Identification of an adaptor-associated kinase, AAK1, as a regulator of clathrin-mediated endocytosis

The mu 2 subunit of the AP2 complex is known to be phosphorylated in vitro by a copurifying kinase, and it has been demonstrated recently that mu 2 phosphorylation is required for transferrin endocytosis (Olusanya, O., P.D. Andrews, J.R. Swedlow, and E. Smythe. 2001. Curr. Biol. 11:896-900). However, the identity of the endogenous kinase responsible for this phosphorylation is unknown. Here we identify and characterize a novel member of the Prk/Ark family of serine/threonine kinases, adaptor-associated kinase (AAK)1. We find that AAK1 copurifies with adaptor protein (AP)2 and that it directly binds the ear domain of alpha-adaptin in vivo and in vitro. In neuronal cells, AAK1 is enriched at presynaptic terminals, whereas in nonneuronal cells it colocalizes with clathrin and AP2 in clathrin-coated pits and at the leading edge of migrating cells. AAK1 specifically phosphorylates the mu subunit in vitro, and stage-specific assays for endocytosis show that mu phosphorylation by AAK1 results in a decrease in AP2-stimulated transferrin internalization. Together, these results provide strong evidence that AAK1 is the endogenous mu 2 kinase and plays a regulatory role in clathrin-mediated endocytosis. These results also lend support to the idea that clathrin-mediated endocytosis is controlled by cycles of phosphorylation/desphosphorylation.     

Functional partnership between amphiphysin and dynamin in clathrin-mediated endocytosis

Amphiphysin, a protein that is highly concentrated in nerve terminals, has been proposed to function as a linker between the clathrin coat and dynamin in the endocytosis of synaptic vesicles. Here, using a cell-free system, we provide direct morphological evidence in support of this hypothesis. Unexpectedly, we also find that amphiphysin-1, like dynamin-1, can transform spherical liposomes into narrow tubules. Moreover, amphiphysin-1 assembles with dynamin-1 into ring-like structures around the tubules and enhances the liposome-fragmenting activity of dynamin-1 in the presence of GTP. These results show that amphiphysin binds lipid bilayers, indicate a potential function for amphiphysin in the changes in bilayer curvature that accompany vesicle budding, and imply a close functional partnership between amphiphysin and dynamin in endocytosis.     

Phosphorylation of beta-arrestin2 regulates its function in internalization of beta(2)-adrenergic receptors

Beta-arrestins mediate agonist-dependent desensitization and internalization of G protein-coupled receptors. Previously, we have shown that phosphorylation of beta-arrestin1 by ERKs at Ser-412 regulates its association with clathrin and its function in promoting clathrin-mediated internalization of the receptor. In this paper we report that beta-arrestin2 is also phosphorylated, predominantly at residues Thr-383 and Ser-361. Isoproterenol stimulation of the beta(2)-adrenergic receptor promotes dephosphorylation of beta-arrestin2. Mutation of beta-arrestin2 phosphorylation sites to aspartic acid decreases the association of beta-arrestin2 with clathrin, thereby reducing its ability to promote internalization of the beta(2)-adrenergic receptor. Its ability to bind and desensitize the beta(2)-adrenergic receptor is, however, unaltered. These results suggest that, analogous to beta-arrestin1, phosphorylation/dephosphorylation of beta-arrestin2 regulates clathrin-mediated internalization of the beta(2)-adrenergic receptor. In contrast to beta-arrestin1, which is phosphorylated by ERK1 and ERK2, phosphorylation of beta-arrestin2 at Thr-383 is shown to be mediated by casein kinase II. Recently, it has been reported that phosphorylation of visual arrestin at Ser-366 prevents its binding to clathrin. Thus it appears that the function of all arrestin family members in mediating internalization of G protein-coupled receptors is regulated by distinct phosphorylation/dephosphorylation mechanisms.     

Phosphorylation of the medium chain subunit of the AP-2 adaptor complex does not influence its interaction with the tyrosine based internalisation motif of TGN38

Tyrosine based motifs conforming to the consensus YXXphi (where phi represents a bulky hydrophobic residue) have been shown to interact with the medium chain subunit of clathrin adaptor complexes. These medium chains are targets for phosphorylation by a kinase activity associated with clathrin coated vesicles. We have used the clathrin coated vesicle associated kinase activity to specifically phosphorylate a soluble recombinant fusion protein of mu2, the medium chain subunit of the plasma membrane associated adaptor protein complex AP-2. We have tested whether this phosphorylation has any effect on the interaction of mu2 with the tyrosine based motif containing protein, TGN38, that has previously been shown to interact with mu2. Phosphorylation of mu2 was shown to have no significant effect on the in vitro interaction of mu2 with the cytosolic domain of TGN38, indicating that reversible phosphorylation of mu2 does not play a role in regulating its direct interaction with tyrosine based internalisation motifs. In addition, although a casein kinase II-like activity has been shown to be associated with clathrin coated vesicles, we show that mu2 is not phosphorylated by casein kinase II implying that another kinase activity is present in clathrin coated vesicles. Furthermore the kinase activity associated with clathrin coated vesicles was shown to be capable of phosphorylating dynamin 1. Phosphorylation of dynamin 1 has previously been shown to regulate its interaction with other proteins involved in clathrin mediated endocytosis.     

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.     

Cluster of differentiation antigen 4 (CD4) endocytosis and adaptor complex binding require activation of the CD4 endocytosis signal by serine phosphorylation

Cluster of differentiation antigen 4 (CD4), the T lymphocyte antigen receptor component and human immunodeficiency virus coreceptor, is down-modulated when cells are activated by antigen or phorbol esters. During down-modulation CD4 dissociates from p56(lck), undergoes endocytosis through clathrin-coated pits, and is then sorted in early endosomes to late endocytic organelles where it is degraded. Previous studies have suggested that phosphorylation and a dileucine sequence are required for down-modulation. Using transfected HeLa cells, in which CD4 endocytosis can be studied in the absence of p56(lck), we show that the dileucine sequence in the cytoplasmic domain is essential for clathrin-mediated CD4 endocytosis. However, this sequence is only functional as an endocytosis signal when neighboring serine residues are phosphorylated. Phosphoserine is required for rapid endocytosis because CD4 molecules in which the cytoplasmic domain serine residues are substituted with glutamic acid residues are not internalized efficiently. Using surface plasmon resonance, we show that CD4 peptides containing the dileucine sequence bind weakly to clathrin adaptor protein complexes 2 and 1. The affinity of this interaction is increased 350- to 700-fold when the peptides also contain phosphoserine residues.     

Accessory factors in clathrin-dependent synaptic vesicle endocytosis

Clathrin-mediated endocytosis is a special form of vesicle budding important for the internalization of receptors and extracellular ligands, for the recycling of plasma membrane components, and for the retrieval of surface proteins destined for degradation. In nerve terminals, clathrin-mediated endocytosis is crucial for synaptic vesicle recycling. Recent structural studies have provided molecular details of coat assembly. In addition, biochemical and genetic studies have identified numerous accessory proteins that assist the clathrin coat in its function at synapses and in other systems. This review summarizes these advances with a special focus on accessory factors and highlights new aspects of clathrin-mediated endocytosis revealed by the study of these factors.     

Clathrin-mediated endocytosis is the dominant mechanism of vesicle retrieval at hippocampal synapses

The maintenance of synaptic transmission requires that vesicles be recycled after releasing neurotransmitter. Several modes of retrieval have been proposed to operate at small synaptic terminals of central neurons, including a fast "kiss-and-run" mechanism that releases neurotransmitter through a fusion pore. Using an improved fluorescent reporter comprising pHluorin fused to synaptophysin, we find that only a slow mode of endocytosis (tau = 15 s) operates at hippocampal synapses when vesicle fusion is triggered by a single nerve impulse or short burst. This retrieval mechanism is blocked by overexpression of the C-terminal fragment of AP180 or by knockdown of clathrin using RNAi, and it is associated with the movement of clathrin and vesicle proteins out of the synapse. These results indicate that clathrin-mediated endocytosis is the major, if not exclusive, mechanism of vesicle retrieval after physiological stimuli.     

Stonin 2 is an AP-2-dependent endocytic sorting adaptor for synaptotagmin internalization and recycling

Clathrin-mediated endocytosis is involved in the internalization, recycling, and degradation of cycling membrane receptors as well as in the biogenesis of synaptic vesicle proteins. While many constitutively internalized cargo proteins are recognized directly by the clathrin adaptor complex AP-2, stimulation-dependent endocytosis of membrane proteins is often facilitated by specialized sorting adaptors. Although clathrin-mediated endocytosis appears to be a major pathway for presynaptic vesicle cycling, no sorting adaptor dedicated to synaptic vesicle membrane protein endocytosis has been indentified in mammals. Here, we show that stonin 2, a mammalian ortholog of Drosophila stoned B, facilitates clathrin/AP-2-dependent internalization of synaptotagmin and targets it to a recycling vesicle pool in living neurons. The ability of stonin 2 to facilitate endocytosis of synaptotagmin is dependent on its association with AP-2, an intact mu-homology domain, and functional AP-2 heterotetramers. Our data identify stonin 2 as an AP-2-dependent endocytic sorting adaptor for synaptotagmin internalization and recycling.     

Major Cdk5-dependent phosphorylation sites of amphiphysin 1 are implicated in the regulation of the membrane binding and endocytosis

Amphiphysin 1 (amph 1) is an endocytic protein enriched in the nerve terminals that functions in the clathrin-mediated endocytosis. It acts as membrane curvature sensor, a linker of clathrin coat proteins, and an enhancer of dynamin Guanosine Triphosphatase (GTPase) activity. Amph 1 undergoes phosphorylation by cyclin-dependent kinase 5 (Cdk5), at five phosphorylation sites, serine 262, 272, 276, 285, and threonine 310, as determined by mass spectrometry (MS). We show here that Cdk5-dependent phosphorylation of amph 1 is enhanced in the presence of lipid membranes. Analysis by tandem liquid chromatograph MS revealed that the phosphorylation occurs at two phosphorylation sites. The phosphorylation was markedly decreased by mutation either Ser276 or Ser285 of amph 1 to alanine (S276A and S285A). Furthermore, mutation of both sites (S276, 285A) completely eliminated the phosphorylation. Functional studies indicated that binding of amph 1 to lipid membrane was attenuated by Cdk5-dependent phosphorylation of wild type amph 1, but not of the S276, 285A form. Interestingly, endocytosis was increased in rat pheochromocytoma cells expressing amph 1 S276, 285A in comparison with wild type. These results suggest that Ser276 and Ser285 are regulatory Cdk5 phosphorylation sites of amph 1 in the lipid-bound state. Phosphorylation at these sites alters binding of amph 1 to lipid membranes, and may be an important regulatory aspect in the regulation of synaptic vesicle endocytosis.     

Regulation of amphiphysin1 by mitogen-activated protein kinase: its significance in nerve growth factor receptor-mediated endocytosis

Amphiphysin1, which can simultaneously bind to dynamin1 and the clathrin adaptor AP-2, is essential for dynamin1 recruitment during receptor-mediated endocytosis, but little is known about its regulatory mechanism. Here, we purified a 120-kDa mitogen-activated protein kinase (MAPK) substrate protein from porcine brains and identified the protein as amphiphysin1. Serine phosphorylation of amphiphysin1 was rapidly induced by nerve growth factor (NGF) in PC12 cells, and the induction was blocked by a MAPK inhibitor. Furthermore, when phosphorylated by MAPK in vitro or by NGF treatment in vivo, amphiphysin1 failed to bind to AP-2, but its association with dynamin1 was unaffected. Consistent with this, mutation of consensus MAPK phosphorylation sites increased amphiphysin1 binding to AP-2 and their intracellular colocalization. Thus, we propose that MAPK phosphorylation of amphiphysin1 controls NGF receptor/TrkA-mediated endocytosis by terminating the amphiphysin1-AP-2 interaction. This perhaps helps to regulate the availability of amphiphysin1-dynamin1 complexes for binding to the endocytic vesicle.     

Overexpression of Dyrk1A causes the defects in synaptic vesicle endocytosis

Trisomy 21-linked Dyrk1A (dual-specificity tyrosine phosphorylation-regulated kinase 1A) overexpression is implicated in pathogenic mechanisms underlying mental retardation in Down syndrome (DS). It is known to phosphorylate multiple substrates including endocytic proteins in vitro, but the functional consequence of Dyrk1A-mediated phosphorylation on endocytosis has never been investigated. Here, we show that overexpression of Dyrk1A causes defects in clathrin-mediated endocytosis and specifically, in the recruitment of endocytic proteins to clathrin-coated pits in fibroblasts. Synaptic vesicle endocytosis also significantly slowed down as a result of Dyrk1A overexpression in cultured hippocampal neurons. These effects are dependent on Dyrk1A kinase activity. The inhibitory effect of Dyrk1A on synaptic vesicle endocytosis was confirmed in neuronal cultures derived from transgenic mice overexpressing Dyrk1A at levels found in DS. Pharmacological blockade of Dyrk1A with epigallocatechin gallate rescued the endocytic phenotypes found in transgenic neurons. Together, our results suggest that aberrant Dyrk1A-mediated phosphorylation of the endocytic machinery perturbs synaptic vesicle endocytosis, which may contribute to synaptic dysfunctions and cognitive deficits associated with DS.     

Phosphorylation of Brain Synaptic and Coated Vesicle Proteins by Endogenous Ca2+/Calmodulin- and cAMP-Dependent Protein Kinases

Phosphorylation of brain synaptic and coated vesicle proteins was stimulated by Ca2+ and calmodulin. As determined by 5-15% sodium dodecylsulfate (SDS) polyacrylamide gel electrophoresis (PAGE), molecular weights (Mr) of the major phosphorylated proteins were 55,000 and 53,000 in synaptic vesicles and 175,000 and 55,000 in coated vesicles. In synaptic vesicles, phosphorylation was inhibited by affinity-purified antibodies raised against a 30,000 Mr protein doublet endogenous to synaptic and coated vesicles. When this doublet, along with clathrin, was extracted from coated vesicles, phosphorylation did not take place, implying that the protein doublet may be closely associated with Ca2+/calmodulin-dependent protein kinase. Affinity-purified antibodies, raised against clathrin used as a control antibody, failed to inhibit Ca2+/calmodulin-dependent phosphorylation in either synaptic or coated vesicles. Immunoelectron cytochemistry revealed that this protein doublet was present in axon terminal synaptic and coated vesicles. Synaptic vesicles also displayed cAMP-dependent kinase activity; coated vesicles did not. The molecular weights of phosphorylated synaptic vesicle proteins in the presence of Mg2+ and cAMP were: 175,000, 100,000, 80,000, 57,000, 55,000, 53,000, 40,000, and 30,000. Based on the different phosphorylation patterns observed in synaptic and coated vesicles, we propose that brain vesicle protein kinase activities may be involved in the regulation of exocytosis and in retrieval of synaptic membrane in presynaptic axon terminals.     

Characterization of the localization and function of NECAP 1 in neurons

NECAPs (adaptin ear-binding clathrin-associated protein) are a new family of clathrin accessory proteins identified through a proteomic analysis of clathrin-coated vesicles (CCVs) from the brain. One member of this family, NECAP 1, is found primarily in tissues from the central nervous system and has been shown to be complexed tightly with a substantial portion of adaptor protein-2 (AP-2) in brain extracts. However, the function and intracellular location of this protein is unknown. In this study, we find that endogenous and epitope-tagged NECAP 1 co-localizes well with clathrin and AP-2 in punctate structures, many of which also contain the presynaptic markers synaptophysin, synaptotagmin or synaptic vesicle protein 2 (SV2). NECAP 1 was also detected by western blot in synaptic vesicle preparations. Overexpression of a truncation mutant of NECAP 1 (BC-NECAP 1) in neurons inhibited transferrin endocytosis but not epidermal growth factor (EGF) endocytosis, and this inhibition was dependent on an AP-2-binding WVQF motif. Moreover, overexpression of BC-NECAP 1 results in inhibition of synaptotagmin endocytosis both in unstimulated neurons and in neurons stimulated with potassium chloride. This inhibition was abrogated by truncation of the WVQF domain. We conclude from these observations that NECAP 1 plays a role in clathrin-mediated neuronal endocytosis, including a role in presynaptic endocytosis.     

Dynamin I phosphorylation and the control of synaptic vesicle endocytosis

The GTPase dynamin I is essential for synaptic vesicle endocytosis in nerve terminals. It is a nerve terminal phosphoprotein that is dephosphorylated on nerve terminal stimulation by the calcium-dependent protein phosphatase calcineurin and then rephosphorylated by cyclin-dependent kinase 5 on termination of the stimulus. Because of its unusual phosphorylation profile, the phosphorylation status of dynamin I was assumed to be inexorably linked to synaptic vesicle endocytosis; however, direct proof of this link has been elusive until very recently. This review will describe current knowledge regarding dynamin I phosphorylation in nerve terminals and how this regulates its biological function with respect to synaptic vesicle endocytosis.     

Human stoned B interacts with AP-2 and synaptotagmin and facilitates clathrin-coated vesicle uncoating

Synaptic vesicle biogenesis involves the recycling of synaptic vesicle components by clathrin-mediated endocytosis from the presynaptic membrane. stoned B, a protein encoded by the stoned locus in Drosophila melanogaster has been shown to regulate vesicle recycling by interacting with synaptotagmin. We report here the identification and characterization of a human homolog of stoned B (hStnB). Human stoned B is a brain-specific protein which co-enriches with other endocytic proteins such as AP-2 in a crude synaptic vesicle fraction and at nerve terminals. A domain with homology to the medium chain of adaptor complexes binds directly to both AP-2 and synaptotagmin and competes with AP-2 for the same binding site within synaptotagmin. Finally we show that the µ2 homology domain of hStnB stimulates the uncoating of both clathrin and AP-2 adaptors from clathrin-coated vesicles. We hypothesize that hStnB regulates synaptic vesicle recycling by facilitating vesicle uncoating.     

Protein phosphorylation during meiotic maturation of Xenopus oocytes: cdc2 protein kinase targets

M-Phase specific protein kinase or cdc2 protein kinase is a component of MPF (M-Phase promoting factor). During meiotic maturation of Xenopus oocytes, cdc2 protein kinase is activated in correlation with MPF activity. A protein phosphorylation cascade takes place involving several protein kinases, among which casein kinase II, and different changes associated with meiosis occur such as germinal vesicle breakdown, chromosome condensation, cytoskeletal reorganization and increase in protein synthesis. Our results provide a biochemical link between cdc2 protein kinase and protein synthesis since they show that the kinase phosphorylates in vitro a p47 protein identified as elongation factor EF1 (gamma subunit) and that the in vitro site of p47 corresponds to the site phosphorylated in vivo. Immunofluorescence showed that the elongation factor (EF1-beta gamma) is localized in the oocyte cortex. Furthermore, they show that cdc2 kinase phosphorylates and activates casein kinase II in vitro, strongly supporting the view that casein kinase II is involved in the phosphorylation cascade originated by cdc2 kinase.     

AP180 and AP-2 interact directly in a complex that cooperatively assembles clathrin.

Clathrin-coated vesicles are involved in protein and lipid trafficking between intracellular compartments in eukaryotic cells. AP-2 and AP180 are the resident coat proteins of clathrin-coated vesicles in nerve terminals, and interactions between these proteins could be important in vesicle dynamics. AP180 and AP-2 each assemble clathrin efficiently under acidic conditions, but neither protein will assemble clathrin efficiently at physiological pH. We find that there is a direct, clathrin-independent interaction between AP180 and AP-2 and that the AP180-AP-2 complex is more efficient at assembling clathrin under physiological conditions than is either protein alone. AP180 is phosphorylated in vivo, and in crude vesicle extracts its phosphorylation is enhanced by stimulation of casein kinase II, which is known to be present in coated vesicles. We find that recombinant AP180 is a substrate for casein kinase II in vitro and that its phosphorylation weakens both the binding of AP-2 by AP180 and the cooperative clathrin assembly activity of these proteins. We have localized the binding site for AP-2 to amino acids 623-680 of AP180. The AP180/AP-2 interaction can be disrupted by a recombinant AP180 fragment containing the AP-2 binding site, and this fragment also disrupts the cooperative clathrin assembly activity of the AP180-AP-2 complex. These results indicate that AP180 and AP-2 interact directly to form a complex that assembles clathrin more efficiently than either protein alone. Phosphorylation of AP180, by modulating the affinity of AP180 for AP-2, may contribute to the regulation of clathrin assembly in vivo.     

Phosphorylation of synaptic vesicle protein 2 modulates binding to synaptotagmin

Synaptic vesicle protein 2 (SV2) is a component of all synaptic vesicles that is required for normal neurotransmission. Here we report that in intact synaptic terminals SV2 is a phosphoprotein. Phosphopeptide mapping studies indicate that a major site of phosphorylation is located on the cytoplasmic amino terminus. SV2 is phosphorylated on serine and threonine but not on tyrosine residues, indicating that it is a substrate for serine/threonine kinases. Phosphopeptide mapping, in gel kinase assays, and surveys of kinase inhibitors suggest that casein kinase I is a primary SV2 kinase. The amino terminus of SV2 was previously shown to mediate its interaction with synaptotagmin, a calcium-binding protein also required for normal neurotransmission. Comparison of synaptotagmin binding with phosphorylated and unphosphorylated SV2 amino-terminal peptides reveals an increase in binding with phosphorylation. These results suggest that the affinity of SV2 for synaptotagmin is modulated by phosphorylation of SV2.     

Phosphorylation of VAMP/Synaptobrevin in Synaptic Vesicles by Endogenous Protein Kinases

Abstract: VAMP/synaptobrevin (SYB), an integral membrane protein of small synaptic vesicles, is specifically cleaved by tetanus neurotoxin and botulinum neurotoxins B, D, F, and G and is thought to play an important role in the docking and/or fusion of synaptic vesicles with the presynaptic membrane. Potential phosphorylation sites for various kinases are present in SYB sequence. We have studied whether SYB is a substrate for protein kinases that are present in nerve terminals and known to modulate neurotransmitter release. SYB can be phosphorylated within the same vesicle by endogenous Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) associated with synaptic vesicles. This phosphorylation reaction occurs rapidly and involves serine and threonine residues in the cytoplasmic region of SYB. Similarly to CaMKII, a casein kinase II (CasKII) activity copurifying with synaptic vesicles is able to phosphorylate SYB selectively on serine residues of the cytoplasmic region. This phosphorylation reaction is markedly stimulated by sphingosine, a sphingolipid known to activate CasKII and to inhibit CaMKII and protein kinase C. The results show that SYB is a potential substrate for protein kinases involved in the regulation of neurotransmitter release and open the possibility that phosphorylation of SYB plays a role in modulating the molecular interactions between synaptic vesicles and the presynaptic membrane.