MuSK induces in vivo acetylcholine receptor clusters in a ligand-independent manner

Muscle-specific receptor tyrosine kinase (MuSK) is required for the formation of the neuromuscular junction. Using direct gene transfer into single fibers, MuSK was expressed extrasynaptically in innervated rat muscle in vivo to identify its contribution to synapse formation. Spontaneous MuSK kinase activity leads, in the absence of its putative ligand neural agrin, to the appearance of ϵ-subunit–specific transcripts, the formation of acetylcholine receptor clusters, and acetylcholinesterase aggregates. Expression of kinase-inactive MuSK did not result in the formation of acetylcholine receptor (AChR) clusters, whereas a mutant MuSK lacking the ectodomain did induce AChR clusters. The contribution of endogenous MuSK was excluded by using genetically altered mice, where the kinase domain of the MuSK gene was flanked by loxP sequences and could be deleted upon expression of Cre recombinase. This allowed the conditional inactivation of endogenous MuSK in single muscle fibers and prevented the induction of ectopic AChR clusters. Thus, the kinase activity of MuSK initiates signals that are sufficient to induce the formation of AChR clusters. This process does not require additional determinants located in the ectodomain.

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Nuclear receptor-binding protein 1: a novel tumour suppressor and pseudokinase

Biochemical Society Transactions ◽

10.1042/bst20130069 ◽

2013 ◽

Vol 41 (4) ◽

pp. 1055-1060 ◽

Cited By ~ 9

Author(s):

Jason S. Kerr ◽

Catherine H. Wilson

Keyword(s):

Nuclear Receptor ◽

Receptor Binding ◽

Kinase Activity ◽

Binding Protein ◽

Kinase Domain ◽

Present Review ◽

Critical Components ◽

Receptor Binding Protein

Pseudokinases are a class of kinases which are structurally designated as lacking kinase activity. Despite the lack of kinase domain sequence conservation, there is increasing evidence that a number of pseudokinases retain kinase activity and/or have critical cellular functions, casting aside previous notions that pseudokinases simply exist as redundant kinases. Moreover, a number of recent studies have implicated pseudokinases as critical components in cancer formation and progression. The present review discusses the interactions and potential functions that nuclear receptor-binding protein 1, a pseudokinase recently described to have a tumour-suppressive role in cancer, may play in cellular homoeostasis and protein regulation. The recent findings highlighted in the present review emphasize the requirement to fully determine the function of pseudokinases in vitro and in vivo, the understanding of which may ultimately uncover new directions for drug discovery.

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Pie1, a Protein Interacting with Mec1, Controls Cell Growth and Checkpoint Responses in Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.21.3.755-764.2001 ◽

2001 ◽

Vol 21 (3) ◽

pp. 755-764 ◽

Cited By ~ 71

Author(s):

Tatsushi Wakayama ◽

Tae Kondo ◽

Seiko Ando ◽

Kunihiro Matsumoto ◽

Katsunori Sugimoto

Keyword(s):

Dna Damage ◽

Cell Growth ◽

Kinase Activity ◽

Critical Role ◽

Kinase Domain ◽

Protein Kinase Activity ◽

Replication Checkpoint ◽

Family Protein ◽

Replication Block

ABSTRACT In eukaryotes, the ATM and ATR family proteins play a critical role in the DNA damage and replication checkpoint controls. These proteins are characterized by a kinase domain related to the phosphatidylinositol 3-kinase, but they have the ability to phosphorylate proteins. In budding yeast, the ATR family protein Mec1/Esr1 is essential for checkpoint responses and cell growth. We have isolated the PIE1 gene in a two-hybrid screen for proteins that interact with Mec1, and we show that Pie1 interacts physically with Mec1 in vivo. Like MEC1, PIE1is essential for cell growth, and deletion of the PIE1 gene causes defects in the DNA damage and replication block checkpoints similar to those observed in mec1Δ mutants. Rad53 hyperphosphorylation following DNA damage and replication block is also decreased in pie1Δ cells, as in mec1Δcells. Pie1 has a limited homology to fission yeast Rad26, which forms a complex with the ATR family protein Rad3. Mutation of the region in Pie1 homologous to Rad26 results in a phenotype similar to that of thepie1Δ mutation. Mec1 protein kinase activity appears to be essential for checkpoint responses and cell growth. However, Mec1 kinase activity is unaffected by the pie1Δ mutation, suggesting that Pie1 regulates some essential function other than Mec1 kinase activity. Thus, Pie1 is structurally and functionally related to Rad26 and interacts with Mec1 to control checkpoints and cell proliferation.

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Negative regulation of Raf-1 by phosphorylation of serine 621.

Molecular and Cellular Biology ◽

10.1128/mcb.16.10.5409 ◽

1996 ◽

Vol 16 (10) ◽

pp. 5409-5418 ◽

Cited By ~ 151

Author(s):

H Mischak ◽

T Seitz ◽

P Janosch ◽

M Eulitz ◽

H Steen ◽

...

Keyword(s):

Catalytic Activity ◽

Kinase Activity ◽

Phosphorylation Site ◽

Kinase Domain ◽

Negative Regulation ◽

Catalytic Function ◽

Dependent Protein ◽

The One

The elevation of cyclic AMP (cAMP) levels in the cell downregulates the activity of the Raf-1 kinase. It has been suggested that this effect is due to the activation of cAMP-dependent protein kinase (PKA), which can directly phosphorylate Raf-1 in vitro. In this study, we confirmed this hypothesis by coexpressing Raf-1 with the constitutively active catalytic subunit of PKA, which could fully reproduce the inhibition previously achieved by cAMP. PKA-phosphorylated Raf-1 exhibits a reduced affinity for GTP-loaded Ras as well as impaired catalytic activity. As the binding to GTP-loaded Ras induces Raf-1 activation in the cell, we examined which mechanism is required for PKA-mediated Raf-1 inhibition in vivo. A Raf-1 point mutant (RafR89L), which is unable to bind Ras, as well as the isolated Raf-1 kinase domain were still fully susceptible to inhibition by PKA, demonstrating that the phosphorylation of the Raf-1 kinase suffices for inhibition. By the use of mass spectroscopy and point mutants, PKA phosphorylation site was mapped to a single site in the Raf-1 kinase domain, serine 621. Replacement of serine 621 by alanine or cysteine or destruction of the PKA consensus motif by changing arginine 618 resulted in the loss of catalytic activity. Notably, a mutation of serine 619 to alanine did not significantly affect kinase activity or regulation by activators or PKA. Changing serine 621 to aspartic acid yielded a Raf-1 protein which, when expressed to high levels in Sf-9 insect cells, retained a very low inducible kinase activity that was resistant to PKA downregulation. The purified Raf-1 kinase domain displayed slow autophosphorylation of serine 621, which correlated with a decrease in catalytic function. The Raf-1 kinase domain activated by tyrosine phosphorylation could be downregulated by PKA. Specific removal of the phosphate residue at serine 621 reactivated the catalytic activity. These results are most consistent with a dual role of serine 621. On the one hand, serine 621 appears essential for catalytic activity; on the other hand, it serves as a phosphorylation site which confers negative regulation.

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Direct gene transfer into rat articular cartilage by in vivo electroporation

The FASEB Journal ◽

10.1096/fj.02-0518com ◽

2003 ◽

Vol 17 (8) ◽

pp. 829-835 ◽

Cited By ~ 26

Author(s):

Lagrent Grossin ◽

Christel Cournil-Henrionnet ◽

Lluis M. Mir ◽

Bertrand Liagre ◽

Dominique Dumas ◽

...

Keyword(s):

Articular Cartilage ◽

Gene Transfer ◽

Direct Gene Transfer ◽

In Vivo Electroporation ◽

Direct Gene

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

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1620315114 ◽

2017 ◽

Vol 114 (5) ◽

pp. E879-E886 ◽

Cited By ~ 19

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.

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Antibodies to insulin receptor tyrosine kinase stimulate its activity towards exogenous substrates without inducing receptor autophosphorylation

Biochemical Journal ◽

10.1042/bj2600749 ◽

1989 ◽

Vol 260 (3) ◽

pp. 749-756 ◽

Cited By ~ 11

Author(s):

V Baron ◽

N Gautier ◽

N Rochet ◽

R Ballotti ◽

B Rossi ◽

...

Keyword(s):

Growth Factor ◽

Tyrosine Kinase ◽

Insulin Receptor ◽

Receptor Tyrosine Kinase ◽

Kinase Activity ◽

Kinase Domain ◽

Tyrosine Kinase Domain ◽

Insulin Receptor Tyrosine Kinase ◽

Substrate Phosphorylation ◽

Exogenous Substrates

Anti-peptide antibodies directed against a highly-conserved sequence of the insulin receptor tyrosine kinase domain have been used to study the relationship between this specific region and kinase activation. Antibodies have been prepared by the injection into a rabbit of a synthetic peptide (P2) corresponding to residues 1110-1125 of the proreceptor. The peptide exhibits 88-95% sequence similarity with the corresponding sequence in the v-ros protein and in receptors for epidermal growth factor and for insulin-like growth factor 1. Two antibodies with different specificities could be separated from total antiserum obtained after immunization with P2. One antibody [anti-(P-Tyr)] cross-reacted with phosphotyrosine and immunoprecipitated solely autophosphorylated receptors. This antibody was shown to increase or decrease the receptor tyrosine kinase activity depending on its concentration. In all circumstances receptor autophosphorylation and substrate phosphorylation were modulated in a parallel fashion. The second antibody (anti-P2) failed to immunoprecipitate the insulin receptor, but was found to interact with both the peptide and the receptor by e.l.i.s.a. assay. Using a tyrosine co-polymer we found that anti-P2 activated the insulin receptor kinase leading to substrate phosphorylation at a level similar to that observed with insulin. This effect was additive to the hormonal effect. In contrast, receptor autophosphorylation was not modified by the anti-peptide. The differential effect of this anti-peptide further supports the idea that receptor autophosphorylation and kinase activity towards exogenous substrates might be independently regulated. Finally, our data suggest that conformational changes in the receptor tyrosine kinase domain may be sufficient for activation of its enzymic activity.

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Interaction of activated Cdc42-associated tyrosine kinase ACK2 with HSP90

Biochemical Journal ◽

10.1042/bj20040623 ◽

2004 ◽

Vol 382 (1) ◽

pp. 199-204 ◽

Cited By ~ 10

Author(s):

Wannian YANG ◽

Jaclyn M. JANSEN ◽

Qiong LIN ◽

Sabrina CANOVA ◽

Richard A. CERIONE ◽

...

Keyword(s):

Tyrosine Kinase ◽

Kinase Activity ◽

Heat Shock Protein 90 ◽

Kinase Domain ◽

Atpase Inhibitor ◽

Sorting Nexin ◽

Downstream Effector ◽

Tyrosine Kinase 2 ◽

Functional Complex

ACK2 (activated Cdc42-associated tyrosine kinase 2) is a specific downstream effector for Cdc42, a member of the Rho family of small G-proteins. ACK2 interacts with clathrin, an endocytic vesicle coating protein, and SH3PX1, a sorting nexin, and is involved in clathrin-mediated endocytosis. While searching for proteins that interact with ACK2, we found that HSP90 (heat-shock protein 90) binds to ACK2. Analysis of a series of truncation mutants of ACK2 has defined the regions within the kinase domain of ACK2 that are required for binding to HSP90. The binding of HSP90 to ACK2 is blocked upon treatment with geldanamycin, an HSP90-specific ATPase inhibitor, and is required for the in vivo kinase activity of ACK2 and its association with Cdc42. Overall, our data suggest a novel mechanism of regulation in which HSP90 serves as a regulatory component in an ACK2 functional complex and plays a role in sustaining its kinase activity.

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A vesicle surface tyrosine kinase regulates phagosome maturation

The Journal of Cell Biology ◽

10.1083/jcb.200701023 ◽

2007 ◽

Vol 178 (3) ◽

pp. 411-423 ◽

Cited By ~ 14

Author(s):

Jun Fang ◽

Joseph A. Brzostowski ◽

Stephen Ou ◽

Nilgun Isik ◽

Vinod Nair ◽

...

Keyword(s):

Tyrosine Kinase ◽

Receptor Tyrosine Kinase ◽

Molecular Mechanisms ◽

Control Mechanism ◽

Kinase Domain ◽

Microbial Pathogens ◽

Phagosome Maturation ◽

Tyrosine Kinase Signaling ◽

Late Endosomes

Phagocytosis is crucial for host defense against microbial pathogens and for obtaining nutrients in Dictyostelium discoideum. Phagocytosed particles are delivered via a complex route from phagosomes to lysosomes for degradation, but the molecular mechanisms involved in the phagosome maturation process are not well understood. Here, we identify a novel vesicle-associated receptor tyrosine kinase-like protein, VSK3, in D. discoideum. We demonstrate how VSK3 is involved in phagosome maturation. VSK3 resides on the membrane of late endosomes/lysosomes with its C-terminal kinase domain facing the cytoplasm. Inactivation of VSK3 by gene disruption reduces the rate of phagocytosis in cells, which is rescued by re-expression of VSK3. We found that the in vivo function of VSK3 depends on the presence of the kinase domain and vesicle localization. Furthermore, VSK3 is not essential for engulfment, but instead, is required for the fusion of phagosomes with late endosomes/lysosomes. Our findings suggest that localized tyrosine kinase signaling on the surface of endosome/lysosomes represents a control mechanism for phagosome maturation.

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The C-terminal fragment of LRRK2 with the G2019S substitution increases the neurotoxicity of mutant A53T α-synuclein in dopaminergic neurons in vivo

10.21203/rs.3.rs-24218/v2 ◽

2020 ◽

Author(s):

Noémie Cresto ◽

Camille Gardier ◽

Marie-Claude Gaillard ◽

Francesco Gubinelli ◽

Pauline Roost ◽

...

Keyword(s):

Kinase Activity ◽

Neuronal Loss ◽

Cell Loss ◽

Kinase Domain ◽

Alpha Synuclein ◽

Significant Loss ◽

Histological Evaluation ◽

Mutant Form ◽

G2019s Mutation

Abstract Background: Alpha-synuclein (α-syn) and leucine-rich repeat kinase 2 (LRRK2) likely play crucial roles both in sporadic and familial forms of Parkinson’s disease (PD). The most prevalent mutation in LRRK2 is the G2019S substitution, which induces neurotoxicity through increased kinase activity. There is likely an interplay between LRRK2 and α-syn involved in the neurodegeneration of dopaminergic (DA) neurons in the substantia nigra (SNpc) in PD. However, the mechanisms underlying this interplay are ill-defined. Here, we investigated whether LRRK2 G2019S can increase the neurotoxicity induced by a mutant form of α-syn (A53T mutation) in DA neurons in vivo . Methods: We used a co-transduction approach with adeno-associated virus (AAV), AAV2/6 vectors encoding human α-syn A53T and the C-terminal portion of LRRK2 (ΔLRRK2), which contains the kinase domain, with either the G2019S mutation (ΔLRRK2 G2019S ) alone or the D1994A mutation (ΔLRRK2 G2019S/D1994A ), which inactivates the kinase activity of LRRK2. The AAVs were co-injected into the rat SNpc and histological evaluation was performed at 6- and 15-weeks post-injection (PI). Results: The majority of SNpc neurons co-expressed ΔLRRK2 and human α-syn A53T after transduction. ΔLRRK2 G2019S alone produced no cell loss at 15-weeks PI. Injection of AAV-α-syn A53T alone or mixed with a control AAV coding for GFP produced a significant loss of DA neurons. Co-injection of AAV-α-syn A53T with AAV-ΔLRRK2 G2019S instead of GFP slightly exacerbated that neuronal loss We also studied the inactive form, ΔLRRK2 G2019S/D1994A at 6 weeks PI. Injection of AAV-ΔLRRK2 G2019S mixed with AAV-α-syn A53T produced a neurotoxic effect that was stronger than that produced by the co-injection of AAV-DLRRK2 G2019S/D1994A and AAV-α-syn A53T . Conclusion: Thus, these results show that mutant LRRK2 may selectively facilitate α-syn toxicity in DA neurons through a cell-autonomous mechanism involving its kinase domain. However, considering that the effect of ΔLRRK2 G2019S upon human α-syn A53T is moderate in our paradigm where pathological proteins are overexpressed, the study supports the hypothesis that the interplay between LRRK2 and α-syn may also implicate non-cell-autonomous mechanisms such as those involved in neuroinflammation and spreading of α-syn aggregated species.

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Src acts as the target of matrine to inhibit the proliferation of cancer cells by regulating phosphorylation signaling pathways

10.22541/au.160619952.26820683/v1 ◽

2020 ◽

Author(s):

Xi Zhang ◽

Hui Xu ◽

Xiaoyang Bi ◽

Guoqing Hou ◽

Andong Liu ◽

...

Keyword(s):

Mass Spectrometry ◽

Cancer Cells ◽

Signaling Pathways ◽

Kinase Activity ◽

Src Kinase ◽

Kinase Domain ◽

Akt Phosphorylation ◽

In Vivo Experiments

Background and Purpose: Identification of accurate targets is essential for a successful development of targeted therapy in cancer. Studies have shown that matrine has antitumor activity against many types of cancers. However, the direct target in cancer cells of its anticancer effect has not been identified. The purpose of this study was to find the molecular target of matrine to inhibit the proliferation of cancer cells and explore its mechanism of action. Experimental Approach: The effect of matrine on the proliferation of cancer cells were examined by MTT assay. Pull-down assay and liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) were performed to explore the target of matrine. A series of in vitro and in vivo experiments were conducted to reveal the mechanisms by which matrine targeted Src to regulate the downstream signaling pathways of Src in cancer cells. Key Results: Herein we showed that matrine inhibited the proliferation of cancer in vitro and in vivo. Pull-down assay with matrine-amino coupling resins (MA beads) and LC-MS/MS identified Src as the target of matrine. Src kinase domain is required for its interaction with matrine and Ala392 in the kinase domain participated in matrine-Src interaction. Intriguingly, matrine was proven to inhibit Src kinase activity in a non-ATP-competitive manner by blocking the autophosphorylation of Tyr419. Matrine down-regulated the phosphorylation levels of MAPK/ERK, JAK2/STAT3 and PI3K/Akt signaling pathways. Conclusions and Implications: Collectively, matrine targeted Src, inhibited kinase activity and down-regulated its downstream MAPK/ERK, JAK2/STAT3 and PI3K/Akt phosphorylation signaling pathways to inhibit the proliferation of cancer cells.

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