Recognition of dileucine-based sorting signals from HIV-1 Nef and LIMP-II by the AP-1 gamma-sigma1 and AP-3 delta-sigma3 hemicomplexes

The sorting of transmembrane proteins to endosomes and lysosomes is mediated by signals present in the cytosolic tails of the proteins. A subset of these signals conform to the [DE]XXXL[LI] consensus motif and mediate sorting via interactions with heterotetrameric adaptor protein (AP) complexes. However, the identity of the AP subunits that recognize these signals remains controversial. We have used a yeast three-hybrid assay to demonstrate that [DE]XXXL[LI]-type signals from the human immunodeficiency virus negative factor protein and the lysosomal integral membrane protein II interact with combinations of the gamma and sigma1 subunits of AP-1 and the delta and sigma3 subunits of AP-3, but not the analogous combinations of AP-2 and AP-4 subunits. The sequence requirements for these interactions are similar to those for binding to the whole AP complexes in vitro and for function of the signals in vivo. These observations reveal a novel mode of recognition of sorting signals involving the gamma/delta and sigma subunits of AP-1 and AP-3.     

Overexpression of the myelin proteolipid protein leads to accumulation of cholesterol and proteolipid protein in endosomes/lysosomes: implications for Pelizaeus-Merzbacher disease

The mouse mutants mocha and pearl are deficient in the AP-3 delta and beta3A subunits, respectively. We have used cells from these mice to investigate both the assembly of AP-3 complexes and AP-3 function. In mocha cells, the beta3 and mu3 subunits coassemble into a heterodimer, whereas the sigma3 subunit remains monomeric. In pearl cells, the delta and sigma3 subunits coassemble into a heterodimer, whereas mu3 gets destroyed. The yeast two hybrid system was used to confirm these interactions, and also to demonstrate that the A (ubiquitous) and B (neuronal-specific) isoforms of beta3 and mu3 can interact with each other. Pearl cell lines were generated that express beta3A, beta3B, a beta3Abeta2 chimera, two beta3A deletion mutants, and a beta3A point mutant lacking a functional clathrin binding site. All six constructs assembled into complexes and were recruited onto membranes. However, only beta3A, beta3B, and the point mutant gave full functional rescue, as assayed by LAMP-1 sorting. The beta3Abeta2 chimera and the beta3A short deletion mutant gave partial functional rescue, whereas the beta3A truncation mutant gave no functional rescue. These results indicate that the hinge and/or ear domains of beta3 are important for function, but the clathrin binding site is not needed.     

Clathrin‐Coated Pits: Vive La Différence?

Because of the discovery of coated pits and vesicles more than 40 years ago and the identification of clathrin as a major component of the coat, it has been assumed that clathrin-coated pits (CCPs) are responsible for the uptake of most plasma membrane receptors undergoing internalization. The recent molecular characterization of clathrin-independent routes of endocytosis confirms that several alternative endocytic pathways operate at the plasma membrane of mammalian cells. This heterogeneous view of endocytosis has been expanded still further by recent studies, suggesting that different subpopulations of CCPs responsible for the internalization of specific sets of cargo may coexist. In the present review, we have discussed the experimental evidence in favor or against the existence of distinct parallel clathrin-dependent pathways at the plasma membrane.     

Role of the mammalian retromer in sorting of the cation-independent mannose 6-phosphate receptor

Disruption of latent TGF-beta binding protein (LTBP)-4 expression in the mouse leads to abnormal lung development and colorectal cancer. Lung fibroblasts from these mice produced decreased amounts of active TGF-beta, whereas secretion of latent TGF-beta was significantly increased. Expression and secretion of TGF-beta2 and -beta3 increased considerably. These results suggested that TGF-beta activation but not secretion would be severely impaired in LTBP-4 -/- fibroblasts. Microarrays revealed increased expression of bone morphogenic protein (BMP)-4 and decreased expression of its inhibitor gremlin. This finding was accompanied by enhanced expression of BMP-4 target genes, inhibitors of differentiation 1 and 2, and increased deposition of fibronectin-rich extracellular matrix. Accordingly, increased expression of BMP-4 and decreased expression of gremlin were observed in mouse lung. Transfection of LTBP-4 rescued the -/- fibroblast phenotype, while LTBP-1 was inefficient. Treatment with active TGF-beta1 rescued BMP-4 and gremlin expression to wild-type levels. Our results indicate that the lack of LTBP-4-mediated targeting and activation of TGF-beta1 leads to enhanced BMP-4 signaling in mouse lung.     

In vitro, insulin receptor catalyses phosphorylation of clathrin heavy chain and a plasma membrane 180,000 molecular weight protein

Insulin receptor mutation studies that the receptor tyrosine kinase activity is necessary for receptor endocytosis, and several insulin receptor-containing tissues have a plasma membrane-associated protein (M_r 180,000, p180) whose tyrosine phosphorylation is receptor catalysed. Since clathrin heavy chain (M_r 180,000 in dodecyl sulphate gel electrophoresis) is a major component of coated vesicles, the latter functioning in receptor endocytosis, we investigated whether insulin receptors can catalyse clathrin phosphorylation and whether p180 is clathrin. Bovine brain triskelion or coated vesicles and ³²P-ATP were added to prephosphorylated insulin receptor preparations (wheat ferm agglutinin-purified human placenta membrane proteins). Antiphosphotyrosine immunoprecipitated a phosphorylated 180,000 molecular weight protein. Insulin (10⁻⁷M) increased the rate of phosphorylation. Monoclonal anti-clathrin antibody immunoprecipitated the phosphorylated 180,000 molecular weight protein, whereas monoclonal anti-insulin receptor antibodies (-IR1, MA10) immunoprecipitated both insulin receptors and the phosphorylated 180,000 molecular weight protein. In the absence of added clathrin, anticlathrin immunoprecipitated no proteins, and -IR1 imunoprecipitated only the insulin receptor. Density gradient (glycerol 7.5–30%, w/v) centrifugation separated human placenta microsomal membrane proteins into endosomal, plasma membrane, cytoplasmic and coated vesicle fractions. Antiphosphotyrosine immunoprecipitated phosphorylated-microsomal proteins that centrifugated into endosomal and plasma membrane fractions. Addition of glycerol gradient fractions to a prephosphorylated insulin receptor preparation, however, gave a tyrosine-phosphorylated 180,000 molecular weight protein when cytoplasmic and coated vesicle fractions were added. Taken together these results suggest: (1) that, in vitro, human placenta insulin receptors can phosphorylate bovine brain and human placenta clathrin heavy chain; (2) that both assembled and unassembled clathrin can be phosphorylated; and (3) that p180, the plasma membrane-associated insulin receptor substrate, is not clathrin heavy chain.     

The kinase Syk as an adaptor controlling sustained calcium signalling and B-cell development

Upon B-cell antigen receptor (BCR) activation, the protein tyrosine kinase Syk phosphorylates the adaptor protein SH2 domain-containing leukocyte protein of 65 kDa (SLP-65), thus coupling the BCR to diverse signalling pathways. Here, we report that SLP-65 is not only a downstream target and substrate of Syk but also a direct binding-partner and activator of this kinase. This positive feedback is mediated by the binding of the SH2 domain of SLP-65 to an autophosphorylated tyrosine of Syk. The mutant B cells that cannot form the Syk/SLP-65 complex are defective in BCR-induced extracellular signal-regulated kinase, nuclear factor kappa B and nuclear factor of activated T cells, but not Akt activation, and are blocked in B-cell development. Furthermore, we show that formation of the Syk/SLP-65 complex is required for sustained Ca(2+) responses in activated B cells. We suggest that after activation and internalization of the BCR, Syk remains active as part of a membrane-bound Syk/SLP-65 complex controlling sustained signalling and calcium influx.     

Identification of the universal cofactor (auxilin 2) in clathrin coat dissociation

Uncoating of clathrin-coated vesicles in neuronal cells requires hsc70 in concert with the cofactor auxilin which contains a J-domain as well as a domain with homology to dual specific phosphatases and tensin, known as PTEN. The question of whether an analogous factor operates in other cell types has until now remained unanswered. Here we show that it is the recently discovered and widely expressed cyclin G-associated protein kinase which fulfils the function of neuronal auxilin in hsc70-mediated clathrin coat dissociation. GAK possesses a J-domain, which stimulates the hsc70 ATPase, it competes with auxilin for clathrin binding and at sufficiently high concentrations acts as a clathrin assembly protein. Moreover, GAK binds to the gamma- and alpha-appendage domains of the adaptor proteins AP-1 and AP-2 in vitro and phosphorylates their medium chains. Cells that transiently overexpress GAK are impaired in respect of receptor-mediated endocytosis. In transfected cells clathrin is dislodged from coated pits/vesicles and co-localizes with GFP-GAK in the form of large aggregates. The cellular distribution of membrane-associated adaptors was unaffected by overexpression of GAK. Our results point to a hsc70/auxilin-based uncoating system as a ubiquitous feature of eukaryotic cells.     

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.