scholarly journals Deciphering the LRRK code: LRRK1 and LRRK2 phosphorylate distinct Rab proteins and are regulated by diverse mechanisms

2021 ◽  
Vol 478 (3) ◽  
pp. 553-578
Author(s):  
Asad U. Malik ◽  
Athanasios Karapetsas ◽  
Raja S. Nirujogi ◽  
Sebastian Mathea ◽  
Deep Chatterjee ◽  
...  
Keyword(s):  
Growth Factor Receptor ◽  
Binding Motif ◽  
Rab Proteins ◽  
Rab Gtpases ◽  
Embryonic Fibroblasts ◽  
Knock Out ◽  
Maximal Stimulation ◽  
Equivalent Site ◽  
Effector Binding ◽  
Stimulation Of

Autosomal dominant mutations in LRRK2 that enhance kinase activity cause Parkinson's disease. LRRK2 phosphorylates a subset of Rab GTPases including Rab8A and Rab10 within its effector binding motif. Here, we explore whether LRRK1, a less studied homolog of LRRK2 that regulates growth factor receptor trafficking and osteoclast biology might also phosphorylate Rab proteins. Using mass spectrometry, we found that in LRRK1 knock-out cells, phosphorylation of Rab7A at Ser72 was most impacted. This residue lies at the equivalent site targeted by LRRK2 on Rab8A and Rab10. Accordingly, recombinant LRRK1 efficiently phosphorylated Rab7A at Ser72, but not Rab8A or Rab10. Employing a novel phospho-specific antibody, we found that phorbol ester stimulation of mouse embryonic fibroblasts markedly enhanced phosphorylation of Rab7A at Ser72 via LRRK1. We identify two LRRK1 mutations (K746G and I1412T), equivalent to the LRRK2 R1441G and I2020T Parkinson's mutations, that enhance LRRK1 mediated phosphorylation of Rab7A. We demonstrate that two regulators of LRRK2 namely Rab29 and VPS35[D620N], do not influence LRRK1. Widely used LRRK2 inhibitors do not inhibit LRRK1, but we identify a promiscuous inhibitor termed GZD-824 that inhibits both LRRK1 and LRRK2. The PPM1H Rab phosphatase when overexpressed dephosphorylates Rab7A. Finally, the interaction of Rab7A with its effector RILP is not affected by LRRK1 phosphorylation and we observe that maximal stimulation of the TBK1 or PINK1 pathway does not elevate Rab7A phosphorylation. Altogether, these findings reinforce the idea that the LRRK enzymes have evolved as major regulators of Rab biology with distinct substrate specificity.

scholarly journals Deciphering the LRRK code: LRRK1 and LRRK2 phosphorylate distinct Rab proteins and are regulated by diverse mechanisms

2020 ◽  
Author(s):  
Asad U. Malik ◽  
Athanasios Karapetsas ◽  
Raja S. Nirujogi ◽  
Sebastian Mathea ◽  
Prosenjit Pal ◽  
...  
Keyword(s):  
Kinase Inhibitor ◽  
Growth Factor Receptor ◽  
Binding Motif ◽  
Rab Proteins ◽  
Rab Gtpases ◽  
Missense Mutations ◽  
Knock Out ◽  
Equivalent Site ◽  
Effector Binding

AbstractMuch attention has focused on LRRK2, as autosomal dominant missense mutations that enhance its kinase activity cause inherited Parkinson’s disease. LRRK2 regulates biology by phosphorylating a subset of Rab GTPases including Rab8A and Rab10 within its effector binding motif. In this study we explore whether LRRK1, a less studied homologue of LRRK2 that regulates growth factor receptor trafficking and osteoclast biology might also phosphorylate Rab proteins. Using mass spectrometry, we found that the endogenous Rab7A protein, phosphorylated at Ser72 was most impacted by LRRK1 knock-out. This residue is not phosphorylated by LRRK2 but lies at the equivalent site targeted by LRRK2 on Rab8A and Rab10. Accordingly, recombinant LRRK1 efficiently phosphorylated Rab7A at Ser72, but not Rab8A or Rab10. Employing a novel phospho-specific antibody, we found that phorbol ester stimulation of mouse embryonic fibroblasts markedly enhanced phosphorylation of Rab7A at Ser72 via LRRK1. We identify two LRRK1 mutations (K746G and I1412T), equivalent to the LRRK2 R1441G and I2020T Parkinson’s mutations, that enhance LRRK1 mediated phosphorylation of Rab7A. We demonstrate that two regulators of LRRK2 namely Rab29 and VPS35[D620N], do not influence LRRK1. Widely used LRRK2 inhibitors do not inhibit LRRK1, but we identify a promiscuous Type-2 tyrosine kinase inhibitor termed GZD-824 that inhibits both LRRK1 and LRRK2. Finally, we show that interaction of Rab7A with its effector RILP is not affected by high stoichiometry LRRK1 phosphorylation. Altogether, these finding reinforce the idea that the LRRK enzymes have evolved as major regulators of Rab biology.

scholarly journals Site-specific monoubiquitination downregulates Rab5 by disrupting effector binding and guanine nucleotide conversion

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Donghyuk Shin ◽  
Wooju Na ◽  
Ji-Hyung Lee ◽  
Gyuhee Kim ◽  
Jiseok Baek ◽  
...  
Keyword(s):  
Egf Receptor ◽  
Regulatory Mechanism ◽  
Rab Proteins ◽  
Rab Gtpases ◽  
Guanine Nucleotide ◽  
Site Specific ◽  
Downstream Effectors ◽  
Specific Manner ◽  
A Site ◽  
Effector Binding

Rab GTPases, which are involved in intracellular trafficking pathways, have recently been reported to be ubiquitinated. However, the functions of ubiquitinated Rab proteins remain unexplored. Here we show that Rab5 is monoubiquitinated on K116, K140, and K165. Upon co-transfection with ubiquitin, Rab5 exhibited abnormalities in endosomal localization and EGF-induced EGF receptor degradation. Rab5 K140R and K165R mutants restored these abnormalities, whereas K116R did not. We derived structural models of individual monoubiquitinated Rab5 proteins (mUbRab5s) by solution scattering and observed different conformational flexibilities in a site-specific manner. Structural analysis combined with biochemical data revealed that interactions with downstream effectors were impeded in mUbRab5K140, whereas GDP release and GTP loading activities were altered in mUbRab5K165. By contrast, mUbRab5K116 apparently had no effect. We propose a regulatory mechanism of Rab5 where monoubiquitination downregulates effector recruitment and GDP/GTP conversion in a site-specific manner.

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 The interaction between two trains of impulses converging on the same motoneurone

1929 ◽  
Vol 105 (738) ◽  
pp. 363-371 ◽  
Keyword(s):  
Mechanical Effect ◽  
The Other ◽  
Muscle Fibres ◽  
Flexor Muscle ◽  
Afferent Pathways ◽  
Afferent Nerves ◽  
Maximal Stimulation ◽  
The Common ◽  
The Relationship ◽  
Stimulation Of

It was shown in an earlier paper (7) that if maximal stimulation of either of two different afferent nerves can reflexly excite fractions of a given flexor muscle, there are generally, within the aggregate of neurones which innervate that muscle, motoneurones which can be caused to discharge by either afferent (i. e., motoneurones common to both fractions). The relationship which two such afferents bear to a common motoneurone was shown, by the isometric method of recording contraction, to be such that the activation of one afferent, at a speed sufficient to cause a maximal motor tetanus when trans­mitted to the muscle fibres, caused exclusion of any added mechanical effect when the other afferent was excited concurrently. This default in mechanical effect was called “occlusion.” Occlusion may conceivably be due to total exclusion of the effect of one afferent pathway on the common motoneurone by the activity of the other; but facilitation of the effect of one path by the activation of the other when the stimuli were minimal suggests that, in some circumstances at least, the effect of each could augment and summate with th at of the other at the place of convergence of two afferent pathways. Further investigation, using the action currents of the muscle as indication of the nerve impulses discharged by the motoneurone units, has now given some information regarding the effect of impulses arriving at the locus of convergence by one afferent path when the unit common to both is already discharging in response to impulses arriving by the other afferent path. Our method has been to excite both afferent nerves in overlapping sequence by series of break shocks at a rapid rate and to examine the action currents of the resulting reflex for evidence of the appearance of the rhythm of the second series in the discharge caused by the first when the two series are both reaching the motoneurone.

scholarly journals Structures ofDrosophila melanogasterRab2 and Rab3 bound to GMPPNP

2015 ◽  
Vol 71 (1) ◽  
pp. 34-40 ◽  
Author(s):  
Jennifer A. Lardong ◽  
Jan H. Driller ◽  
Harald Depner ◽  
Christoph Weise ◽  
Astrid Petzoldt ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Intracellular Trafficking ◽  
Large Family ◽  
Cysteine Residue ◽  
Effector Proteins ◽  
Rab Proteins ◽  
Rab Gtpases ◽  
Ras Proteins ◽  
Transport Vesicles ◽  
Switch Regions

Rab GTPases belong to the large family of Ras proteins. They act as key regulators of membrane organization and intracellular trafficking. Functionally, they act as switches. In the active GTP-bound form they can bind to effector proteins to facilitate the delivery of transport vesicles. Upon stimulation, the GTP is hydrolyzed and the Rab proteins undergo conformational changes in their switch regions. This study focuses on Rab2 and Rab3 fromDrosophila melanogaster. Whereas Rab2 is involved in vesicle transport between the Golgi and the endoplasmatic reticulum, Rab3 is a key player in exocytosis, and in the synapse it is involved in the assembly of the presynaptic active zone. Here, high-resolution crystal structures of Rab2 and Rab3 in complex with GMPPNP and Mg2+are presented. In the structure of Rab3 a modified cysteine residue is observed with an enigmatic electron density attached to its thiol function.

scholarly journals Phos-tag analysis of Rab10 phosphorylation by LRRK2: a powerful assay for assessing kinase function and inhibitors

2016 ◽  
Vol 473 (17) ◽  
pp. 2671-2685 ◽  
Author(s):  
Genta Ito ◽  
Kristina Katsemonova ◽  
Francesca Tonelli ◽  
Pawel Lis ◽  
Marco A.S. Baptista ◽  
...  
Keyword(s):  
Autosomal Dominant ◽  
Mouse Lung ◽  
Rab Gtpase ◽  
Leucine Rich Repeat ◽  
Serine Residue ◽  
Mouse Embryonic Fibroblasts ◽  
Embryonic Fibroblasts ◽  
The Impact ◽  
Effector Binding

Autosomal dominant mutations that activate the leucine-rich repeat kinase 2 (LRRK2) cause inherited Parkinson's disease. Recent work has revealed that LRRK2 directly phosphorylates a conserved threonine/serine residue in the effector-binding switch-II motif of a number of Rab GTPase proteins, including Rab10. Here we describe a facile and robust method to assess phosphorylation of endogenous Rab10 in mouse embryonic fibroblasts (MEFs), lung and spleen-derived B-cells, based on the ability of the Phos-tag reagent to retard the electrophoretic mobility of LRRK2-phosphorylated Rab10. We exploit this assay to show that phosphorylation of Rab10 is ablated in kinase-inactive LRRK2[D2017A] knockin MEFs and mouse lung, demonstrating that LRRK2 is the major Rab10 kinase in these cells/tissue. We also establish that the Phos-tag assay can be deployed to monitor the impact that activating LRRK2 pathogenic (G2019S and R1441G) knockin mutations have on stimulating Rab10 phosphorylation. We show that upon addition of LRRK2 inhibitors, Rab10 is dephosphorylated within 1–2 min, markedly more rapidly than the Ser935 and Ser1292 biomarker sites that require 40–80 min. Furthermore, we find that phosphorylation of Rab10 is suppressed in LRRK2[S910A+S935A] knockin MEFs indicating that phosphorylation of Ser910 and Ser935 and potentially 14-3-3 binding play a role in facilitating the phosphorylation of Rab10 by LRRK2 in vivo. The Rab Phos-tag assay has the potential to significantly aid with evaluating the effect that inhibitors, mutations and other factors have on the LRRK2 signalling pathway.

The role of host cell Rab GTPases in influenza A virus infections

2021 ◽  
Author(s):  
Jing Wu ◽  
Jiaqi Gu ◽  
Li Shen ◽  
Xiaonan Jia ◽  
Yiqian Yin ◽  
...  
Keyword(s):  
Influenza A Virus ◽  
Influenza A ◽  
Respiratory Infections ◽  
Small Gtpase ◽  
Regulatory Mechanisms ◽  
Rab Proteins ◽  
Rab Gtpases ◽  
Virus Infections ◽  
Adaptation Mechanisms

Influenza A virus (IAV) is a crucial cause of respiratory infections in humans worldwide. Therefore, studies should clarify adaptation mechanisms of IAV and critical factors of the viral pathogenesis in human hosts. GTPases of the Rab family are the largest branch of the Ras-like small GTPase superfamily, and they regulate almost every step during vesicle-mediated trafficking. Evidence has shown that Rab proteins participate in the lifecycle of IAV. In this mini-review, we outline the regulatory mechanisms of different Rab proteins in the lifecycle of IAV. Understanding the role of Rab proteins in IAV infections is important to develop broad-spectrum host-targeted antiviral strategies.

Phosphorylation of Nck in response to a variety of receptors, phorbol myristate acetate, and cyclic AMP

1992 ◽  
Vol 12 (12) ◽  
pp. 5816-5823
Author(s):  
D Park ◽  
S G Rhee
Keyword(s):  
Monoclonal Antibodies ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Growth Factor Receptor ◽  
Immunoglobulin M ◽  
Rat Tissues ◽  
A431 Cells ◽  
Sh3 Domains ◽  
Epidermal Growth ◽  
Stimulation Of

The 47-kDa protein coimmunoprecipitated with phospholipase C (PLC)-gamma 1 by anti-PLC-gamma 1 monoclonal antibodies is proved to be Nck, a protein composed almost exclusively of one SH2 and three SH3 domains. Nck and PLC-gamma 1 are recognized by certain anti-PLC-gamma 1 monoclonal antibodies because Nck and PLC-gamma 1 share an epitope that likely is located in their SH3 domains. Nck is widely distributed in rat tissues, with an especially high level of expression in testes. The expression levels of Nck remains unchanged during the development of rat brain, whereas PLC-gamma 1 decreases during the same developmental period. Stimulation of A431 cells with epidermal growth factor elicits the tight association of Nck with the epidermal growth factor receptor and phosphorylation of Nck on both serine and tyrosine residues. The phosphorylation of Nck is also enhanced in response to stimulation of the nerve growth factor receptor in PC12 cells, the T-cell receptor complex in Jurkat cells, the membrane immunoglobulin M in Daudi cells, and the low-affinity immunoglobulin G receptor (Fc gamma RII) in U937 cells. The phosphorylation of Nck was also enhanced following treatment of A431 cells with phorbol 12-myristate 13-acetate or forskolin. These results suggest that Nck is a target for a variety of protein kinases that might modulate the postulated role of Nck as an adaptor for the physical and functional coordination of signalling proteins.

EFFECT OF VARIOUS PREPARATIONS OF PITUITARY AND DIENCEPHALON ON THE IN VITRO SECRETION OF ALDOSTERONE AND CORTICOSTERONE BY THE RAT ADRENAL GLAND

1961 ◽  
Vol 39 (5) ◽  
pp. 901-913 ◽  
Author(s):  
O. J. Lucis ◽  
I. Dyrenfurth ◽  
E. H. Venning
Keyword(s):  
Adrenal Gland ◽  
Linear Relationship ◽  
Dose Response ◽  
Response Curve ◽  
Maximum Effect ◽  
Percentage Increase ◽  
Aldosterone Secretion ◽  
Maximal Stimulation ◽  
Stimulation Of

Purified corticotropin and ACTH peptides increased the secretion of aldosterone, corticosterone, and an unidentified compound RT4in incubated rat adrenal tissue. When the response was expressed as a percentage increase above that of the control tissue, the increases in corticosterone and compound RT4followed a sigmoid log dose – response curve. The maximum effect on aldosterone was obtained at a time when the response curve for corticosterone assumed a linear relationship between the response and the logarithm of the dose of ACTH. This dose level was considerably less than that required for maximal stimulation of corticosterone.The capacity of the ACTH peptides α1+α2and δ′ for stimulating aldosterone secretion could be greatly diminished by allowing solutions of these fractions to stand at 5 °C for 1 week. These solutions still retained their ability to stimulate corticosterone secretion.Saline suspensions and extracts of fresh hog diencephalon contained a factor which selectively stimulated aldosterone secretion.

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