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.

Lithium regulates PKC-mediated intracellular cross-talk and gene expression in the CNS in vivo.

It has become increasingly appreciated that the long-term treatment of complex neuropsychiatric disorders like bipolar disorder (BD) involves the strategic regulation of signaling pathways and gene expression in critical neuronal circuits. Accumulating evidence from our laboratories and others has identified the family of protein kinase C (PKC) isozymes as a shared target in the brain for the long-term action of both lithium and valproate (VPA) in the treatment of BD. In rats chronically treated with lithium at therapeutic levels, there is a reduction in the levels of frontal cortical and hippocampal membrane-associated PKC alpha and PKC epsilon. Using in vivO microdialysis, we have investigated the effects of chronic lithium on the intracellular cross-talk between PKC and the cyclic AMP (cAMP) generating system in vivo. We have found that activation of PKC produces an increase in dialysate cAMP levels in both prefrontal cortex and hippocampus, effects which are attenuated by chronic lithium administration. Lithium also regulates the activity of another major signaling pathway the c-Jun N-terminal kinase pathway--in a PKC-dependent manner. Both Li and VPA, at therapeutically relevant concentrations, increase the DNA binding of activator protein 1 (AP-1) family of transcription factors in cultured cells in vitro, and in rat brain ex vivo. Furthermore, both agents increase the expression of an AP-1 driven reporter gene, as well as the expression of several endogenous genes known to be regulated by AP-1. Together, these results suggest that the PKC signaling pathway and PKC-mediated gene expression may be important mediators of lithium's long-term therapeutic effects in a disorder as complex as BD.

The epsins define a family of proteins that interact with components of the clathrin coat and contain a new protein module.

Epsin (epsin 1) is an interacting partner for the EH domain-containing region of Eps15 and has been implicated in conjunction with Eps15 in clathrin-mediated endocytosis. We report here the characterization of a similar protein (epsin 2), which we have cloned from human and rat brain libraries. Epsin 1 and 2 are most similar in their NH(2)-terminal region, which represents a module (epsin NH(2) terminal homology domain, ENTH domain) found in a variety of other proteins of the data base. The multiple DPW motifs, typical of the central region of epsin 1, are only partially conserved in epsin 2. Both proteins, however, interact through this central region with the clathrin adaptor AP-2. In addition, we show here that both epsin 1 and 2 interact with clathrin. The three NPF motifs of the COOH-terminal region of epsin 1 are conserved in the corresponding region of epsin 2, consistent with the binding of both proteins to Eps15. Epsin 2, like epsin 1, is enriched in brain, is present in a brain-derived clathrin-coated vesicle fraction, is concentrated in the peri-Golgi region and at the cell periphery of transfected cells, and partially colocalizes with clathrin. High overexpression of green fluorescent protein-epsin 2 mislocalizes components of the clathrin coat and inhibits clathrin-mediated endocytosis. The epsins define a new protein family implicated in membrane dynamics at the cell surface.

ADP-ribosylation factor 1 transiently activates high-affinity adaptor protein complex AP-1 binding sites on Golgi membranes.

Association of the Golgi-specific adaptor protein complex 1 (AP-1) with the membrane is a prerequisite for clathrin coat assembly on the trans-Golgi network (TGN). The AP-1 adaptor is efficiently recruited from cytosol onto the TGN by myristoylated ADP-ribosylation factor 1 (ARF1) in the presence of the poorly hydrolyzable GTP analog guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS). Substituting GTP for GTPgammaS, however, results in only poor AP-1 binding. Here we show that both AP-1 and clathrin can be recruited efficiently onto the TGN in the presence of GTP when cytosol is supplemented with ARF1. Optimal recruitment occurs at 4 microM ARF1 and with 1 mM GTP. The AP-1 recruited by ARF1.GTP is released from the Golgi membrane by treatment with 1 M Tris-HCl (pH 7) or upon reincubation at 37 degreesC, whereas AP-1 recruited with GTPgammaS or by a constitutively active point mutant, ARF1(Q71L), remains membrane bound after either treatment. An incubation performed with added ARF1, GTP, and AlFn, used to block ARF GTPase-activating protein activity, results in membrane-associated AP-1, which is largely insensitive to Tris extraction. Thus, ARF1. GTP hydrolysis results in lower-affinity binding of AP-1 to the TGN. Using two-stage assays in which ARF1.GTP first primes the Golgi membrane at 37 degreesC, followed by AP-1 binding on ice, we find that the high-affinity nucleating sites generated in the priming stage are rapidly lost. In addition, the AP-1 bound to primed Golgi membranes during a second-stage incubation on ice is fully sensitive to Tris extraction, indicating that the priming stage has passed the ARF1.GTP hydrolysis point. Thus, hydrolysis of ARF1.GTP at the priming sites can occur even before AP-1 binding. Our finding that purified clathrin-coated vesicles contain little ARF1 supports the concept that ARF1 functions in the coat assembly process rather than during the vesicle-uncoating step. We conclude that ARF1 is a limiting factor in the GTP-stimulated recruitment of AP-1 in vitro and that it appears to function in a stoichiometric manner to generate high-affinity AP-1 binding sites that have a relatively short half-life.

Tyrosine hydroxylase gene promoter activity is regulated by both cyclic AMP-responsive element and AP1 sites following calcium influx. Evidence for cyclic amp-responsive element binding protein-independent regulation.

Membrane depolarization of PC12 cells using 50 mM KCl leads to induction of tyrosine hydroxylase (TH) mRNA. This induction of TH mRNA is apparently due to increased TH gene promoter activity mediated by the influx of Ca2+. In PC12 cells transiently transfected with a chimeric gene expressing chloramphenicol acetyltransferase (CAT) driven by the proximal TH gene 5'-flanking region, 50 mM KCl increases TH gene promoter activity 3-4-fold. Promoter analysis utilizing TH-CAT constructs containing mutagenized sequences indicates that this response to the depolarization-mediated influx of Ca2+ is primarily dependent on both the TH cAMP-responsive element (CRE) and TH activating protein-1 (AP1) site. Minimal promoter constructs that contain a single copy of either the TH CRE or TH AP1 site fused upstream of the TH gene basal promoter are only modestly responsive or nonresponsive, respectively, to depolarization. However, both these constructs are strongly responsive to the calcium ionophore, A23187. Gel shift assays indicate that TH AP1 complex formation is dramatically increased after treatment with either 50 mM KCl or A23187. Using antibodies to transcription factors of the Fos and Jun families, we show that the nuclear proteins comprising the inducible TH AP1 complex include c-Fos, c-Jun, JunB, and JunD. In cAMP-responsive element binding protein (CREB)-deficient cell lines that express antisense RNA complementary to CREB mRNA, the response of the TH gene promoter to cyclic AMP is dramatically inhibited, but the response to A23187 remains robust. This result indicates that transcription factors other than CREB can participate in the Ca2+-mediated regulation of the TH gene. In summary, our results support the hypothesis that regulation of the TH gene by Ca2+ is mediated by mechanisms involving both the TH CRE and TH AP1 sites and that transcription factors other than or in addition to CREB participate in this response.

Transcriptional activation of the Cu,Zn-superoxide dismutase gene through the AP2 site by ginsenoside Rb2 extracted from a medicinal plant, Panax ginseng.

We report here that the ginseng saponins induce the transcription of Cu,Zn-superoxide dismutase gene (SOD1), which is one of the major antioxidant enzymes. Total saponins and panaxatriol did not elevate the level of SOD1, but panaxadiol significantly increased SOD1. Among the panaxadiol fractions, ginsenoside Rb2 was a more specific and more remarkable inducer of the SOD1 gene than ginsenoside Rb1. Deletion analyses of the SOD1 promoter revealed that the proximal promoter is responsible for this induction. Mobility shift assays with cis-elements in the proximal promoter region showed that specific binding of the AP2 transcription factor was significantly increased by treatment with ginsenoside Rb2. Mutations of the AP2 binding sites in the heterologous promoter and natural context systems abolished the transcriptional activation by ginsenoside Rb2. These results suggest that the SOD1 gene was greatly activated by ginsenoside Rb2 through transcription factor AP2 binding sites and its induction.