Characterization of CAP1 and ECA4 adaptors participating in clathrin-mediated endocytosis

2022 ◽  
Author(s):  
Maciek Adamowski ◽  
Ivana Matijević ◽  
Jiří Friml
Keyword(s):  
Fluorescent Protein ◽  
Phosphorylation Site ◽  
Large Family ◽  
Adaptor Proteins ◽  
Serine Phosphorylation ◽  
Binding Motif ◽  
Double Knockout ◽  
Coated Pits ◽  
Knockout Mutants ◽  
Golgi Network

Formation of endomembrane vesicles is crucial in all eukaryotic cells and relies on vesicle coats such as clathrin. Clathrin-coated vesicles form at the plasma membrane and the trans-Golgi Network. They contain adaptor proteins, which serve as binding bridges between clathrin, vesicle membranes, and cargoes. A large family of monomeric ANTH/ENTH/VHS adaptors is present in A. thaliana. Here, we characterize two homologous ANTH-type clathrin adaptors, CAP1 and ECA4, in clathrin-mediated endocytosis (CME). CAP1 and ECA4 are recruited to sites at the PM identified as clathrin-coated pits (CCPs), where they occasionally exhibit early bursts of high recruitment. Subcellular binding preferences of N- and C-terminal fluorescent protein fusions of CAP1 identified a functional adaptin-binding motif in the unstructured tails of CAP1 and ECA4. In turn, no function can be ascribed to a double serine phosphorylation site conserved in these proteins. Double knockout mutants do not exhibit deficiencies in general development or CME, but a contribution of CAP1 and ECA4 to these processes is revealed in crosses into sensitized endocytic mutant backgrounds. Overall, our study documents a contribution of CAP1 and ECA4 to CME in A. thaliana and opens questions about functional redundancy among non-homologous vesicle coat components.

scholarly journals Kinase activity of TBK1 is required for its binding to STING, but not for its recruitment to the Golgi

2021 ◽  
Author(s):  
Haruka Kemmoku ◽  
Yoshihiko Kuchitsu ◽  
Kojiro Mukai ◽  
Tomohiko Taguchi
Keyword(s):  
Kinase Activity ◽  
Fluorescent Protein ◽  
Super Resolution ◽  
Cell System ◽  
Interferon Response ◽  
Type I ◽  
Double Knockout ◽  
Binding Interface ◽  
A Cell ◽  
Golgi Network

AbstractStimulator of interferon genes (STING) is an innate immune protein for DNA pathogens. In response to the emergence of DNA in the cytosol, STING relocates from the endoplasmic reticulum (ER) to the Golgi and induces the type I interferon response through cytosolic TANK-binding kinase 1 (TBK1). The molecular mechanism underlying TBK1 activation by STING remains poorly understood. Here we report a cell system by which STING and TBK1 are simultaneously monitored. The system utilizes STING/TBK1-double knockout (KO) cells, fluorescent protein-tagged TBK1 and STING, and super-resolution microscopy. After STING stimulation, TBK1 is directly recruited to the trans-Golgi network (TGN), not to the other parts of the Golgi. The recruitment of TBK1 does not require its kinase activity. C-terminal STING variants (ΔC9 and L373A), in which the TBK1-STING binding interface is mutated or deleted, induce the recruitment of TBK1. These results indicate that the kinase activity of TBK1 or the C-terminal motif of STING is not required for its recruitment to TGN, but rather for the formation of the stable STING signalling complex at TGN.

scholarly journals Importance of Constitutive Activity and Arrestin-Independent Mechanisms for Intracellular Trafficking of the Ghrelin Receptor

2007 ◽  
Vol 21 (12) ◽  
pp. 3100-3112 ◽  
Author(s):  
Nicholas D. Holliday ◽  
Birgitte Holst ◽  
Elena A. Rodionova ◽  
Thue W. Schwartz ◽  
Helen M. Cox
Keyword(s):  
Fluorescent Protein ◽  
Adaptor Proteins ◽  
Dominant Negative ◽  
Hek293 Cells ◽  
Constitutive Activity ◽  
Double Knockout ◽  
Ghrelin Receptor ◽  
Naturally Occurring ◽  
Clathrin Adaptor ◽  
Green Fluorescent

Abstract The ghrelin receptor (GhrelinR) and its related orphan GPR39 each display constitutive signaling, but only GhrelinRs undergo basal internalization. Here we investigate these differences by considering the roles of the C tail receptor domains for constitutive internalization and activity. Furthermore the interaction between phosphorylated receptors and β-arrestin adaptor proteins has been examined. Replacement of the FLAG-tagged GhrelinR C tail with the equivalent GPR39 domain (GhR-39 chimera) preserved Gq signaling. However in contrast to the GhrelinR, GhR-39 receptors exhibited no basal and substantially decreased agonist-induced internalization in transiently transfected HEK293 cells. Internalized GhrelinR and GhR-39 were predominantly localized to recycling compartments, identified with transferrin and the monomeric G proteins Rab5 and Rab11. Both the inverse agonist [d-Arg1, d-Phe5, d-Trp7,9, Leu11] substance P and a naturally occurring mutant GhrelinR (A204E) with eliminated constitutive activity inhibited basal GhrelinR internalization. Surprisingly, we found that noninternalizing GPR39 was highly phosphorylated and that basal and agonist-induced phosphorylation of the GhR-39 chimera was elevated compared with GhrelinRs. Moreover, basal GhrelinR endocytosis occurred without significant phosphorylation, and it was not prevented by cotransfection of a dominant-negative β-arrestin1(319–418) fragment or by expression in β-arrestin1/2 double-knockout mouse embryonic fibroblasts. In contrast, agonist-stimulated GhrelinRs recruited the clathrin adaptor green fluorescent protein-tagged β-arrestin2 to endosomes, coincident with increased receptor phosphorylation. Thus, GhrelinR internalization to recycling compartments depends on C-terminal motifs and constitutive activity, but the high levels of GPR39 phosphorylation, and of the GhR-39 chimera, are not sufficient to drive endocytosis. In addition, basal GhrelinR internalization occurs independently of β-arrestins.

scholarly journals Itk promotes the integration of TCR and CD28 costimulation, through its direct substrates, SLP-76 and Gads

2020 ◽  
Author(s):  
Enas Hallumi ◽  
Rose Shalah ◽  
Wan-Lin Lo ◽  
Jasmin Corso ◽  
Ilana Oz ◽  
...  
Keyword(s):  
Signaling Pathways ◽  
Phosphorylation Site ◽  
Transcriptional Response ◽  
Adaptor Proteins ◽  
Binding Motif ◽  
Costimulatory Receptor ◽  
Signaling Complex ◽  
Cd28 Costimulation ◽  
Regulatory Module ◽  
Signaling Function

AbstractThe costimulatory receptor, CD28, synergizes with the T cell antigen receptor (TCR) to promote IL-2 production, cell survival and proliferation. Despite their profound synergy, the obligatory interdependence of the signaling pathways initiated by these two receptors is not well understood. Upon TCR stimulation, Gads, a Grb2-family adaptor, bridges the interaction of two additional adaptors, LAT and SLP-76, to form a TCR-induced effector signaling complex. SLP-76 binds the Tec-family tyrosine kinase, Itk, which phosphorylates SLP-76 at Y173 and PLC-γ1 at Y783. Here we identified Gads Y45 as an additional TCR-inducible, Itk-mediated phosphorylation site. Y45 is found within the N-terminal SH3 domain of Gads, an evolutionarily conserved domain with no known binding partners or signaling function. Gads Y45 phosphorylation depended on the interaction of Gads with SLP-76 and on the preferentially-paired binding of Gads to phospho-LAT. Three Itk-related features, Gads Y45, SLP-76 Y173, and a proline-rich Itk SH3-binding motif on SLP-76, were selectively required for activation of the CD28 RE/AP transcriptional element from the IL-2 promoter, but were not required to activate NFAT. This study illuminates a new regulatory module, in which Itk-targeted phosphorylation sites on two adaptor proteins, SLP-76 and Gads, control the transcriptional response to TCR/CD28 costimulation, thus enforcing the obligatory interdependence of the TCR and CD28 signaling pathways.

scholarly journals PKC-phosphorylation of Liprin-α3 triggers phase separation and controls presynaptic active zone structure

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Javier Emperador-Melero ◽  
Man Yan Wong ◽  
Shan Shan H. Wang ◽  
Giovanni de Nola ◽  
Hajnalka Nyitrai ◽  
...  
Keyword(s):  
Phase Separation ◽  
Active Zone ◽  
Neurotransmitter Release ◽  
Hippocampal Neurons ◽  
Phosphorylation Site ◽  
Double Knockout ◽  
Zone Structure ◽  
Presynaptic Nerve Terminal ◽  
Condensate Formation ◽  
And Function

AbstractThe active zone of a presynaptic nerve terminal defines sites for neurotransmitter release. Its protein machinery may be organized through liquid–liquid phase separation, a mechanism for the formation of membrane-less subcellular compartments. Here, we show that the active zone protein Liprin-α3 rapidly and reversibly undergoes phase separation in transfected HEK293T cells. Condensate formation is triggered by Liprin-α3 PKC-phosphorylation at serine-760, and RIM and Munc13 are co-recruited into membrane-attached condensates. Phospho-specific antibodies establish phosphorylation of Liprin-α3 serine-760 in transfected cells and mouse brain tissue. In primary hippocampal neurons of newly generated Liprin-α2/α3 double knockout mice, synaptic levels of RIM and Munc13 are reduced and the pool of releasable vesicles is decreased. Re-expression of Liprin-α3 restored these presynaptic defects, while mutating the Liprin-α3 phosphorylation site to abolish phase condensation prevented this rescue. Finally, PKC activation in these neurons acutely increased RIM, Munc13 and neurotransmitter release, which depended on the presence of phosphorylatable Liprin-α3. Our findings indicate that PKC-mediated phosphorylation of Liprin-α3 triggers its phase separation and modulates active zone structure and function.

scholarly journals Co-relation with novel phosphorylation sites of IκBα and necroptosis in breast cancer cells

BMC Cancer ◽  
2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Sung Hoon Choi ◽  
Hee-Sub Yoon ◽  
Shin-Ae Yoo ◽  
Sung Ho Yun ◽  
Joo-Hee Park ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cancer Cells ◽  
Breast Cancer Cells ◽  
Phosphorylation Site ◽  
Aurora Kinase ◽  
Serine Phosphorylation ◽  
Flight Mass Spectrometry ◽  
Iκb Kinase ◽  
Orbitrap Mass Spectrometer ◽  
Nf Kappab

Abstract Background Phosphorylation of NF-kappaB inhibitor alpha (IκBα) is key to regulation of NF-κB transcription factor activity in the cell. Several sites of IκBα phosphorylation by members of the IκB kinase family have been identified, but phosphorylation of the protein by other kinases remains poorly understood. We investigated a new phosphorylation site on IκBα and identified its biological function in breast cancer cells. Methods Previously, we observed that aurora kinase (AURK) binds IκBα in the cell. To identify the domains of IκBα essential for phosphorylation by AURK, we performed kinase assays with a series of IκBα truncation mutants. AURK significantly promoted activation of IκBα at serine 32 but not serine 36; by contrast, IκB kinase (IKK) family proteins activated both of these residues. We also confirmed phosphorylation of IκBα by matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/TOF MS) and nano-liquid chromatography hybrid quadrupole orbitrap mass spectrometer (nanoLC-MS/MS; Q-Exactive). Results We identified two novel sites of serine phosphorylation, S63 and S262. Alanine substitution of S63 and S262 (S63A and S262A) of IκBα inhibited proliferation and suppressed p65 transcription activity. In addition, S63A and/or S262A of IκBα regulated apoptotic and necroptotic effects in breast cancer cells. Conclusions Phosphorylation of IκBα by AURK at novel sites is related to the apoptosis and necroptosis pathways in breast cancer cells.

scholarly journals LKB1, a novel serine/threonine protein kinase and potential tumour suppressor, is phosphorylated by cAMP-dependent protein kinase (PKA) and prenylated in vivo

2000 ◽  
Vol 345 (3) ◽  
pp. 673-680 ◽  
Author(s):  
Sean P. COLLINS ◽  
Junewai L. REOMA ◽  
David M. GAMM ◽  
Michael D. UHLER
Keyword(s):  
Protein Kinase ◽  
Cellular Localization ◽  
Fluorescent Protein ◽  
Phosphorylation Site ◽  
Multiple Cancer ◽  
Dependent Protein Kinase ◽  
Rnase Protection ◽  
Camp Dependent Protein Kinase ◽  
Increased Risk ◽  
Dependent Protein

Peutz-Jeghers syndrome (PJS) is an autosomal dominant disease characterized by melanocytic macules, hamartomatous polyps and an increased risk for numerous cancers. The human LKB1 (hLKB1) gene encodes a serine/threonine protein kinase that is deficient in the majority of patients with PJS. The murine LKB1 (mLKB1) cDNA was isolated, sequenced and shown to produce a 2.4-kb transcript encoding a 436 amino acid protein with 90% identity with hLKB1. RNA blot and RNase-protection analysis revealed that mLKB1 mRNA is expressed in all tissues and cell lines examined. The widespread expression of LKB1 transcripts is consistent with the elevated risk of multiple cancer types in PJS patients. The predicted LKB1 protein sequence terminates with a conserved prenylation motif (Cys433-Lys-Gln-Gln436) directly downstream from a consensus cAMP-dependent protein kinase (PKA) phosphorylation site (Arg428-Arg-Leu-Ser431). The expression of enhanced green fluorescent protein (EGFP)-mLKB1 chimaeras demonstrated that LKB1 possesses a functional prenylation motif that is capable of targeting EGFP to cellular membranes. Mutation of Cys433 to an alanine residue, but not phosphorylation by PKA, blocked membrane localization. These findings suggest that PKA does phosphorylate LKB1, although this phosphorylation does not alter the cellular localization of LKB1.

scholarly journals Increased oncogenic potential of ErbB is associated with the loss of a COOH-terminal domain serine phosphorylation site.

1992 ◽  
Vol 267 (12) ◽  
pp. 7967-7970
Author(s):  
S.J. Theroux ◽  
C Taglienti-Sian ◽  
N Nair ◽  
J.L. Countaway ◽  
H.L. Robinson ◽  
...  
Keyword(s):  
Phosphorylation Site ◽  
Serine Phosphorylation ◽  
Oncogenic Potential ◽  
Terminal Domain

scholarly journals Insight into the Dual Functions of Bacterial Enhancer-Binding Protein Rrp2 of Borrelia burgdorferi

2016 ◽  
Vol 198 (10) ◽  
pp. 1543-1552 ◽  
Author(s):  
Yanping Yin ◽  
Youyun Yang ◽  
Xuwu Xiang ◽  
Qian Wang ◽  
Zhang-Nv Yang ◽  
...  
Keyword(s):  
Dna Binding ◽  
Borrelia Burgdorferi ◽  
Binding Protein ◽  
Phosphorylation Site ◽  
Binding Motif ◽  
Cell Replication ◽  
Additional Function ◽  
Content Type ◽  
Terminal Domain ◽  
Enhancer Binding Protein

ABSTRACTIt is well established that the RpoN-RpoS sigma factor (σ54-σS) cascade plays an essential role in differential gene expression during the enzootic cycle ofBorrelia burgdorferi, the causative agent of Lyme disease. The RpoN-RpoS pathway is activated by the response regulator/σ54-dependent activator (also called bacterial enhancer-binding protein [bEBP]) Rrp2. One unique feature of Rrp2 is that this activator is essential for cell replication, whereas RpoN-RpoS is dispensable for bacterial growth. How Rrp2 controls cell replication, a function that is independent of RpoN-RpoS, remains to be elucidated. In this study, by generating a series of conditionalrrp2mutant strains, we demonstrated that the N-terminal receiver domain of Rrp2 is required for spirochetal growth. Furthermore, a D52A point mutation at the phosphorylation site within the N terminus of Rrp2 abolished cell replication. Mutation of the ATPase motif within the central domain of Rrp2 did not affect spirochetal replication, indicating that phosphorylation-dependent ATPase activity of Rrp2 for σ54activation is not required for cell growth. However, deletion of the C-terminal domain or a 16-amino-acid truncation of the helix-turn-helix (HTH) DNA-binding motif within the C-terminal domain of Rrp2 abolished spirochetal replication. It was shown that constitutive expression ofrpoSis deleterious to borrelial growth. We showed that the essential nature of Rrp2 is not due to an effect onrpoS. These data suggest that phosphorylation-dependent oligomerization and DNA binding of Rrp2 likely function as a repressor, independently of the activation of σ54, controlling an essential step of cell replication inB. burgdorferi.IMPORTANCEBacterial enhancer-binding proteins (bEBPs) are a unique group of transcriptional activators specifically required for σ54-dependent gene transcription. This work demonstrates that theB. burgdorferibEBP, Rrp2, has an additional function that is independent of σ54, that of its essentiality for spirochetal growth, and such a function is dependent on its N-terminal signal domain and C-terminal DNA-binding domain. These findings expand our knowledge on bEBP and provide a foundation to further study the underlying mechanism of this new function of bEBP.

scholarly journals The Actin-Binding Protein UNC-115/abLIM Controls Formation of Lamellipodia and Filopodia and Neuronal Morphogenesis in Caenorhabditis elegans

2005 ◽  
Vol 25 (12) ◽  
pp. 5158-5170 ◽  
Author(s):  
Yieyie Yang ◽  
Erik A. Lundquist
Keyword(s):  
Caenorhabditis Elegans ◽  
Molecular Mechanisms ◽  
Binding Protein ◽  
Phosphorylation Site ◽  
Serine Phosphorylation ◽  
Actin Binding ◽  
Axon Pathfinding ◽  
Neuronal Morphogenesis ◽  
Actin Binding Protein ◽  
C Elegans

ABSTRACT The roles of actin-binding proteins in development and morphogenesis are not well understood. The actin-binding protein UNC-115 has been implicated in cytoskeletal signaling downstream of Rac in Caenorhabditis elegans axon pathfinding, but the cellular role of UNC-115 in this process remains undefined. Here we report that UNC-115 overactivity in C. elegans neurons promotes the formation of neurites and lamellipodial and filopodial extensions similar to those induced by activated Rac and normally found in C. elegans growth cones. We show that UNC-115 activity in neuronal morphogenesis is enhanced by two molecular mechanisms: when ectopically driven to the plasma membrane by the myristoylation sequence of c-Src, and by mutation of a putative serine phosphorylation site in the actin-binding domain of UNC-115. In support of the hypothesis that UNC-115 modulates actin cytoskeletal organization, we show that UNC-115 activity in serum-starved NIH 3T3 fibroblasts results in the formation of lamellipodia and filopodia. We conclude that UNC-115 is a novel regulator of the formation of lamellipodia and filopodia in neurons, possibly in the growth cone during axon pathfinding.

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