Kinase activity of mutant LRRK2 manifests differently in hetero-dimeric vs. homo-dimeric complexes

2019 ◽  
Vol 476 (3) ◽  
pp. 559-579 ◽  
Author(s):  
Emmanouela Leandrou ◽  
Eliana Markidi ◽  
Anna Memou ◽  
Katerina Melachroinou ◽  
Elisa Greggio ◽  
...  
Keyword(s):  
Kinase Activity ◽  
Cultured Cells ◽  
Kinase Domain ◽  
Small Gtpase ◽  
Wild Type ◽  
Risk Variant ◽  
Dimeric Complexes ◽  
Mutant Lrrk2 ◽  
Mutant Forms

Abstract The Parkinson's disease (PD) protein leucine-rich repeat kinase 2 (LRRK2) exists as a mixture of monomeric and dimeric species, with its kinase activity highly concentrated in the dimeric conformation of the enzyme. We have adapted the proximity biotinylation approach to study the formation and activity of LRRK2 dimers isolated from cultured cells. We find that the R1441C and I2020T mutations both enhance the rate of dimer formation, whereas, the G2019S kinase domain mutant is similar to WT, and the G2385R risk factor variant de-stabilizes dimers. Interestingly, we find a marked departure in the kinase activity between G2019S–LRRK2 homo-dimers and wild-type-G2019S hetero-dimers. While the homo-dimeric G2019S–LRRK2 exhibits the typical robust enhancement of kinase activity, hetero-dimers comprised of wild-type (WT) and G2019S–LRRK2 exhibit kinase activity similar to WT. Dimeric complexes of specific mutant forms of LRRK2 show reduced stability following an in vitro kinase reaction, in LRRK2 mutants for which the kinase activity is similar to WT. Phosphorylation of the small GTPase Rab10 follows a similar pattern in which hetero-dimers of WT and mutant LRRK2 show similar levels of phosphorylation of Rab10 to WT homo-dimers; while the levels of pRab10 are significantly increased in cells expressing mutant homo-dimers. Interestingly, while the risk variant G2385R leads to a de-stabilization of LRRK2 dimers, those dimers possess significantly elevated kinase activity. The vast majority of familial LRRK2-dependent PD cases are heterozygous; thus, these findings raise the possibility that a crucial factor in disease pathogenesis may be the accumulation of homo-dimeric mutant LRRK2.

scholarly journals Phosphorylation by PKC and PKA regulate the kinase activity and downstream signaling of WNK4

2017 ◽  
Vol 114 (5) ◽  
pp. E879-E886 ◽  
Author(s):  
Maria Castañeda-Bueno ◽  
Juan Pablo Arroyo ◽  
Junhui Zhang ◽  
Jeremy Puthumana ◽  
Orlando Yarborough ◽  
...  
Keyword(s):  
Kinase Activity ◽  
Cultured Cells ◽  
Kinase Domain ◽  
Tandem Mass ◽  
Volume Depletion ◽  
Kinase Activation ◽  
Downstream Signaling ◽  
Electrolyte Homeostasis

With-no-lysine kinase 4 (WNK4) regulates electrolyte homeostasis and blood pressure. WNK4 phosphorylates the kinases SPAK (Ste20-related proline alanine-rich kinase) and OSR1 (oxidative stress responsive kinase), which then phosphorylate and activate the renal Na-Cl cotransporter (NCC). WNK4 levels are regulated by binding to Kelch-like 3, targeting WNK4 for ubiquitylation and degradation. Phosphorylation of Kelch-like 3 by PKC or PKA downstream of AngII or vasopressin signaling, respectively, abrogates binding. We tested whether these pathways also affect WNK4 phosphorylation and activity. By tandem mass spectrometry and use of phosphosite-specific antibodies, we identified five WNK4 sites (S47, S64, S1169, S1180, S1196) that are phosphorylated downstream of AngII signaling in cultured cells and in vitro by PKC and PKA. Phosphorylation at S64 and S1196 promoted phosphorylation of the WNK4 kinase T-loop at S332, which is required for kinase activation, and increased phosphorylation of SPAK. Volume depletion induced phosphorylation of these sites in vivo, predominantly in the distal convoluted tubule. Thus, AngII, in addition to increasing WNK4 levels, also modulates WNK4 kinase activity via phosphorylation of sites outside the kinase domain.

scholarly journals Twitchin kinase inhibits muscle activity

2017 ◽  
Vol 28 (12) ◽  
pp. 1591-1600 ◽  
Author(s):  
Yohei Matsunaga ◽  
Hyundoo Hwang ◽  
Barbara Franke ◽  
Rhys Williams ◽  
McKenna Penley ◽  
...  
Keyword(s):  
Muscle Activity ◽  
Kinase Activity ◽  
Kinase Domain ◽  
Wild Type ◽  
Competitive Fitness ◽  
Binding Partners ◽  
Fibronectin Domains ◽  
First Time

Muscle sarcomeres contain giant polypeptides composed of multiple immunoglobulin and fibronectin domains and one or two protein kinase domains. Although binding partners for a number of this family’s kinase domains have been identified, the catalytic necessity of these kinase domains remains unknown. In addition, various members of this kinase family are suspected pseudokinases with no or little activity. Here we address catalytic necessity for the first time, using the prototypic invertebrate representative twitchin (UNC-22) from Caenorhabditis elegans. In in vitro experiments, change of a conserved lysine (K) that is involved in ATP coordination to alanine (A) resulted in elimination of kinase activity without affecting the overall structure of the kinase domain. The same mutation, unc-22(sf21), was generated in the endogenous twitchin gene. The unc-22(sf21) worms have well-organized sarcomeres. However, unc-22(sf21) mutants move faster than wild-type worms and, by optogenetic experiments, contract more. Wild-type nematodes exhibited greater competitive fitness than unc-22(sf21) mutants. Thus the catalytic activity of twitchin kinase has a role in vivo, where it inhibits muscle activity and is likely maintained by selection.

The Bcr-Abl SH2-Kinase Domain Interface Is Critical for Leukemogenesis and An Additional Therapeutic Target in CML.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 37-37
Author(s):  
Oliver D. Hantschel ◽  
Florian Grebien ◽  
Ines Kaupe ◽  
Boris Kovacic ◽  
John Wojcik ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Signaling Pathways ◽  
Kinase Activity ◽  
Critical Factor ◽  
Kinase Domain ◽  
Sh2 Domain ◽  
Wild Type ◽  
Tyrosine Kinase Activity ◽  
Downstream Signaling

Abstract Abstract 37 We previously showed that the Abl SH2 domain is an allosteric activator of c-Abl tyrosine kinase activity and substrate phosphorylation (Filippakopoulos et al. (2008) Cell 134(5), 793-803). This effect is exerted directly by docking of the SH2 domain onto the N-lobe of the kinase domain in the active conformation of c-Abl. We also showed that the same structural mechanism is a critical factor for full activation of the oncogenic fusion kinase Bcr-Abl. Disruption of binding of the SH2 domain to the kinase domain in Bcr-Abl by the Ile164Glu mutation in the SH2 domain, led to a strong reduction in in vitro tyrosine kinase activity and Bcr-Abl autophosphorylation. Unexpectedly, we observed a differential attenuation of downstream signaling pathways upon disruption of the SH2-kinase domain interface, indicating different activation thresholds of Bcr-Abl downstream signaling pathways. Here, we show that disrupting the SH2-kinase domain interface abrogates the transforming capacity of Bcr-Abl. Cells expressing the Bcr-Abl Ile164Glu mutant were unable to generate cytokine-independent colonies in vitro. Furthermore, mice transplanted with Bcr-Abl Ile164Glu expressing bone marrow cells did not develop the characteristic MPD-like disease that is caused by wild-type Bcr-Abl. Mice that received Bcr-Abl Ile164Glu cells showed normal survival, blood counts and histology after more than 100 days post-transplant, despite the presence of Bcr-Abl Ile164Glu-expressing cells in all blood lineages. This shows that the formation of the SH2-kinase domain interface is strictly necessary for Bcr-Abl to cause CML. Together with our data that show sensitization to imatinib inhibition of Bcr-Abl Ile164Glu as compared to Bcr-Abl wild-type, this argues for the SH2-kinase domain interface as an additional drug target on Bcr-Abl that may synergize with tyrosine kinase inhibitors and may be useful to inhibit tyrosine kinase inhibitor resistant Bcr-Abl clones. To address possibilities to interfere with the SH2-kinase domain interface, we are using an engineered binding protein that binds to the Abl SH2 domain with high-affinity and specificity and supposedly disrupts the interface with the kinase domain, resulting in a decrease in Bcr-Abl kinase activity. In conclusion, we provide strong evidence that the structural positioning of the SH2 domain is a crucial factor for constitutive activity, signal transduction and leukemogenicity of Bcr-Abl. Besides oligomerization via the N-terminal coiled-coiled domain and loss of the auto-inhibitory N-terminal myristoyl group, the proper positioning of the SH2 domain appears to be another critical factor that is required for constitutive activation of Bcr-Abl. Inhibitors of the SH2-kinase domain interface of Bcr-Abl may comprise alternative or additional points of pharmacological intervention for the treatment of imatinib-sensitive or -resistant CML or Ph+ acute lymphocytic leukemia. Disclosures: No relevant conflicts of interest to declare.

scholarly journals The N-terminal LIM domain negatively regulates the kinase activity ofLIM-kinase 1

1999 ◽  
Vol 343 (1) ◽  
pp. 99-105 ◽  
Author(s):  
Kyoko NAGATA ◽  
Kazumasa OHASHI ◽  
Neng YANG ◽  
Kensaku MIZUNO
Keyword(s):  
Kinase Activity ◽  
Cultured Cells ◽  
Pdz Domain ◽  
Limited Proteolysis ◽  
Direct Interaction ◽  
Kinase Domain ◽  
Wild Type ◽  
Lim Domain ◽  
Lim Kinase ◽  
Lim Domains

LIM-kinase 1 (LIMK1, where LIM is an acronym of the three gene products Lin-11, Isl-1 and Mec-3) is a serine/threonine kinase that phosphorylates cofilin and regulates actin cytoskeletal reorganization. LIMK1 contains two LIM domains and a PDZ (an acronym of the three proteins PSD-95, Dlg and ZO-1) domain in the N-terminal half and a kinase domain in the C-terminal half. In this study we examined the role of the extra-catalytic region in the regulation of kinase activity of LIMK1. Limited proteolysis of LIMK1 resulted in the production of the 35-40-kDa kinase core fragments with 3.5-5.5-fold increased kinase activity. The LIMK1 mutants with deleted LIM domains (δLIM) or conserved cysteines in the two LIM domains replaced with glycines (dmLIMK1) had 3-7-fold higher kinase activitiesin vitro, compared with the wild-type LIMK1. The C-terminal kinase fragment of LIMK1 bound to the LIM domain but not to the PDZ domain. Furthermore, the LIM fragment dose-dependently inhibited the kinase catalytic activity of the kinase core fragment of LIMK1. Taken together, these results suggest that the N-terminal LIM domain negatively regulates the kinase activity of LIMK1 by direct interaction with the C-terminal kinase domain. In addition, expression of the δLIM mutant in cultured cells induced punctate accumulation of actin filaments, an event distinct from the pattern of actin organization induced by expression of the wild-type LIMK1, suggesting that the LIM domain plays a role in the function of LIMK1in vivo.

scholarly journals Nanoparticle Delivered Human Biliverdin Reductase-Based Peptide Increases Glucose Uptake by Activating IRK/Akt/GSK3 Axis: The Peptide Is Effective in the Cell and Wild-Type and Diabetic Ob/Ob Mice

2016 ◽  
Vol 2016 ◽  
pp. 1-15 ◽  
Author(s):  
Peter E. M. Gibbs ◽  
Tihomir Miralem ◽  
Nicole Lerner-Marmarosh ◽  
Mahin D. Maines
Keyword(s):  
Amino Acids ◽  
Cell Culture ◽  
Glucose Uptake ◽  
Animal Studies ◽  
Cultured Cells ◽  
Kinase Domain ◽  
Terminal Segment ◽  
Wild Type ◽  
Stimulation Of

Insulin’s stimulation of glucose uptake by binding to the IRK extracellular domain is compromised in diabetes. We have recently described an unprecedented approach to stimulating glucose uptake. KYCCSRK (P2) peptide, corresponding to the C-terminal segment of hBVR, was effective in binding to and inducing conformational change in the IRK intracellular kinase domain. Although myristoylated P2, made of L-amino acids, was effective in cell culture, its use for animal studies was unsuitable. We developed a peptidase-resistant formulation of the peptide that was efficient in both mice and cell culture systems. The peptide was constructed of D-amino acids, in reverse order, and blocked at both termini. Delivery of the encapsulated peptide to HepG2 and HSKM cells was confirmed by its prolonged effect on stimulation of glucose uptake (>6 h). The peptide improved glucose clearance in both wild-type and Ob/Ob mice; it lowered blood glucose levels and suppressed glucose-stimulated insulin secretion. IRK activity was stimulated in the liver of treated mice and in cultured cells. The peptide potentiated function of IRK’s downstream effector, Akt-GSK3-(α,β)axis. Thus, P2-based approach can be used for improving glucose uptake by cells. Also, it allows for screening peptidesin vitroand in animal models for treatment of diabetes.

Transmembrane prolyl 4-hydroxylase is a fourth prolyl 4-hydroxylase regulating EPO production and erythropoiesis

Blood ◽  
2012 ◽  
Vol 120 (16) ◽  
pp. 3336-3344 ◽  
Author(s):  
Anu Laitala ◽  
Ellinoora Aro ◽  
Gail Walkinshaw ◽  
Joni M. Mäki ◽  
Maarit Rossi ◽  
...  
Keyword(s):  
Cultured Cells ◽  
Mrna Level ◽  
Hypoxia Inducible Factor ◽  
Mrna Levels ◽  
Wild Type ◽  
Α Subunit ◽  
Hepcidin Expression ◽  
In The Wild ◽  
Hepcidin Mrna

AbstractAn endoplasmic reticulum transmembrane prolyl 4-hydroxylase (P4H-TM) is able to hydroxylate the α subunit of the hypoxia-inducible factor (HIF) in vitro and in cultured cells, but nothing is known about its roles in mammalian erythropoiesis. We studied such roles here by administering a HIF-P4H inhibitor, FG-4497, to P4h-tm−/− mice. This caused larger increases in serum Epo concentration and kidney but not liver Hif-1α and Hif-2α protein and Epo mRNA levels than in wild-type mice, while the liver Hepcidin mRNA level was lower in the P4h-tm−/− mice than in the wild-type. Similar, but not identical, differences were also seen between FG-4497–treated Hif-p4h-2 hypomorphic (Hif-p4h-2gt/gt) and Hif-p4h-3−/− mice versus wild-type mice. FG-4497 administration increased hemoglobin and hematocrit values similarly in the P4h-tm−/− and wild-type mice, but caused higher increases in both values in the Hif-p4h-2gt/gt mice and in hematocrit value in the Hif-p4h-3−/− mice than in the wild-type. Hif-p4h-2gt/gt/P4h-tm−/− double gene-modified mice nevertheless had increased hemoglobin and hematocrit values without any FG-4497 administration, although no such abnormalities were seen in the Hif-p4h-2gt/gt or P4h-tm−/− mice. Our data thus indicate that P4H-TM plays a role in the regulation of EPO production, hepcidin expression, and erythropoiesis.

scholarly journals Phosphoinositides Regulate Membrane-dependent Actin Assembly by Latex Bead Phagosomes

2002 ◽  
Vol 13 (4) ◽  
pp. 1190-1202 ◽  
Author(s):  
Hélène Defacque ◽  
Evelyne Bos ◽  
Boyan Garvalov ◽  
Cécile Barret ◽  
Christian Roy ◽  
...  
Keyword(s):  
Binding Sites ◽  
Kinase Activity ◽  
Kinase Inhibitors ◽  
Membrane Surface ◽  
Latex Bead ◽  
Actin Assembly ◽  
Wild Type ◽  
Membrane Surfaces ◽  
Organelle Membrane

Actin assembly on membrane surfaces is an elusive process in which several phosphoinositides (PIPs) have been implicated. We have reconstituted actin assembly using a defined membrane surface, the latex bead phagosome (LBP), and shown that the PI(4,5)P2-binding proteins ezrin and/or moesin were essential for this process ( Defacque et al., 2000b ). Here, we provide several lines of evidence that both preexisting and newly synthesized PI(4,5)P2, and probably PI(4)P, are essential for phagosomal actin assembly; only these PIPs were routinely synthesized from ATP during in vitro actin assembly. Treatment of LBP with phospholipase C or with adenosine, an inhibitor of type II PI 4-kinase, as well as preincubation with anti-PI(4)P or anti-PI(4,5)P2 antibodies all inhibited this process. Incorporation of extra PI(4)P or PI(4,5)P2 into the LBP membrane led to a fivefold increase in the number of phagosomes that assemble actin. An ezrin mutant mutated in the PI(4,5)P2-binding sites was less efficient in binding to LBPs and in reconstituting actin assembly than wild-type ezrin. Our data show that PI 4- and PI 5-kinase, and under some conditions also PI 3-kinase, activities are present on LBPs and can be activated by ATP, even in the absence of GTP or cytosolic components. However, PI 3-kinase activity is not required for actin assembly, because the process was not affected by PI 3-kinase inhibitors. We suggest that the ezrin-dependent actin assembly on the LBP membrane may require active turnover of D4 and D5 PIPs on the organelle membrane.

Arabidopsis MLK3, a Plant-specific Casein Kinase 1, Negatively Regulated Flowering and Phosphorylated Histone H3 in vivo

2019 ◽  
Author(s):  
Zhen Wang ◽  
Junmei Kang ◽  
Shangang Jia ◽  
Tiejun Zhang ◽  
Zhihai Wu ◽  
...  
Keyword(s):  
Kinase Activity ◽  
Histone H3 ◽  
Casein Kinase ◽  
Kinase Assay ◽  
Wild Type ◽  
Serine Threonine Kinase ◽  
Altered Expression ◽  
Casein Kinase 1 ◽  
Threonine Kinase

Abstract Background: Casein kinase 1 (CK1) family members are highly conserved serine/threonine kinase present in most eukaryotes with multiple biological functions. Arabidopsis MUT9-like kinases ( MLKs ) belong to a clade CK1 specific to the plant kingdom and have been implicated collectively in modulating flowering related processes. Three of the four MLKs ( MLK1/2/4 ) have been characterized, however, little is known about MLK3 , the most divergent MLKs. Results: We demonstrated that compared with wild type, mlk3 , a truncated MLK3 , flowered slightly early under long day conditions and ectopic expression of MLK3 rescued the morphological defects of mlk3 , indicating that MLK3 negatively regulates flowering. GA 3 application accelerated flowering of both wild type and mlk3 , suggesting that mlk3 had normal GA response. The recombinant MLK3-GFP was localized in the nucleus exclusively. In vitro kinase assay revealed that the nuclear protein MLK3 phosphorylated histone 3 at threonine 3 (H3T3ph). Mutation of a conserved catalytic residue (Lysine 175) abolished the kinase activity and resulted in failure to complement the early flowering phenotype of mlk3 . Interestingly, the global level of H3T3 phosphorylation in mlk3 did not differ significantly from wild type, suggesting the redundant roles of MLKs in flowering regulation. The transcriptomic analysis demonstrated that 425 genes significantly altered expression level in mlk3 relative to wild type. The mlk3 mlk4 double mutant generated by crossing mlk3 with mlk4 , a loss-of-function mutant of MLK4 showing late flowering, flowered between the two parental lines, suggesting that MLK3 played an antagonistic role to MLK4 in plant transition to flowering. Conclusions: A serine/threonine kinase encoding gene MLK3 is a casein kinase 1 specific to the plant species and represses flowering slightly. MLK3 located in nucleus catalyzes the phosphorylation of histone H3 at threonine 3 in vitro and an intact lysine residue (K175) is indispensible for the kinase activity. This study sheds new light on the delicate control of flowering by the plant-specific CK1 in Arabidopsis.

scholarly journals Mammalian Dynamin-like Protein DLP1 Tubulates Membranes

2001 ◽  
Vol 12 (9) ◽  
pp. 2894-2905 ◽  
Author(s):  
Yisang Yoon ◽  
Kelly R. Pitts ◽  
Mark A. McNiven
Keyword(s):  
Cultured Cells ◽  
Immunogold Labeling ◽  
Wild Type ◽  
Tubule Formation ◽  
Defective Mutant ◽  
Ring Structures ◽  
Membrane Tubules ◽  
Large Gtpases

Dynamins are large GTPases with mechanochemical properties that are known to constrict and tubulate membranes. A recently identified mammalian dynamin-like protein (DLP1) is essential for the proper cellular distribution of mitochondria and the endoplasmic reticulum in cultured cells. In this study, we investigated the ability of DLP1 to remodel membranes similar to conventional dynamin. We found that the expression of a GTPase-defective mutant, DLP1-K38A, in cultured cells led to the formation of large cytoplasmic aggregates. Electron microscopy (EM) of cells expressing DLP1-K38A revealed that these aggregates were comprised of membrane tubules of a consistent diameter. High-magnification EM revealed the presence of many regular striations along individual membrane tubules, and immunogold labeling confirmed the association of DLP1 with these structures. Biochemical experiments with the use of recombinant DLP1 and labeled GTP demonstrated that DLP1-K38A binds but does not hydrolyze or release GTP. Furthermore, the affinity of DLP1-K38A for membrane is increased compared with wild-type DLP1. To test whether DLP1 could tubulate membrane in vitro, recombinant DLP1 was combined with synthetic liposomes and nucleotides. We found that DLP1 protein alone assembled into sedimentable macromolecular structures in the presence of guanosine-5′-O-(3-thio)triphosphate (GTPγS) but not GTP. EM of the GTPγS-treated DLP1 revealed clusters of stacked helical ring structures. When liposomes were included with DLP1, formation of long membrane tubules similar in size to those formed in vivo was observed. Addition of GTPγS greatly enhanced membrane tubule formation, suggesting the GTP-bound form of DLP1 deforms liposomes into tubules as the DLP1-K38A does in vivo. These results provide the first evidence that the dynamin family member, DLP1, is able to tubulate membranes both in living cells and in vitro. Furthermore, these findings also indicate that despite the limited homology to conventional dynamins (35%) these proteins remodel membranes in a similar manner.

scholarly journals Adp Ribosylation Factor-like Protein 2 (Arl2) Regulates the Interaction of Tubulin-Folding Cofactor D with Native Tubulin

2000 ◽  
Vol 149 (5) ◽  
pp. 1087-1096 ◽  
Author(s):  
Arunashree Bhamidipati ◽  
Sally A. Lewis ◽  
Nicholas J. Cowan
Keyword(s):  
Cultured Cells ◽  
Gtpase Activating Protein ◽  
Adp Ribosylation ◽  
Chaperone Protein ◽  
Ras Family ◽  
Mutant Forms ◽  
Adp Ribosylation Factor ◽  
Β Tubulin

The ADP ribosylation factor-like proteins (Arls) are a family of small monomeric G proteins of unknown function. Here, we show that Arl2 interacts with the tubulin-specific chaperone protein known as cofactor D. Cofactors C, D, and E assemble the α/β- tubulin heterodimer and also interact with native tubulin, stimulating it to hydrolyze GTP and thus acting together as a β-tubulin GTPase activating protein (GAP). We find that Arl2 downregulates the tubulin GAP activity of C, D, and E, and inhibits the binding of D to native tubulin in vitro. We also find that overexpression of cofactors D or E in cultured cells results in the destruction of the tubulin heterodimer and of microtubules. Arl2 specifically prevents destruction of tubulin and microtubules by cofactor D, but not by cofactor E. We generated mutant forms of Arl2 based on the known properties of classical Ras-family mutations. Experiments using these altered forms of Arl2 in vitro and in vivo demonstrate that it is GDP-bound Arl2 that interacts with cofactor D, thereby averting tubulin and microtubule destruction. These data establish a role for Arl2 in modulating the interaction of tubulin-folding cofactors with native tubulin in vivo.

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