Regulation of clathrin-dependent endocytosis by diacylglycerol kinase δ: importance of kinase activity and binding to AP2α

2007 ◽  
Vol 409 (2) ◽  
pp. 471-479 ◽  
Author(s):  
Takumi Kawasaki ◽  
Takeshi Kobayashi ◽  
Takehiko Ueyama ◽  
Yasuhito Shirai ◽  
Naoaki Saito
Keyword(s):  
Catalytic Domain ◽  
Intracellular Localization ◽  
Adaptor Protein ◽  
Diacylglycerol Kinase ◽  
Wild Type ◽  
Coated Pits ◽  
Genome Wide ◽  
A Subunit ◽  
Transferrin Uptake ◽  
Interfering Rna

DGKδ (diacylglycerol kinase δ), which phosphorylates DAG (diacylglycerol) and converts it into PA (phosphatidic acid), has an important role in signal transduction. In the present study, we have demonstrated the molecular mechanism of DGKδ-mediated regulation of clathrin-dependent endocytosis that controls the internalization, recycling and degradation of receptors. Involvement of DGKδ in the regulation of clathrin-dependent endocytosis was previously proposed following genome-wide RNAi (RNA interference) screening. Clathrin-coated pits are mainly formed by clathrin and AP-2 (adaptor protein 2) complex. These proteins assemble a polyhedral lattice at the membrane and gather several endocytic accessory proteins. As the intracellular localization of DGKδ2 overlapped with clathrin-coated pits, we predicted the possible regulation of clathrin-dependent endocytosis by DGKδ2 and its interaction with some endocytosis-regulatory proteins. DGKδ2 contained the DXF-type binding motifs, and DGKδ2 bound to AP2α, a subunit of the AP-2 complex. DGKδ2 interacted with the platform subdomain in the AP2α ear domain via F369DTFRIL and D746PF sequences in the catalytic domain of DGKδ2. For further insight into the role for DGKδ2 in clathrin-dependent endocytosis, we measured the transferrin and EGF (epidermal growth factor) uptake-expressing wild-type or mutant DGKδ2 under knockdown of endogenous DGKδ. Mutants lacking binding ability to AP2α as well as kinase-negative mutants could not compensate for the uptake of transferrin inhibited by siRNA (small interfering RNA) treatment, whereas overexpression of wild-type DGKδ2 completely recovered the transferrin uptake. These results demonstrate that binding between DGKδ2 and AP2α is involved in the transferrin internalization and that DGK activity is also necessary for the regulation of the endocytic process.

scholarly journals Genome-wide screens identify specific drivers of mutant hTERT promoters

2022 ◽  
Vol 119 (3) ◽  
pp. e2105171119
Author(s):  
Raghuvaran Shanmugam ◽  
Mert Burak Ozturk ◽  
Joo-Leng Low ◽  
Semih Can Akincilar ◽  
Joelle Yi Heng Chua ◽  
...  
Keyword(s):  
Small Interfering Rna ◽  
Regulatory Region ◽  
Telomerase Activity ◽  
Chromatin Interaction ◽  
Mediator Complex ◽  
Wild Type ◽  
Distal Regulatory Region ◽  
Genome Wide ◽  
Promoter Mutations ◽  
Interfering Rna

Cancer-specific hTERT promoter mutations reported in 19% of cancers result in enhanced telomerase activity. Understanding the distinctions between transcriptional regulation of wild-type (WT) and mutant (Mut) hTERT promoters may open up avenues for development of inhibitors which specially block hTERT expression in cancer cells. To comprehensively identify physiological regulators of WT- or Mut-hTERT promoters, we generated several isogenic reporter cells driven by endogenous hTERT loci. Genome-wide CRISPR-Cas9 and small interfering RNA screens using these isogenic reporter lines identified specific regulators of Mut-hTERT promoters. We validate and characterize one of these hits, namely, MED12, a kinase subunit of mediator complex. We demonstrate that MED12 specifically drives expression of hTERT from the Mut-hTERT promoter by mediating long-range chromatin interaction between the proximal Mut-hTERT promoter and T-INT1 distal regulatory region 260 kb upstream. Several hits identified in our screens could serve as potential therapeutic targets, inhibition of which may specifically block Mut-hTERT promoter driven telomerase reactivation in cancers.

scholarly journals The C-terminal part of diacylglycerol kinase α lacking zinc fingers serves as a catalytic domain

1996 ◽  
Vol 318 (2) ◽  
pp. 583-590 ◽  
Author(s):  
Fumio SAKANE ◽  
Masahiro KAI ◽  
Ikuo WADA ◽  
Shin-ichi IMAI ◽  
Hideo KANOH
Keyword(s):  
Enzyme Activity ◽  
Binding Sites ◽  
Enzyme Activities ◽  
Catalytic Domain ◽  
Zinc Fingers ◽  
Diacylglycerol Kinase ◽  
Site Directed Mutagenesis ◽  
Atp Binding ◽  
Wild Type

All mammalian diacylglycerol kinase (DGK) isoenzymes so far cloned consist of four conserved regions, namely C1, C2 (tandem EF-hand structures), C3 (tandem cysteine-rich zinc finger sequences) and the C-terminal C4 domains. To determine the catalytic domain we expressed in COS-7 cells various truncation mutants of pig DGKα and assessed their enzyme activities. We found that the C4 domain lacking the whole N-terminal region including the zinc fingers possessed DGK activity that was dependent on the concentrations of diacylglycerol and ATP very similarly, as did the wild-type DGKα. Furthermore the DGK activity of the wild-type DGK and that expressed by the C4 domain were similarly activated by anionic amphiphiles such as phosphatidylserine, phosphatidylinositol and deoxycholate. It was also shown that a DGK mutant consisting of the zinc fingers and the C4 domain has enzymological properties very similar to those expressed by the C4 domain alone. We also confirmed that the intact DGKs α, β and γ expressed in COS-7 cells displayed no detectable phorbol ester binding. These results show that the C4 domain of DGK is the catalytic region that is responsible for the enzyme activities sensitive to different activators. We cannot exclude the possibility that the N-terminal portion including the zinc fingers can still interact with diacylglycerol and activators without affecting the enzyme activity measured in vitro. However, it is quite likely that the DGK zinc fingers do not serve as diacylglycerol-binding sites, in contrast with those present in other proteins such as protein kinases C and n-chimaerin. Site-directed mutagenesis of all six putative ATP binding sites (Lys248, Lys383, Lys395, Lys483, Lys492, and Lys554) did not significantly affect the enzyme activity. We therefore suggest that DGK does not contain a typical P-loop of ATP binding sites.

scholarly journals A genome-wide almanac of co-essential modules assigns function to uncharacterized genes

2019 ◽  
Author(s):  
Michael Wainberg ◽  
Roarke A. Kamber ◽  
Akshay Balsubramani ◽  
Robin M. Meyers ◽  
Nasa Sinnott-Armstrong ◽  
...  
Keyword(s):  
Protein Complexes ◽  
Functional Modules ◽  
Web Tool ◽  
Coated Pits ◽  
Robust Identification ◽  
Genome Wide ◽  
A Genome ◽  
Gene Functions ◽  
Key Enzyme ◽  
Transferrin Uptake

SUMMARYA central remaining question in the post-genomic era is how genes interact to form biological pathways. Measurements of gene dependency across hundreds of cell lines have been used to cluster genes into ‘co-essential’ pathways, but this approach has been limited by ubiquitous false positives. Here, we develop a statistical method that enables robust identification of gene co-essentiality and yields a genome-wide set of functional modules. This almanac recapitulates diverse pathways and protein complexes and predicts the functions of 102 uncharacterized genes. Validating top predictions, we show that TMEM189 encodes plasmanylethanolamine desaturase, the long-sought key enzyme for plasmalogen synthesis. We also show that C15orf57 binds the AP2 complex, localizes to clathrin-coated pits, and enables efficient transferrin uptake. Finally, we provide an interactive web tool for the community to explore the results (coessentiality.net). Our results establish co-essentiality profiling as a powerful resource for biological pathway identification and discovery of novel gene functions.

Regulation of the Raf-1 kinase domain by phosphorylation and 14-3-3 association

2000 ◽  
Vol 351 (1) ◽  
pp. 151-159 ◽  
Author(s):  
Michele T. YIP-SCHNEIDER ◽  
Wenyan MIAO ◽  
Amy LIN ◽  
Darlene S. BARNARD ◽  
Guri TZIVION ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Protein Kinase ◽  
Phorbol Esters ◽  
Catalytic Domain ◽  
Adaptor Protein ◽  
Kinase Domain ◽  
Phosphorylation Sites ◽  
Regulatory Domain ◽  
Wild Type

The Raf-1 kinase domain is kept in an inactive state by the N-terminal regulatory domain. Activation of the kinase domain occurs following release from the N-terminal repression and possible catalytic upregulation. To distinguish the regulatory mechanisms that directly influence the catalytic activity of the enzyme from those which act through the inhibitory domain, the catalytic domain of Raf-1 (CR3) was expressed in COS-7 cells. The role of phosphorylation in the direct regulation of this domain was determined by substituting non-phosphorylatable amino acids for known serine and tyrosine phosphorylation sites. The intrinsic activity of each mutant protein was determined as well as stimulation by v-Src and phorbol esters. Both v-Src and phorbol esters were potent activators of CR3, requiring the serine 338/339 (p21-activated protein kinase, Pak) and tyrosine 340/341 (Src) phosphorylation sites for full stimulation of CR3. In contrast, loss of the serine 497/499 protein kinase C phosphorylation sites had little effect on CR3 activation by either v-Src or phorbol esters. Loss of serine 621, a 14-3-3 adaptor-protein-binding site, prevented activation of CR3 by v-Src or phorbol esters and partially decreased the high basal activity of the kinase fragment. When co-expressed in COS-7 cells, 14-3-3 associated strongly with full-length Raf-1, weakly with wild-type CR3 and not at all with the A621 and D621 CR3 mutants. The role of 14-3-3 in maintaining the activity of the catalytic domain of Raf-1 was investigated further by performing peptide-competition studies with wild-type CR3, wild-type CR3 and v-Src or constitutively active CR3 (CR3[YY340/341DD]). In each case, incubation of the proteins with a phosphoserine-621 Raf-1 peptide, which we show displaced Raf-1 and CR3[YY340/341DD] from 14-3-3, was found to substantially reduce catalytic activity. Taken together, our results support a model of Raf regulation in which the activity of the Raf-1 catalytic domain is directly upregulated by phosphorylation, following relief of inhibition by the N-terminal regulatory domain upon Ras-GTP binding. Moreover, the presence of serine 621 in the free catalytic fragment is required for full CR3 activation by stimulatory factors, and the continuous presence of 14-3-3 at this site is necessary for retaining activity once the kinase is activated.

scholarly journals Diacylglycerol Kinase alpha in X Linked Lymphoproliferative Disease Type 1

2021 ◽  
Vol 22 (11) ◽  
pp. 5816
Author(s):  
Suresh Velnati ◽  
Sara Centonze ◽  
Federico Girivetto ◽  
Gianluca Baldanzi
Keyword(s):  
T Cell ◽  
T Cell Receptor ◽  
Adaptor Protein ◽  
Diacylglycerol Kinase ◽  
Cell Receptor ◽  
Lymphoproliferative Disease ◽  
Disease Type ◽  
T Cell Response ◽  
Receptor Signalling

Diacylglycerol kinases are intracellular enzymes that control the balance between the secondary messengers diacylglycerol and phosphatidic acid. DGKα and DGKζ are the prominent isoforms that restrain the intensity of T cell receptor signalling by metabolizing PLCγ generated diacylglycerol. Thus, their activity must be tightly controlled to grant cellular homeostasis and refine immune responses. DGKα is specifically inhibited by strong T cell activating signals to allow for full diacylglycerol signalling which mediates T cell response. In X-linked lymphoproliferative disease 1, deficiency of the adaptor protein SAP results in altered T cell receptor signalling, due in part to persistent DGKα activity. This activity constrains diacylglycerol levels, attenuating downstream pathways such as PKCθ and Ras/MAPK and decreasing T cell restimulation induced cell death. This is a form of apoptosis triggered by prolonged T cell activation that is indeed defective in CD8+ cells of X-linked lymphoproliferative disease type 1 patients. Accordingly, inhibition or downregulation of DGKα activity restores in vitro a correct diacylglycerol dependent signal transduction, cytokines production and restimulation induced apoptosis. In animal disease models, DGKα inhibitors limit CD8+ expansion and immune-mediated tissue damage, suggesting the possibility of using inhibitors of diacylglycerol kinase as a new therapeutic approach.

scholarly journals Genome-Wide Identification and Genetic Variations of the Starch Synthase Gene Family in Rice

Plants ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 1154
Author(s):  
Hongjia Zhang ◽  
Seong-Gyu Jang ◽  
San Mar Lar ◽  
Ah-Rim Lee ◽  
Fang-Yuan Cao ◽  
...  
Keyword(s):  
Gene Family ◽  
Catalytic Domain ◽  
Amylose Content ◽  
Expression Patterns ◽  
Starch Synthase ◽  
Eating Quality ◽  
Segmental Duplications ◽  
Genome Wide ◽  
The Relationship

Starch is a major ingredient in rice, and the amylose content of starch significantly impacts rice quality. OsSS (starch synthase) is a gene family related to the synthesis of amylose and amylopectin, and 10 members have been reported. In the present study, a synteny analysis of a novel family member belonging to the OsSSIV subfamily that contained a starch synthase catalytic domain showed that three segmental duplications and multiple duplications were identified in rice and other species. Expression data showed that the OsSS gene family is involved in diverse expression patterns. The prediction of miRNA targets suggested that OsSS are possibly widely regulated by miRNA functions, with miR156s targeted to OsSSII-3, especially. Haplotype analysis exhibited the relationship between amylose content and diverse genotypes. These results give new insight and a theoretical basis for the improved amylose content and eating quality of rice.

scholarly journals Isoform-specific knockdown and expression of adaptor protein ShcA using small interfering RNA

2002 ◽  
Vol 363 (1) ◽  
pp. 1 ◽  
Author(s):  
Malgorzata KISIELOW ◽  
Sandra KLEINER ◽  
Michiaki NAGASAWA ◽  
Amir FAISAL ◽  
Yoshikuni NAGAMINE
Keyword(s):  
Small Interfering Rna ◽  
Adaptor Protein ◽  
Interfering Rna

scholarly journals Genome-Wide Analysis of Wild-Type Epstein–Barr Virus Genomes Derived from Healthy Individuals of the 1000 Genomes Project

2014 ◽  
Vol 6 (4) ◽  
pp. 846-860 ◽  
Author(s):  
Gabriel Santpere ◽  
Fleur Darre ◽  
Soledad Blanco ◽  
Antonio Alcami ◽  
Pablo Villoslada ◽  
...  
Keyword(s):  
Epstein Barr Virus ◽  
Healthy Individuals ◽  
Wild Type ◽  
1000 Genomes Project ◽  
Genome Wide Analysis ◽  
Barr Virus ◽  
1000 Genomes ◽  
Genome Wide ◽  
Epstein Barr ◽  
Virus Genomes

scholarly journals Phosphatidylinositol-(4,5)-bisphosphate regulates clathrin-coated pit initiation, stabilization, and size

2011 ◽  
Vol 22 (14) ◽  
pp. 2588-2600 ◽  
Author(s):  
Costin N. Antonescu ◽  
François Aguet ◽  
Gaudenz Danuser ◽  
Sandra L. Schmid
Keyword(s):  
Mammalian Cells ◽  
Lipid Binding ◽  
Coated Pits ◽  
Inositol Phosphatase ◽  
Major Mechanism ◽  
Synaptojanin 1 ◽  
Phosphatidylinositol 4 ◽  
Sirna Knockdown ◽  
Interfering Rna ◽  
Pit Initiation

Clathrin-mediated endocytosis (CME) is the major mechanism for internalization in mammalian cells. CME initiates by recruitment of adaptors and clathrin to form clathrin-coated pits (CCPs). Nearly half of nascent CCPs abort, whereas others are stabilized by unknown mechanisms and undergo further maturation before pinching off to form clathrin-coated vesicles (CCVs). Phosphatidylinositol-(4,5)-bisphosphate (PIP2), the main lipid binding partner of endocytic proteins, is required for CCP assembly, but little is currently known about its contribution(s) to later events in CCV formation. Using small interfering RNA (siRNA) knockdown and overexpression, we have analyzed the effects of manipulating PIP2 synthesis and turnover on CME by quantitative total internal reflection fluorescence microscopy and computational analysis. Phosphatidylinositol-4-phosphate-5-kinase cannot be detected within CCPs but functions in initiation and controls the rate and extent of CCP growth. In contrast, the 5′-inositol phosphatase synaptojanin 1 localizes to CCPs and controls early stabilization and maturation efficiency. Together these results suggest that the balance of PIP2 synthesis in the bulk plasma membrane and its local turnover within CCPs control multiple stages of CCV formation.

scholarly journals Activation of the Epidermal Growth Factor (EGF) Receptor Induces Formation of EGF Receptor- and Grb2-Containing Clathrin-Coated Pits

2006 ◽  
Vol 26 (2) ◽  
pp. 389-401 ◽  
Author(s):  
Lene E. Johannessen ◽  
Nina Marie Pedersen ◽  
Ketil Winther Pedersen ◽  
Inger Helene Madshus ◽  
Espen Stang
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Egf Receptor ◽  
Point Mutations ◽  
Adaptor Protein ◽  
Mitogen Activated Protein Kinase ◽  
Coated Pits ◽  
Α Subunit ◽  
Sh3 Domains ◽  
Epidermal Growth

ABSTRACT In HeLa cells depleted of adaptor protein 2 complex (AP2) by small interfering RNA (siRNA) to the μ2 or α subunit or by transient overexpression of an AP2 sequestering mutant of Eps15, endocytosis of the transferrin receptor (TfR) was strongly inhibited. However, epidermal growth factor (EGF)-induced endocytosis of the EGF receptor (EGFR) was inhibited only in cells where the α subunit had been knocked down. By immunoelectron microscopy, we found that in AP2-depleted cells, the number of clathrin-coated pits was strongly reduced. When such cells were incubated with EGF, new coated pits were formed. These contained EGF, EGFR, clathrin, and Grb2 but not the TfR. The induced coated pits contained the α subunit, but labeling density was reduced compared to control cells. Induction of clathrin-coated pits required EGFR kinase activity. Overexpression of Grb2 with inactivating point mutations in N- or C-terminal SH3 domains or in both SH3 domains inhibited EGF-induced formation of coated pits efficiently, even though Grb2 SH3 mutations did not block activation of mitogen-activated protein kinase (MAPK) or phosphatidylinositol 3-kinase (PI3K). Our data demonstrate that EGFR-induced signaling and Grb2 are essential for formation of clathrin-coated pits accommodating the EGFR, while activation of MAPK and PI3K is not required.

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