scholarly journals Anterograde trafficking of ciliary MAP kinase–like ICK/CILK1 by the intraflagellar transport machinery is required for intraciliary retrograde protein trafficking

2020 ◽  
Vol 295 (38) ◽  
pp. 13363-13376 ◽  
Author(s):  
Kentaro Nakamura ◽  
Tatsuro Noguchi ◽  
Mariko Takahara ◽  
Yoshihiro Omori ◽  
Takahisa Furukawa ◽  
...  
Keyword(s):  
Protein Trafficking ◽  
Intraflagellar Transport ◽  
Extracellular Vesicle ◽  
Mitogen Activated Protein Kinase ◽  
Ciliary Protein ◽  
Mitogen Activated Protein ◽  
Exogenous Expression ◽  
Bidirectional Movement ◽  
Dependent Transport ◽  
G Protein Coupled

ICK (also known as CILK1) is a mitogen-activated protein kinase–like kinase localized at the ciliary tip. Its deficiency is known to result in the elongation of cilia and causes ciliopathies in humans. However, little is known about how ICK is transported to the ciliary tip. We here show that the C-terminal noncatalytic region of ICK interacts with the intraflagellar transport (IFT)–B complex of the IFT machinery and participates in its transport to the ciliary tip. Furthermore, total internal reflection fluorescence microscopy demonstrated that ICK undergoes bidirectional movement within cilia, similarly to IFT particles. Analysis of ICK knockout cells demonstrated that ICK deficiency severely impairs the retrograde trafficking of IFT particles and ciliary G protein–coupled receptors. In addition, we found that in ICK knockout cells, ciliary proteins are accumulated at the bulged ciliary tip, which appeared to be torn off and released into the environment as an extracellular vesicle. The exogenous expression of various ICK constructs in ICK knockout cells indicated that the IFT-dependent transport of ICK, as well as its kinase activity and phosphorylation at the canonical TDY motif, is essential for ICK function. Thus, we unequivocally show that ICK transported to the ciliary tip is required for retrograde ciliary protein trafficking and consequently for normal ciliary function.

scholarly journals CCRK/CDK20 regulates ciliary retrograde protein trafficking via interacting with BROMI/TBC1D32

PLoS ONE ◽  
2021 ◽  
Vol 16 (10) ◽  
pp. e0258497
Author(s):  
Tatsuro Noguchi ◽  
Kentaro Nakamura ◽  
Yuuki Satoda ◽  
Yohei Katoh ◽  
Kazuhisa Nakayama
Keyword(s):  
Extracellular Vesicles ◽  
Protein Trafficking ◽  
Hedgehog Signaling ◽  
Intraflagellar Transport ◽  
Wild Type ◽  
Ciliary Membrane ◽  
Ciliary Protein ◽  
Exogenous Expression ◽  
Turnaround Process

CCRK/CDK20 was reported to interact with BROMI/TBC1D32 and regulate ciliary Hedgehog signaling. In various organisms, mutations in the orthologs of CCRK and those of the kinase ICK/CILK1, which is phosphorylated by CCRK, are known to result in cilia elongation. Furthermore, we recently showed that ICK regulates retrograde ciliary protein trafficking and/or the turnaround event at the ciliary tips, and that its mutations result in the elimination of intraflagellar transport (IFT) proteins that have overaccumulated at the bulged ciliary tips as extracellular vesicles, in addition to cilia elongation. However, how these proteins cooperate to regulate ciliary protein trafficking has remained unclear. We here show that the phenotypes of CCRK-knockout (KO) cells closely resemble those of ICK-KO cells; namely, the overaccumulation of IFT proteins at the bulged ciliary tips, which appear to be eliminated as extracellular vesicles, and the enrichment of GPR161 and Smoothened on the ciliary membrane. The abnormal phenotypes of CCRK-KO cells were rescued by the exogenous expression of wild-type CCRK but not its kinase-dead mutant or a mutant defective in BROMI binding. These results together indicate that CCRK regulates the turnaround process at the ciliary tips in concert with BROMI and probably via activating ICK.

scholarly journals Intraflagellar transport-A complex mediates ciliary entry and retrograde trafficking of ciliary G protein–coupled receptors

2017 ◽  
Vol 28 (3) ◽  
pp. 429-439 ◽  
Author(s):  
Tomoaki Hirano ◽  
Yohei Katoh ◽  
Kazuhisa Nakayama
Keyword(s):  
G Protein ◽  
Protein Interactions ◽  
Protein Trafficking ◽  
Intraflagellar Transport ◽  
G Protein Coupled Receptors ◽  
Protein Protein Interactions ◽  
Retrograde Trafficking ◽  
The Core ◽  
Ciliary Protein ◽  
G Protein Coupled

Cilia serve as cellular antennae where proteins involved in sensory and developmental signaling, including G protein–coupled receptors (GPCRs), are specifically localized. Intraflagellar transport (IFT)-A and -B complexes mediate retrograde and anterograde ciliary protein trafficking, respectively. Using a visible immunoprecipitation assay to detect protein–protein interactions, we show that the IFT-A complex is divided into a core subcomplex, composed of IFT122/IFT140/IFT144, which is associated with TULP3, and a peripheral subcomplex, composed of IFT43/IFT121/IFT139, where IFT139 is most distally located. IFT139-knockout (KO) and IFT144-KO cells demonstrated distinct phenotypes: IFT139-KO cells showed the accumulation of IFT-A, IFT-B, and GPCRs, including Smoothened and GPR161, at the bulged ciliary tips; IFT144-KO cells showed failed ciliary entry of IFT-A and GPCRs and IFT-B accumulation at the bulged tips. These observations demonstrate the distinct roles of the core and peripheral IFT-A subunits: IFT139 is dispensable for IFT-A assembly but essential for retrograde trafficking of IFT-A, IFT-B, and GPCRs; in contrast, IFT144 is essential for functional IFT-A assembly and ciliary entry of GPCRs but dispensable for anterograde IFT-B trafficking. Thus the data presented here demonstrate that the IFT-A complex mediates not only retrograde trafficking but also entry into cilia of GPCRs.

scholarly journals Cooperation of the IFT-A complex with the IFT-B complex is required for ciliary retrograde protein trafficking and GPCR import

2021 ◽  
Vol 32 (1) ◽  
pp. 45-56
Author(s):  
Takuya Kobayashi ◽  
Yamato Ishida ◽  
Tomoaki Hirano ◽  
Yohei Katoh ◽  
Kazuhisa Nakayama
Keyword(s):  
G Protein ◽  
Protein Trafficking ◽  
Intraflagellar Transport ◽  
G Protein Coupled Receptors ◽  
Retrograde Trafficking ◽  
Ciliary Protein ◽  
G Protein Coupled

Little is known about how the IFT-A and IFT-B complexes of the intraflagellar transport machinery cooperate to mediate ciliary protein trafficking. We show that IFT144-IFT122 and IFT88-IFT52-IFT46 contribute to the IFT-A-IFT-B interface and that the interaction of IFT-A with IFT-B is required for retrograde trafficking of the IFT machinery and ciliary entry of G protein-coupled receptors.

scholarly journals Interaction of WDR60 intermediate chain with TCTEX1D2 light chain of the dynein-2 complex is crucial for ciliary protein trafficking

2018 ◽  
Vol 29 (13) ◽  
pp. 1628-1639 ◽  
Author(s):  
Yuki Hamada ◽  
Yuta Tsurumi ◽  
Shohei Nozaki ◽  
Yohei Katoh ◽  
Kazuhisa Nakayama
Keyword(s):  
Cell Lines ◽  
Light Chain ◽  
Protein Trafficking ◽  
Intraflagellar Transport ◽  
Immunoprecipitation Assay ◽  
Established Cell Lines ◽  
Ciliary Protein ◽  
Established Cell ◽  
Exogenous Expression ◽  
Auxiliary Role

The dynein-2 complex mediates trafficking of ciliary proteins by powering the intraflagellar transport (IFT) machinery containing IFT-A and IFT-B complexes. Although 11 subunits are known to constitute the dynein-2 complex, with several light-chain subunits shared by the dynein-1 complex, the overall architecture of the dynein-2 complex has not been fully clarified. Utilizing the visible immunoprecipitation assay, we demonstrated the interaction modes among the dynein-2 subunits, including previously undefined interactions, such as that between WDR60 and the TCTEX1D2–DYNLT1/DYNLT3 dimer. The dynein-2 complex can be divided into three subcomplexes, namely DYNC2H1–DYNC2LI1, WDR34–DYNLL1/DYNLL2–DYNLRB1/DYNLRB2, and WDR60–TCTEX1D2–DYNLT1/DYNLT3. We established cell lines lacking WDR60 or TCTEX1D2, both of which are dynein-2–specific subunits encoded by ciliopathy-causing genes, and found that both WDR60-knockout (KO) and TCTEX1D2-KO cells show defects in retrograde ciliary protein trafficking, with WDR60-KO cells demonstrating more severe defects probably due to failed assembly of the dynein-2 complex. The exogenous expression of a WDR60 mutant lacking TCTEX1D2 binding partially restored retrograde trafficking to a level comparable to that of TCTEX1D2-KO cells. Thus, our results demonstrated that WDR60 plays a major role and TCTEX1D2 plays an auxiliary role in the dynein-2 complex to mediate retrograde ciliary protein trafficking.

scholarly journals G Protein-Coupled Receptor-Mediated Mitogen-Activated Protein Kinase Activation through Cooperation of Gαq and Gαi Signals

2000 ◽  
Vol 20 (18) ◽  
pp. 6837-6848 ◽  
Author(s):  
Andree Blaukat ◽  
Ana Barac ◽  
Michael J. Cross ◽  
Stefan Offermanns ◽  
Ivan Dikic
Keyword(s):  
Protein Kinase ◽  
G Protein ◽  
Tyrosine Kinases ◽  
Synergistic Effects ◽  
Mitogen Activated Protein Kinase ◽  
General Mechanism ◽  
Mitogen Activated Protein ◽  
Novel Concept ◽  
G Protein Coupled ◽  
Stimulation Of

ABSTRACT G protein-coupled receptors (GPCRs) have been shown to stimulate extracellular regulated kinases (ERKs) through a number of linear pathways that are initiated by Gq/11 or Giproteins. We studied signaling to the ERK cascade by receptors that simultaneously activate both G protein subfamilies. In HEK293T cells, bradykinin B2 receptor (B2R)-induced stimulation of ERK2 and transcriptional activity of Elk1 are dependent on Gαq-mediated protein kinase C (PKC) and on Gαi-induced Ras activation, while they are independent of Gβγ subunits, phosphatidylinositol 3-kinase, and tyrosine kinases. Similar results were obtained with m1 and m3muscarinic receptors in HEK293T cells and with the B2R in human and mouse fibroblasts, indicating a general mechanism in signaling toward the ERK cascade. Furthermore, the bradykinin-induced activation of ERK is strongly reduced in Gαq/11-deficient fibroblasts. In addition, we found that constitutively active mutants of Gαq/11 or Gαi proteins alone poorly stimulate ERK2, whereas a combination of both led to synergistic effects. We conclude that dually coupled GPCRs require a cooperation of Gαi- and Gq/11-mediated pathways for efficient stimulation of the ERK cascade. Cooperative signaling by multiple G proteins thus might represent a novel concept implicated in the regulation of cellular responses by GPCRs.

scholarly journals Neutrophils Stimulated with a Variety of Chemoattractants Exhibit Rapid Activation of p21-Activated Kinases (Paks): Separate Signals Are Required for Activation and Inactivation of Paks

1998 ◽  
Vol 18 (12) ◽  
pp. 7130-7138 ◽  
Author(s):  
RiYun Huang ◽  
Jian P. Lian ◽  
Dwight Robinson ◽  
John A. Badwey
Keyword(s):  
G Proteins ◽  
Platelet Activating Factor ◽  
Cell Types ◽  
Mitogen Activated Protein Kinase ◽  
Transient Activation ◽  
Anaphylatoxin C5a ◽  
Mitogen Activated Protein ◽  
Fmlp Receptor ◽  
Rapid Activation ◽  
G Protein Coupled

ABSTRACT Activation of the p21-activated protein kinases (Paks) was compared in neutrophils stimulated with a wide variety of agonists that bind to receptors coupled to heterotrimeric G proteins. Neutrophils stimulated with sulfatide, a ligand for the L-selectin receptor, or the chemoattractant fMet-Leu-Phe (fMLP), platelet-activating factor, leukotriene B4, interleukin-8, or the chemokine RANTES exhibited a rapid and transient activation of the 63- and 69-kDa Paks. These kinases exhibited maximal activation with each of these agonists within 15 s followed by significant inactivation at 3 min. In contrast, neutrophils treated with the chemoattractant and anaphylatoxin C5a exhibited a prolonged activation (>15 min) of these Paks even though the receptor for this ligand may activate the same overall population of complex G proteins as the fMLP receptor. Addition of fMLP to neutrophils already stimulated with C5a resulted in the inactivation of the 63- and 69-kDa Paks. Optimal activation of Paks could be observed at concentrations of these agonists that elicited only shape changes and chemotaxis in neutrophils. While all of the agonists listed above triggered quantitatively similar activation of the 63- and 69-kDa Paks, fMLP was far superior to the other stimuli in triggering activation of the c-Jun N-terminal kinase (JNK) and the p38 mitogen-activated protein kinase (MAPK). These data indicate that separate signals are required for activation and inactivation of Paks and that, in contrast to other cell types, activated Pak does not trigger activation of JNK or p38-MAPK in neutrophils. These results are consistent with the recent hypothesis that G-protein-coupled receptors may initiate signals independent of those transmitted by the α and βγ subunits of complex G proteins.

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