Differential requirements for AP-2 in clathrin-mediated endocytosis

AP-2 complexes are key components in clathrin-mediated endocytosis (CME). They trigger clathrin assembly, interact directly with cargo molecules, and recruit a number of endocytic accessory factors. Adaptor-associated kinase (AAK1), an AP-2 binding partner, modulates AP-2 function by phosphorylating its mu2 subunit. Here, we examined the effects of adenoviral-mediated overexpression of WT AAK1, kinase-dead, and truncation mutants in HeLa cells, and show that AAK1 also regulates AP-2 function in vivo. WT AAK1 overexpression selectively blocks transferrin (Tfn) receptor and LRP endocytosis. Inhibition was kinase independent, but required the full-length AAK1 as truncation mutants were not inhibitory. Although changes in mu2 phosphorylation were not detected, AAK1 overexpression significantly decreased the phosphorylation of large adaptin subunits and the normally punctate AP-2 distribution was dispersed, suggesting that AAK1 overexpression inhibited Tfn endocytosis by functionally sequestering AP-2. Surprisingly, clathrin distribution and EGF uptake were unaffected by AAK1 overexpression. Thus, AP-2 may not be stoichiometrically required for coat assembly, and may have a more cargo-selective function in CME than previously thought.     

Phosphorylation of the AP2 mu subunit by AAK1 mediates high affinity binding to membrane protein sorting signals

During receptor-mediated endocytosis, AP2 complexes act as a bridge between the cargo membrane proteins and the clathrin coat by binding to sorting signals via the mu 2 subunit and to clathrin via the beta subunit. Here we show that binding of AP2 to sorting signals in vitro is regulated by phosphorylation of the mu 2 subunit of AP2. Phosphorylation of mu 2 enhances the binding affinity of AP2 for sorting motifs as much as 25-fold compared with dephosphorylated AP2. The recognition of sorting signals was not affected by the phosphorylation status of the alpha or beta 2 subunit, suggesting that phosphorylation of mu 2 is critical for regulation of AP2 binding to sorting signals. Phosphorylation of mu 2 occurs at a single threonine residue (Thr-156) and is mediated by the newly discovered adaptor-associated kinase, AAK1, which copurifies with AP2. We propose that phosphorylation of the AP2 mu 2 subunit by AAK1 ensures high affinity binding of AP2 to sorting signals of cargo membrane proteins during the initial steps of receptor-mediated endocytosis.     

AAK1-mediated micro2 phosphorylation is stimulated by assembled clathrin

AAK1, the adaptor-associated kinase 1, phosphorylates the μ2 subunit of AP2 and regulates the recruitment of AP2 to tyrosine-based internalization motifs found on membrane-bound receptors. AAK1 overexpression specifically inhibits the AP2-dependent internalization of transferrin receptor and LDL-receptor related protein by functionally sequestering AP2 (Conner and Schmid. J Cell Biol 2003; 162: 773). However, while AAK1 stably associates with AP2 and specifically targets the μ2 subunit in vitro , μ2 phosphorylation in vivo was not altered by overexpression of either wild-type or kinase-inactive AAK1. These results suggested that AAK1 might be tightly regulated in the cell. Here, we report that AAK1 is an atypical kinase that is rate limited by its stable association with AP2 and that clathrin stimulates μ2 phosphorylation by AAK1. Efficient stimulation of AAK1 by clathrin involves multiple interactions between several domains on AAK1 and both heavy and light chains on clathrin. Importantly, incubation of AAK1 with clathrin cages resulted in even greater stimulation when compared to that of unassembled clathrin triskelia. Collectively, our observations indicate that clathrin function is not limited to structural and/or mechanical roles in endocytic vesicle formation: the stimulatory effects of clathrin on AAK1 activity argue that it also plays a regulatory role by modulating the activity of AP2 complexes through activation of AAK1. We suggest a model in which AAK1 is specifically activated in coated pits to enhance cargo recruitment and efficient internalization.     

A novel AAK1 splice variant functions at multiple steps of the endocytic pathway

Phosphorylation is a critical step in regulating receptor transport through the endocytic pathway. AAK1 is a serine/threonine kinase that is thought to coordinate the recruitment of AP-2 to receptors containing tyrosine-based internalization motifs by phosphorylating the micro2 subunit. Here we have identified a long form of AAK1 (AAK1L) that contains an extended C-terminus that encodes an additional clathrin-binding domain (CBD2) consisting of multiple low-affinity interaction motifs. Protein interaction studies demonstrate that AAK1L CBD2 directly binds clathrin. However, in vitro kinase assays reveal little difference between AAK1 isoforms in their basal or clathrin-stimulated kinase activity toward the AP-2 micro2 subunit. However, overexpression of AAK1L CBD2 impairs transferrin endocytosis, confirming an endocytic role for AAK1. Surprisingly, CBD2 overexpression or AAK1 depletion by RNA interference significantly impairs transferrin recycling from the early/sorting endosome. These observations suggest that AAK1 functions at multiple steps of the endosomal pathway by regulating transferrin internalization and its rapid recycling back to the plasma membrane from early/sorting endosome.     

Clathrin promotes incorporation of cargo into coated pits by activation of the AP2 adaptor micro2 kinase

Endocytic cargo such as the transferrin receptor is incorporated into clathrin-coated pits by associating, via tyrosine-based motifs, with the AP2 complex. Cargo-AP2 interactions occur via the mu2 subunit of AP2, which needs to be phosphorylated for endocytosis to occur. The most likely role for mu2 phosphorylation is in cargo recruitment because mu2 phosphorylation enhances its binding to internalization motifs. Here, we investigate the control of mu2 phosphorylation. We identify clathrin as a specific activator of the mu2 kinase and, in permeabilized cells, we show that ligand sequestration, driven by exogenous clathrin, results in elevated levels of mu2 phosphorylation. Furthermore, we show that AP2 containing phospho-mu2 is mainly associated with assembled clathrin in vivo, and that the level of phospho-mu2 is strongly reduced in a chicken B cell line depleted of clathrin heavy chain. Our results imply a central role for clathrin in the regulation of cargo selection via the modulation of phospho-mu2 levels.     

Phosphorylation of threonine 156 of the mu2 subunit of the AP2 complex is essential for endocytosis in vitro and in vivo

The clathrin-coated pit is the major port of entry for many receptors and pathogens and is the paradigm for membrane-based sorting events in higher cells [1]. Recently, it has been possible to reconstitute in vitro the events leading to assembly, invagination, and budding off of clathrin-coated vesicles, allowing dissection of the machinery required for sequestration of receptors into these structures [2-6]. The AP2 adaptor complex is a key element of this machinery linking receptors to the coat lattice, and it has previously been reported that AP2 can be phosphorylated both in vitro and in vivo [7-10]. However, the physiological significance of this has never been established. Here, we show that phosphorylation of a single threonine residue (Thr156) of the mu2 subunit of the AP2 complex is essential for efficient endocytosis of transferrin both in an in vitro coated-pit budding assay and in living cells.     

EphrinB-EphB signalling regulates clathrin-mediated endocytosis through tyrosine phosphorylation of synaptojanin 1

Recent studies show that Eph receptors act mainly through the regulation of actin reorganization. Here, we show a novel mode of action for EphB receptors. We identify synaptojanin 1 - a phosphatidylinositol 5'-phosphatase that is involved in clathrin-mediated endocytosis - as a physiological substrate for EphB2. EphB2 causes tyrosine phosphorylation in the proline-rich domain of synaptojanin 1, and inhibits both the interaction with endophilin and the 5'-phosphatase activity of synaptojanin 1. Treatment with the EphB ligand, ephrinB2, elevates the cellular level of phosphatidylinositol 4,5-bisphosphate and promotes transferrin uptake. A kinase inactive mutant of EphB2 and a phosphorylation site mutant of synaptojanin 1 both neutralize the increase of transferrin uptake after ephrinB2 treatment. These mutants also inhibit AMPA glutamate receptor endocytosis in hippocampal neurons. Interestingly, incorporated transferrin does not reach endosomes, suggesting dual effects of EphB signalling on the early and late phases of clathrin-mediated endocytosis. Our results indicate that ephrinB-EphB signalling regulates clathrin-mediated endocytosis in various cellular contexts by influencing protein interactions and phosphoinositide turnover through tyrosine phosphorylation of synaptojanin 1.     

Nerve growth factor-induced phosphorylation of amphiphysin-1 by casein kinase 2 regulates clathrin-amphiphysin interactions

Amphiphysins interact directly with clathrin and have a function in clathrin-mediated synaptic vesicle recycling and clathrin-mediated endocytosis. The neuronal isoform amphiphysin-1 is a serine/threonine phosphoprotein that is dephosphorylated upon stimulation of synaptic vesicle endocytosis. Rephosphorylation was stimulated by nerve growth factor. We analysed the regulation of amphiphysin-clathrin interactions by phosphorylation. The N-terminal domain of clathrin bound to unphosphorylated amphiphysin-1, but not to the phosphorylated protein. A search for possible phosphorylation sites revealed two casein kinase 2 consensus motifs in close proximity to the clathrin binding sites in amphiphysin-1 and -2. We mutagenized these residues (T350 and T387) to glutamate, mimicking a constitutive phosphorylation. The double mutant showed a strong reduction in clathrin binding. The assumption that casein kinase 2 phosphorylates amphiphysin-1 at T350 and T387 was corroborated by experiments showing that: (i) casein kinase 2 phosphorylated these residues directly in vitro, (ii) when expressed in HeLa cells, the glutamate mutant showed reduced phosphorylation, and (iii) casein kinase 2 inhibitors blocked nerve growth factor-induced phosphorylation of endogenous amphiphysin-1 in PC12 cells. These observations are consistent with the hypothesis that, upon activation by nerve growth factor, casein kinase 2 phosphorylates amphiphysin-1 and thereby regulates the endocytosis of clathrin-coated vesicles via the interaction between clathrin and amphiphysin.     

Regulation of the clathrin-coated vesicle cycle by reversible phosphorylation

Reversible phosphorylation has long been an attractive mechanism to control cycles of coat assembly and disassembly during clathrin-mediated endocytosis. Many of the coat proteins are phosphorylated in vivo and in vitro. Our work has focused on the role of phosphorylation of the mu2 subunit of AP-2 (adaptor protein 2), which appears to be necessary for efficient cargo recruitment. Studies to probe the regulation of mu2 phosphorylation demonstrated that clathrin is a specific activator of the mu2 kinase, and, in permeabilized cells, cargo sequestration, driven by exogenously added clathrin, results in elevated levels of m2 phosphorylation. Furthermore, phosphorylated mu2 is mainly associated with assembled clathrin in vivo and its steady-state level is strongly reduced in cells depleted of clathrin heavy chain. Our results imply a central role for clathrin in the regulation of cargo selection via modulation of phospho-mu2 levels. This is therefore a novel regulatory role for clathrin that is independent of its structural role and that provides elegant spatial control of AP-2 and cargo interactions, ensuring that AP-2 is only activated at the correct cellular location and in the correct functional context. Ongoing studies are exploring further the roles of reversible phosphorylation in the coated vesicle cycle.     

Ligand-induced clathrin-mediated endocytosis of the keratinocyte growth factor receptor occurs independently of either phosphorylation or recruitment of eps15

Keratinocyte growth factor receptor (KGFR) is a receptor tyrosine kinase expressed on epithelial cells. Following ligand binding, KGFR is rapidly activated and internalized by clathrin-mediated endocytosis. Among the possible receptor substrates which could be involved in the regulation of KGFR endocytosis and down-modulation, we analyzed here the eps15 protein in view of the proposed general role of eps15 in regulating clathrin-mediated endocytosis as well as that of eps15 tyrosine phosphorylation in the control of regulated endocytosis. Immunoprecipitation and Western blot analysis showed that activated KGFR was not able to phosphorylate eps15, suggesting that eps15 is not a receptor substrate. Double immunofluorescence and confocal microscopy revealed that activated KGFR, differently from epidermal growth factor receptor (EGFR), did not induce recruitment of eps15 to the cell plasma membrane. Microinjection of a monoclonal antibody directed against the C-terminal DPF domain which contains the AP2 binding region of eps15 led to inhibition of both pathways of receptor-mediated endocytosis, the EGFR ligand-induced endocytosis and the transferrin constitutive endocytosis, but did not appear to block the KGFR ligand-induced internalization. Taken together our results indicate that the clathrin-mediated uptake of KGFR is not mediated by eps15.     

Inhibition of the receptor-binding function of clathrin adaptor protein AP-2 by dominant-negative mutant mu2 subunit and its effects on endocytosis

Although interactions between the mu2 subunit of the clathrin adaptor protein complex AP-2 and tyrosine-based internalization motifs have been implicated in the selective recruitment of cargo molecules into coated pits, the functional significance of this interaction for endocytosis of many types of membrane proteins remains unclear. To analyze the function of mu2-receptor interactions, we constructed an epitope-tagged mu2 that incorporates into AP-2 and is targeted to coated pits. Mutational analysis revealed that Asp176 and Trp421 of mu2 are involved in the interaction with internalization motifs of TGN38 and epidermal growth factor (EGF) receptor. Inducible overexpression of mutant mu2, in which these two residues were changed to alanines, resulted in metabolic replacement of endogenous mu2 in AP-2 complexes and complete abrogation of AP-2 interaction with the tyrosine-based internalization motifs. As a consequence, endocytosis of the transferrin receptor was severely impaired. In contrast, internalization of the EGF receptor was not affected. These results demonstrate the potential usefulness of the dominant-interfering approach for functional analysis of the adaptor protein family, and indicate that clathrin-mediated endocytosis may proceed in both a mu2-dependent and -independent manner.     

Receptor-mediated endocytosis involves tyrosine phosphorylation of cortactin

Efficient internalization of cell surface receptors requires actin polymerization mediated by Arp2/3 complex and cortactin, a prominent substrate of the protein-tyrosine kinase Src. However, the significance of cortactin tyrosine phosphorylation in endocytosis is unknown. We found that overexpression of a cortactin mutant deficient in tyrosine phosphorylation decreased transferrin uptake. Suppression of cortactin expression by RNA interference also reduced transferrin internalization. Such inhibition was effectively rescued by overexpressing wild-type cortactin but not a cortactin mutant deficient in tyrosine phosphorylation or a mutant with deletion of the Src homology 3 domain. Likewise, purified phosphorylation-null cortactin failed to restore the formation of clathrin-coated vesicles in a cortactin-depleted cell extract. In vitro analysis revealed that Src-mediated phosphorylation enhanced the association of cortactin with dynamin-2 in a tyrosine phosphorylation-dependent manner. Quantitative analysis demonstrated that Src enhances the affinity of cortactin for dynamin-2 by more than 3-fold. On the other hand, Src-treated dynamin-2 had no effect on its interaction with cortactin. These data indicate that Src kinase is implicated in clathrin-mediated endocytosis by phosphorylation of cortactin.     

Myriocin, an inhibitor of serine palmitoyl transferase, impairs the uptake of transferrin and low-density lipoprotein in mammalian cells

The role of sphingolipids in clathrin-mediated endocytosis is only poorly understood in mammalian cells. Thus the relationship between sphingolipid de novo synthesis and clathrin-mediated endocytosis of transferrin were studied in L929 fibroblasts and two other cell lines. Endocytosis was measured using live cell imaging with fluorescent transferrin or (125)I-transferrin. Lipids were primarily measured using electrospray ionization tandem mass spectrometry. At physiological temperature, transferrin uptake was significantly decreased by the inhibitor of serine palmitoyl transferase myriocin. Myriocin inhibited also the uptake of low-density lipoproteins. The endocytosis inhibition by myriocin could be released by the addition of sphingoid base and by the protein phosphorylation effectors phorbol-12-myristate, 13-acetate (PMA) and okadaic acid. Myriocin influenced not only sphingolipids but also the glycerophospholipid profile. The study of phosphatidylcholine species shows adaptations to more saturated, alkylated and longer fatty acid moieties. The reported results imply that in mammalian cells, at 37°C, sphingolipid de novo synthesis is required for clathrin-mediated endocytosis.     

The adaptor complex 2 directly interacts with the alpha 1b-adrenergic receptor and plays a role in receptor endocytosis

Using the yeast two-hybrid system, we identified the mu 2 subunit of the clathrin adaptor complex 2 as a protein interacting with the C-tail of the alpha 1b-adrenergic receptor (AR). Direct association between the alpha 1b-AR and mu 2 was demonstrated using a solid phase overlay assay. The alpha 1b-AR/mu 2 interaction occurred inside the cells, as shown by the finding that the transfected alpha 1b-AR and the endogenous mu 2 could be coimmunoprecipitated from HEK-293 cell extracts. Mutational analysis of the alpha 1b-AR revealed that the binding site for mu 2 does not involve canonical YXX Phi or dileucine motifs but a stretch of eight arginines on the receptor C-tail. The binding domain of mu 2 for the receptor C-tail involves both its N terminus and the subdomain B of its C-terminal portion. The alpha 1b-AR specifically interacted with mu 2, but not with the mu 1, mu 3, or mu 4 subunits belonging to other AP complexes. The deletion of the mu 2 binding site in the C-tail markedly decreased agonist-induced receptor internalization as demonstrated by confocal microscopy as well as by the results of a surface receptor biotinylation assay. The direct association of the adaptor complex 2 with a G protein-coupled receptor has not been reported so far and might represent a common mechanism underlying clathrin-mediated receptor endocytosis.     

Type Igamma661 phosphatidylinositol phosphate kinase directly interacts with AP2 and regulates endocytosis

Clathrin-coated vesicles mediate sorting and intracellular transport of membrane-bound proteins. The formation of these coats is initiated by the assembly of adaptor proteins (AP), which specifically bind to membrane cargo proteins via recognition of endocytic sorting motifs. The lipid signaling molecule phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) is critical for this process, as it serves as both a targeting and regulatory factor. PI(4,5)P(2) is synthesized by type I phosphatidylinositol phosphate kinases (PIPKI). We have discovered a direct interaction between the mu2-subunit of the AP2 complex and PIPKIgamma661 via a yeast two-hybrid screen. This interaction was confirmed using both the mu2-subunit in glutathione S-transferase pulldowns and via coimmunoprecipitation of endogenous PIPKIgamma661 with the AP2 complex from HEK293 cells. The interaction is mediated, in vivo, by a tyrosine-based motif in the 26-amino acid tail of PIPKIgamma661. Because AP2 regulates endocytosis of transferrin receptor from the plasma membrane, we also examined a role for PIPKIgamma661 using a flow cytometry endocytosis assay. We observed that stable expression of wild type PIPKIgamma661 in Madin-Darby canine kidney cells enhanced transferrin uptake, whereas stable expression of kinase-dead PIPKIgamma661 had an inhibitory effect. Neither condition affected the overall cellular level of PI(4,5)P(2). RNA interference-based knockdown of PIPKIgamma661 in HeLa cells also had an inhibitory effect on transferrin endocytosis using the same assay system. Collectively, this evidence implies an important role for PIPKIgamma661 in the AP2-mediated endocytosis of transferrin.     

Phosphorylation of dynamin II at serine-764 is associated with cytokinesis

Calcineurin is a phosphatase that is activated at the last known stage of mitosis, abscission. Among its many substrates, it dephosphorylates dynamin II during cytokinesis at the midbody of dividing cells. However, dynamin II has several cellular roles including clathrin-mediated endocytosis, centrosome cohesion and cytokinesis. It is not known whether dynamin II phosphorylation plays a role in any of these functions nor have the phosphosites involved in cytokinesis been directly identified. We now report that dynamin II from rat lung is phosphorylated to a low stoichiometry on a single major site, Ser-764, in the proline-rich domain. Phosphorylation on Ser-764 also occurred in asynchronously growing HeLa cells and was greatly increased upon mitotic entry. Tryptic phospho-peptides isolated by TiO₂ chromatography revealed only a single phosphosite in mitotic cells. Mitotic phosphorylation was abolished by roscovitine, suggesting the mitotic kinase is cyclin-dependent kinase 1. Cyclin-dependent kinase 1 phosphorylated full length dynamin II and Glutathione-S-Transferase-tagged-dynamin II-proline-rich domain in vitro, and mutation of Ser-764 to alanine reduced proline-rich domain phosphorylation by 80%, supporting that there is only a single major phosphosite. Ser-764 phosphorylation did not affect clathrin-mediated endocytosis or bulk endocytosis using penetratin-based phospho-deficient or phospho-mimetic peptides or following siRNA depletion/rescue experiments. Phospho-dynamin II was enriched at the mitotic centrosome, but this targeting was unaffected by the phospho-deficient or phospho-mimetic peptides. In contrast, the phospho-mimetic peptide displaced endogenous dynamin II, but not calcineurin, from the midbody and induced cytokinesis failure. Therefore, phosphorylation of dynamin II primarily occurs on a single site that regulates cytokinesis downstream of calcineurin, rather than regulating endocytosis or centrosome function.     

The adaptor-associated kinase 1, AAK1, is a positive regulator of the Notch pathway

The Notch pathway is involved in cell-cell signaling during development and adulthood from invertebrates to higher eukaryotes. Activation of the Notch receptor by its ligands relies upon a multi-step processing. The extracellular part of the receptor is removed by a metalloprotease of the ADAM family and the remaining fragment is cleaved within its transmembrane domain by a presenilin-dependent γ-secretase activity. γ-Secretase processing of Notch has been shown to depend upon monoubiquitination as well as clathrin-mediated endocytosis (CME). We show here that AAK1, the adaptor-associated kinase 1, directly interacts with the membrane-tethered active form of Notch released by metalloprotease cleavage. Active AAK1 acts upstream of the γ-secretase cleavage by stabilizing both the membrane-tethered activated form of Notch and its monoubiquitinated counterpart. We propose that AAK1 acts as an adaptor for Notch interaction with components of the clathrin-mediated pathway such as Eps15b. Moreover, transfected AAK1 increases the localization of activated Notch to Rab5-positive endocytic vesicles, while AAK1 depletion or overexpression of Numb, an inhibitor of the pathway, interferes with this localization. These results suggest that after ligand-induced activation of Notch, the membrane-tethered form can be directed to different endocytic pathways leading to distinct fates.     

在笼型蛋白和脂筏介导的两种内吞途径中nephrin的磷酸化修饰动力学不同

研究肾小球裂隙膜的主要成分nephrin分子在细胞内的转运途径及不同转运途径对nephrin磷酸化的影响.分别应用笼型蛋白介导的内吞(clathrin-mediated endocytosis,CME)和脂筏介导的内吞(raft-mediated endocytosis,RME)标记物转铁蛋白和霍乱毒素B亚基对nephrin的内吞过程进行分析,并进一步应用两种内吞途径阻断物EPS15Δ和Dyn2aK44A,研究阻断nephrin的内吞途径对其磷酸化水平的影响.结果显示,nephrin通过笼型蛋白和脂筏介导的两种内吞途径以不同速率进行内吞;与Src酪氨酸激酶家族成员Fyn共表达时,细胞内nephrin酪氨酸磷酸化被增强,而在Src家族激酶抑制剂PP2的作用下,nephrin酪氨酸磷酸化被减弱,表明nephrin的磷酸化过程是Fyn依赖的;内吞20min时,笼型蛋白介导的内吞途径的特异性阻断物EPS15Δ降低了nephrin磷酸化水平、笼型蛋白和脂筏介导的内吞途径的通用抑制剂Dyn2aK44A则增加了nephrin的磷酸化水平,综上结果表明:单独阻断脂筏介导的内吞可引起nephrin的磷酸化水平增加,脂筏介导的内吞对nephrin磷酸化过程起下调作用.     

Activation of the epidermal growth factor (EGF) receptor induces formation of EGF receptor- and Grb2-containing clathrin-coated pits

In HeLa cells depleted of adaptor protein 2 complex (AP2) by small interfering RNA (siRNA) to the mu2 or alpha 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 alpha 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 alpha 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.     

Regulation of clathrin-dependent endocytosis by diacylglycerol kinase delta: importance of kinase activity and binding to AP2alpha

DGKdelta (diacylglycerol kinase delta), 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 DGKdelta-mediated regulation of clathrin-dependent endocytosis that controls the internalization, recycling and degradation of receptors. Involvement of DGKdelta 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 DGKdelta2 overlapped with clathrin-coated pits, we predicted the possible regulation of clathrin-dependent endocytosis by DGKdelta2 and its interaction with some endocytosis-regulatory proteins. DGKdelta2 contained the DXF-type binding motifs, and DGKdelta2 bound to AP2alpha, a subunit of the AP-2 complex. DGKdelta2 interacted with the platform subdomain in the AP2alpha ear domain via F369DTFRIL and D746PF sequences in the catalytic domain of DGKdelta2. For further insight into the role for DGKdelta2 in clathrin-dependent endocytosis, we measured the transferrin and EGF (epidermal growth factor) uptake-expressing wild-type or mutant DGKdelta2 under knockdown of endogenous DGKdelta. Mutants lacking binding ability to AP2alpha 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 DGKdelta2 completely recovered the transferrin uptake. These results demonstrate that binding between DGKdelta2 and AP2alpha is involved in the transferrin internalization and that DGK activity is also necessary for the regulation of the endocytic process.