinary exosomes from male and female subjects with a LRRK2 mutation. Male LRRK2 mutation carriers without PD had intermediate pS1292-LRRK2 levels compared to male carriers with PD and controls. However, female LRRK2 mutation carriers without PD had the same pS1292-LRRK2 levels compared to female carriers with PD. pS1292-LRRK2 levels in CSF exosomes were near saturated in most subjects, ten-fold higher on average than pS1292-LRRK2 levels in urinary exosomes, irrespective of LRRK2 mutation status or PD diagnosis. These results provide insights into the effects of LRRK2 mutations in both the periphery and brain in a well-characterized clinical population and show that LRRK2 protein in brain exosomes may be much more active than in the periphery in most subjects.
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Urinary LRRK2 phosphorylation predicts parkinsonian phenotypes in G2019S LRRK2 carriers
Neurology, 2016Co-Authors: Kyle B. Fraser, Mark S. Moehle, Roy N. Alcalay, Andrew B WestAbstract:Objective: To test whether phosphorylated Ser-1292 LRRK2 levels in urine exosomes predicts LRRK2 mutation carriers ( LRRK2 +) and noncarriers ( LRRK2 −) with Parkinson disease (PD+) and without Parkinson disease (PD−). Methods: LRRK2 protein was purified from urinary exosomes collected from participants in 2 independent cohorts. The first cohort included 14 men ( LRRK2 +/PD+, n = 7; LRRK2− /PD+, n = 4; LRRK2− /PD−, n = 3). The second cohort included 62 men ( LRRK2− /PD−, n = 16; LRRK2+ /PD−, n = 16; LRRK2 +/PD+, n = 14; LRRK2− /PD+, n = 16). The ratio of Ser(P)-1292 LRRK2 to total LRRK2 was compared between LRRK2 +/PD+ and LRRK2− in the first cohort and between LRRK2 G2019S carriers with and without PD in the second cohort. Results: LRRK2 +/PD+ had higher ratios of Ser(P)-1292 LRRK2 to total LRRK2 than LRRK2− /PD− (4.8-fold, p LRRK2− /PD+ (4.6-fold, p p Conclusion: Elevated ratio of phosphorylated Ser-1292 LRRK2 to total LRRK2 in urine exosomes predicted LRRK2 mutation status and PD risk among LRRK2 mutation carriers. Future studies may explore whether interventions that reduce this ratio may also reduce PD risk.
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Phosphorylated pS1292 LRRK2 to Total LRRK2 Concentration Ratio in Urine Exosomes Distinguishes LRRK2-PD and Idiopathic PD (S7.002)
Neurology, 2015Co-Authors: Roy N. Alcalay, Kyle B. Fraser, Andrew B WestAbstract:Objective: To measure total LRRK2 and phosphorylated pS1292-LRRK2 levels in urine exosomes of LRRK2 G2019S carriers with PD (LRRK2+/PD+), and non-carriers with (LRRK2-/PD+), and without PD (LRRK2-/PD-). Background: LRRK2 G2019S mutations may increase Parkinson’s disease (PD) risk through a gain of kinase function including autophosphorylation at the serine-1292 residue near the LRRK2 GTPase domain. Phosphorylated pS1292-LRRK2 may reflect LRRK2 kinase activity. Methods: 14 Male participants included LRRK2+/PD+ (n=7), LRRK2-/PD+ (n=4), and LRRK2-/PD- (n=3). Fresh urine was collected and frozen in a -80oC freezer. Urinary exosomes were purified from ~40 ml samples. Monoclonal anti-LRRK2/Dardarin clone acquired from NeuroMab were used to measure total LRRK2, and novel polyclonal antibodies generated in rabbits against pS1292-LRRK2 were used to quantify total-LRRK2 and pS1292-LRRK2 in Western blots. Total LRRK2, pS1292-LRRK2 and the ratio between total LRRK2 and pS1292-LRRK2 were compared between G2019S carriers with PD and non-carriers with and without PD. Results: All three groups were similar in age (mean=67.0; SD=7.5). The ratio of pS1292-LRRK2 to total LRRK2 was significantly different between LRRK2+/PD+ (mean=0.90; SD=0.28; range 0.60-1.46) and both LRRK2-/PD+ (mean=0.20; SD=0.03; range 0.16-0.24, p
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lrrk2 inhibition attenuates microglial inflammatory responses
The Journal of Neuroscience, 2012Co-Authors: Mark S. Moehle, Philip J Webber, Nour Sukar, Tara M Desilva, David G. Standaert, Rita M. Cowell, Andrew B WestAbstract:Missense mutations in leucine-rich repeat kinase 2 (LRRK2) cause late-onset Parkinson9s disease (PD), and common genetic variation in LRRK2 modifies susceptibility to Crohn9s disease and leprosy. High levels of LRRK2 expression in peripheral monocytes and macrophages suggest a role for LRRK2 in these cells, yet little is known about LRRK2 expression and function in immune cells of the brain. Here, we demonstrate a role for LRRK2 in mediating microglial proinflammatory responses and morphology. In a murine model of neuroinflammation, we observe robust induction of LRRK2 in microglia. Experiments with toll-like receptor 4 (TLR4)-stimulated rat primary microglia show that inflammation increases LRRK2 activity and expression, while inhibition of LRRK2 kinase activity or knockdown of protein attenuates TNFα secretion and nitric oxide synthase (iNOS) induction. LRRK2 inhibition blocks TLR4 stimulated microglial process outgrowth and impairs ADP stimulated microglial chemotaxis. However, actin inhibitors that phenocopy inhibition of process outgrowth and chemotaxis fail to modify TLR4 stimulation of TNFα secretion and inducible iNOS induction, suggesting that LRRK2 acts upstream of cytoskeleton control as a stress-responsive kinase. These data demonstrate LRRK2 in regulating responses in immune cells of the brain and further implicate microglial involvement in late-onset PD.
Subburaman Mohan - One of the best experts on this subject based on the ideXlab platform.
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A small molecular inhibitor of LRRK1 identified by homology modeling and virtual screening suppresses osteoclast function, but not osteoclast differentiation, in vitro.
Aging, 2019Co-Authors: Canjun Zeng, Subburaman Mohan, Helen Goodluck, Sandi Shen, Weirong XingAbstract:We used TGFβ activation kinase 1 as a template to build a 3D structure of the human LRRK1 kinase domain (hLRRK1 KD) and performed small molecule docking. One of the chemicals (IN04) that docked into the pocket was chosen for evaluation of biological effects on osteoclasts (OCs) in vitro. INO4 at 16 nM completely blocked ATP binding to hLRRK1 KD in an in vitro pulldown assay. In differentiation and pit assays, while the number of OCs on bone slices were comparable for OCs treated with IN04 and DMSO, IN04 treatment of OCs significantly impaired their ability to resorb bone. The area of pits on bone slices was reduced by 43% at 5 μM and 83% at 10 μM as compared to DMSO. Individual pits appeared smaller and shallower. F-actin staining revealed that DMSO-treated OCs displayed clear actin rings, and F-actin forms a peripheral sealing zone. By contrast, IN04-treated OCs showed disarranged F-actin in the cytoplasm, and F-actin failed to form a sealing zone on bone slices. IN04 treatment had no effects on OC-derived coupling factor production nor on osteoblast nodule formation. Our data indicate IN04 is a potent inhibitor of LRRK1, suppressing OC function with no effect on OC formation.
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LRRK1 regulation of actin assembly in osteoclasts involves serine 5 phosphorylation of L-plastin.
Journal of cellular biochemistry, 2018Co-Authors: Helen Goodluck, Subburaman Mohan, Canjun Zeng, Songqin Pan, Elizabeth M. Todd, Sharon Celeste Morley, Xuezhong Qin, Weirong XingAbstract:Mice with disruption of LRRK1 and patients with nonfunctional mutant LRRK1 exhibit severe osteopetrosis phenotypes because of osteoclast cytoskeletal dysfunction. To understand how LRRK1 regulates osteoclast function by modulating cytoskeleton rearrangement, we examined the proteins that are differentially phosphorylated in wild-type mice and LRRK1-deficient osteoclasts by metal affinity purification coupled liquid chromatography/mass spectrometry (LC/MS) analyses. One of the candidates that we identified by LC/MS is L-plastin, an actin bundling protein. We found that phosphorylation of L-plastin at serine (Ser) residues 5 was present in wild-type osteoclasts but not in LRRK1-deficient cells. Western blot analyses with antibodies specific for Ser5 phosphorylated L-plastin confirmed the reduced L-plastin Ser5 phosphorylation in LRRK1 knockout (KO) osteoclasts. micro computed tomography (Micro-CT) analyses revealed that the trabecular bone volume of the distal femur was increased by 27% in the 16 to 21-week-old L-plastin KO females as compared with the wild-type control mice. The ratio of bone volume to tissue volume and connectivity density were increased by 44% and 47% (both P
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LRRK1 regulation of actin assembly in osteoclasts involves serine 5 phosphorylation of l plastin
Journal of Cellular Biochemistry, 2018Co-Authors: Helen Goodluck, Subburaman Mohan, Canjun Zeng, Songqin Pan, Elizabeth M. Todd, Sharon Celeste Morley, Xuezhong QinAbstract:Mice with disruption of LRRK1 and patients with nonfunctional mutant LRRK1 exhibit severe osteopetrosis phenotypes because of osteoclast cytoskeletal dysfunction. To understand how LRRK1 regulates osteoclast function by modulating cytoskeleton rearrangement, we examined the proteins that are differentially phosphorylated in wild-type mice and LRRK1-deficient osteoclasts by metal affinity purification coupled liquid chromatography/mass spectrometry (LC/MS) analyses. One of the candidates that we identified by LC/MS is L-plastin, an actin bundling protein. We found that phosphorylation of L-plastin at serine (Ser) residues 5 was present in wild-type osteoclasts but not in LRRK1-deficient cells. Western blot analyses with antibodies specific for Ser5 phosphorylated L-plastin confirmed the reduced L-plastin Ser5 phosphorylation in LRRK1 knockout (KO) osteoclasts. micro computed tomography (Micro-CT) analyses revealed that the trabecular bone volume of the distal femur was increased by 27% in the 16 to 21-week-old L-plastin KO females as compared with the wild-type control mice. The ratio of bone volume to tissue volume and connectivity density were increased by 44% and 47% (both P < 0.05), respectively, in L-plastin KO mice. Our data suggest that targeted disruption of L-plastin increases trabecular bone volume, and phosphorylation of Ser5 in L-plastin in the LRRK1 signaling pathway may in part contribute to actin assembly in mature osteoclasts.
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Role and mechanism of action of leucine-rich repeat kinase 1 in bone.
Bone research, 2017Co-Authors: Weirong R Xing, Helen Goodluck, Canjun Zeng, Subburaman MohanAbstract:Leucine-rich repeat kinase 1 (LRRK1) plays a critical role in regulating cytoskeletal organization, osteoclast activity, and bone resorption with little effect on bone formation parameters. Deficiency of LRRK1 in mice causes a severe osteopetrosis in the metaphysis of the long bones and vertebrae bones, which makes LRRK1 an attractive alternative drug target for the treatment of osteoporosis and other high-turnover bone diseases. This review summarizes recent advances on the functions of the LRRK1-related family members, LRRK1 deficiency-induced skeletal phenotypes, LRRK1 structure-function, potential biological substrates and interacting proteins, and the mechanisms of LRRK1 action in osteoclasts.
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Targeted disruption of leucine‐rich repeat kinase 1 but not leucine‐rich repeat kinase 2 in mice causes severe osteopetrosis
Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research, 2013Co-Authors: Weirong Xing, Jeff Liu, Shaohong Cheng, Peter Vogel, Subburaman Mohan, Robert BrommageAbstract:To assess the roles of LRRK1 and Lrrk2, we examined skeletal phenotypes in LRRK1 and Lrrk2 knockout (KO) mice. LRRK1 KO mice exhibit severe osteopetrosis caused by dysfunction of multinucleated osteoclasts, reduced bone resorption in endocortical and trabecular regions, and increased bone mineralization. LRRK1 KO mice have lifelong accumulation of bone and respond normally to the anabolic actions of teriparatide treatment, but are resistant to ovariectomy-induced bone boss. Precursors derived from LRRK1 KO mice differentiate into multinucleated cells in response to macrophage colony-stimulating factor (M-CSF)/receptor activator of NF-κB ligand (RANKL) treatment, but these cells fail to form peripheral sealing zones and ruffled borders, and fail to resorb bone. The phosphorylation of cellular Rous sarcoma oncogene (c-Src) at Tyr-527 is significantly elevated whereas at Tyr-416 is decreased in LRRK1-deficient osteoclasts. The defective osteoclast function is partially rescued by overexpression of the constitutively active form of Y527F c-Src. Immunoprecipitation assays in osteoclasts detected a physical interaction of LRRK1 with C-terminal Src kinase (Csk). Lrrk2 KO mice do not show obvious bone phenotypes. Precursors derived from Lrrk2 KO mice differentiate into functional multinucleated osteoclasts. Our finding of osteopetrosis in LRRK1 KO mice provides convincing evidence that LRRK1 plays a critical role in negative regulation of bone mass in part through modulating the c-Src signaling pathway in mice.
Shin Kedashiro - One of the best experts on this subject based on the ideXlab platform.
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plk1 dependent activation of LRRK1 regulates spindle orientation by phosphorylating cdk5rap2
Nature Cell Biology, 2015Co-Authors: Hiroshi Hanafusa, Shin Kedashiro, Motohiro Tezuka, Motoki Funatsu, Fumiko Toyoshima, Satoshi Usami, Kunihiro MatsumotoAbstract:Hanafusa and Matsumato and colleagues find that LRRK1 is a substrate for PLK1 in mitosis. Phosphorylated LRRK1 in turn phosphorylates CDK5RAP2 to promote centrosomal microtubule nucleation and correct spindle orientation.
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PLK1-dependent activation of LRRK1 regulates spindle orientation by phosphorylating CDK5RAP2
Nature Cell Biology, 2015Co-Authors: Hiroshi Hanafusa, Shin Kedashiro, Motohiro Tezuka, Motoki Funatsu, Fumiko Toyoshima, Satoshi Usami, Kunihiro MatsumotoAbstract:Hanafusa and Matsumato and colleagues find that LRRK1 is a substrate for PLK1 in mitosis. Phosphorylated LRRK1 in turn phosphorylates CDK5RAP2 to promote centrosomal microtubule nucleation and correct spindle orientation. Correct formation of the cell division axis requires the initial precise orientation of the mitotic spindle. Proper spindle orientation depends on centrosome maturation, and Polo-like kinase 1 (PLK1) is known to play a crucial role in this process. However, the molecular mechanisms that function downstream of PLK1 are not well understood. Here we show that LRRK1 is a PLK1 substrate that is phosphorylated on Ser 1790. PLK1 phosphorylation is required for CDK1-mediated activation of LRRK1 at the centrosomes, and this in turn regulates mitotic spindle orientation by nucleating the growth of astral microtubules from the centrosomes. Interestingly, LRRK1 in turn phosphorylates CDK5RAP2(Cep215), a human homologue of Drosophila Centrosomin (Cnn), in its γ-tubulin-binding motif, thus promoting the interaction of CDK5RAP2 with γ-tubulin. LRRK1 phosphorylation of CDK5RAP2 Ser 140 is necessary for CDK5RAP2-dependent microtubule nucleation. Thus, our findings provide evidence that LRRK1 regulates mitotic spindle orientation downstream of PLK1 through CDK5RAP2-dependent centrosome maturation.
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Phosphorylation of CLIP-170 by LRRK1 regulates EGFR trafficking by promoting recruitment of p150Glued to MT plus-ends
Journal of cell science, 2015Co-Authors: Shin Kedashiro, Kunihiro Matsumoto, Strahil Iv. Pastuhov, Tomoki Nishioka, Takashi Watanabe, Kozo Kaibuchi, Hiroshi HanafusaAbstract:Ligand-induced activation of the EGF receptor (EGFR) initiates trafficking events that relocalize the receptors from the cell surface to intracellular endocytic compartments. Early endosomes containing activated EGFR migrate along microtubules as they mature into late endosomes. We have recently shown that LRRK1, which is related to the familial Parkinsonism gene product Park8/LRRK2, regulates this EGFR transport in a manner dependent on LRRK1 kinase activity. However, the downstream targets of LRRK1 that may modulate this transport function have not been identified. Here, we identify CLIP-170, a microtubule plus-end protein, as a substrate of LRRK1. LRRK1 phosphorylates CLIP-170 at Thr-1384, located in its C-terminal zinc knuckle motif, and this promotes its association with dynactin–dynein complexes. We find that LRRK1 phosphorylation of CLIP-170 causes the accumulation of p150 Glued , a subunit of dynactin, at microtubule plus-ends, thereby facilitating the migration of EGFR-containing endosomes. Thus, our findings provide new mechanistic insights into the dynein-driven transport of EGFR.
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LRRK1-phosphorylated CLIP-170 regulates EGFR trafficking by recruiting p150Glued to microtubule plus ends.
Journal of Cell Science, 2015Co-Authors: Shin Kedashiro, Kunihiro Matsumoto, Strahil Iv. Pastuhov, Tomoki Nishioka, Takashi Watanabe, Kozo Kaibuchi, Hiroshi HanafusaAbstract:ABSTRACT The binding of ligand to epidermal growth factor receptor (EGFR) causes the receptor to become activated and stimulates the endocytosis of EGFR. Early endosomes containing activated EGFR migrate along microtubules as they mature into late endosomes. We have recently shown that LRRK1, which is related to the familial Parkinsonism gene product Park8 (also known as LRRK2), regulates this EGFR transport in a manner dependent on LRRK1 kinase activity. However, the downstream targets of LRRK1 that might modulate this transport function have not been identified. Here, we identify CLIP-170 (also known as CLIP1), a microtubule plus-end protein, as a substrate of LRRK1. LRRK1 phosphorylates CLIP-170 at Thr1384, located in its C-terminal zinc knuckle motif, and this promotes the association of CLIP-170 with dynein–dynactin complexes. We find that LRRK1-mediated phosphorylation of CLIP-170 causes the accumulation of p150 Glued (also known as DCTN1) a subunit of dynactin, at microtubule plus ends, thereby facilitating the migration of EGFR-containing endosomes. Thus, our findings provide new mechanistic insights into the dynein-driven transport of EGFR.
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Leucine-rich repeat kinase LRRK1 regulates endosomal trafficking of the EGF receptor
Nature communications, 2011Co-Authors: Hiroshi Hanafusa, Shin Kedashiro, Kouki Ishikawa, Atsuki Nara, Tsukasa Saigo, Shun-ichiro Iemura, Tohru Natsume, Masayuki Komada, Hiroshi Shibuya, Kunihiro MatsumotoAbstract:Activation of the epidermal growth factor receptor (EGFR) not only initiates multiple signal-transduction pathways, including the MAP kinase (MAPK) pathway, but also triggers trafficking events that relocalize receptors from the cell surface to intracellular endocytic compartments. In this paper, we demonstrate that leucine-rich repeat kinase LRRK1, which contains a MAPKKK-like kinase domain, forms a complex with activated EGFR through an interaction with Grb2. Subsequently, LRRK1 and epidermal growth factor (EGF) are internalized and co-localized in early endosomes. LRRK1 regulates EGFR transport from early to late endosomes and regulates the motility of EGF-containing early endosomes in a manner dependent on its kinase activity. Furthermore, LRRK1 serves as a scaffold facilitating the interaction of EGFR with the endosomal sorting complex required for transport-0 complex, thus enabling efficient sorting of EGFR to the inner vesicles of multivesicular bodies. Our findings provide the first evidence that a MAPKKK-like protein regulates the endosomal trafficking of EGFR.
