A novel 450-kDa coiled-coil protein, CG-NAP (centrosome and Golgi localized PKN-associated protein), was identified as a protein that interacted with the regulatory region of the protein kinase PKN, having a catalytic domain homologous to that of protein kinase C. CG-NAP contains two sets of putative RII (regulatory subunit of protein kinase A)-binding motif. Indeed, CG-NAP tightly bound to RII␣ in HeLa cells. Furthermore, CG-NAP was coimmunoprecipitated with the catalytic subunit of protein phosphatase 2A (PP2A), when one of the B subunit of PP2A (PR130) was exogenously expressed in COS7 cells. CG-NAP also interacted with the catalytic subunit of protein phosphatase 1 in HeLa cells. Immunofluorescence analysis of HeLa cells revealed that CG-NAP was localized to centrosome throughout the cell cycle, the midbody at telophase, and the Golgi apparatus at interphase, where a certain population of PKN and RII␣ were found to be accumulated. These data indicate that CG-NAP serves as a novel scaffolding protein that assembles several protein kinases and phosphatases on centrosome and the Golgi apparatus, where physiological events, such as cell cycle progression and intracellular membrane traffic, may be regulated by phosphorylation state of specific protein substrates.

Alix (ALG-2-interacting protein X) is a 95-kDa protein that interacts with an EF-hand type Ca2؉ -binding protein, ALG-2 (apoptosis-linked gene 2), through its C-terminal proline-rich region. In this study, we searched for proteins that interact with human Alix⌬C (a truncated form not containing the C-terminal region) by using a yeast two-hybrid screen, and we identified two similar human proteins, CHMP4a and CHMP4b

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder. We screened 751 familial ALS patient whole-exome sequences and identified six mutations including p.D40G in the ANXA11 gene in 13 individuals. The p.D40G mutation was absent from 70,000 control whole-exome sequences. This mutation segregated with disease in two kindreds and was present in another two unrelated cases (P = 0.0102), and all mutation carriers shared a common founder haplotype. Annexin A11–positive protein aggregates were abundant in spinal cord motor neurons and hippocampal neuronal axons in an ALS patient carrying the p.D40G mutation. Transfected human embryonic kidney cells expressing ANXA11 with the p.D40G mutation and other N-terminal mutations showed altered binding to calcyclin, and the p.R235Q mutant protein formed insoluble aggregates. We conclude that mutations in ANXA11 are associated with ALS and implicate defective intracellular protein trafficking in disease pathogenesis

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The mechanistic target of rapamycin complex 1 (mTORC1) is an essential regulator of cell growth and metabolism through the modulation of protein and lipid synthesis, lysosome biogenesis, and autophagy. The activity of mTORC1 is dynamically regulated by several environmental cues, including amino acid availability, growth factors, energy levels, and stresses, to coordinate cellular status with environmental conditions. Dysregulation of mTORC1 activity is closely associated with various diseases, including diabetes, cancer, and neurodegenerative disorders. The discovery of Rag GTPases has greatly expanded our understanding of the regulation of mTORC1 activity by amino acids, especially leucine and arginine. In addition to Rag GTPases, other factors that also contribute to the modulation of mTORC1 activity have been identified. In this review, we discuss the mechanisms of regulation of mTORC1 activity by particular amino acids.

CHMP6 (charged multivesicular body protein 6) is a human orthologue of yeast Vps (vacuolar protein sorting) 20, a component of ESCRT (endosomal sorting complex required for transport)-III. Various CHMP6 orthologues in organisms ranging from yeast to humans contain the N-myristoylation consensus sequence at each N-terminus. Metabolic labelling of HEK-293 (human embryonic kidney) cells showed the incorporation of [3H]myristate into CHMP6 fused C-terminally to GFP (green fluorescent protein) (CHMP6–GFP). Interactions of CHMP6 with another ESCRT-III component CHMP4b/Shax [Snf7 (sucrose non-fermenting 7) homologue associated with Alix] 1, one of three paralogues of human Vps32/Snf7, and with EAP20 (ELL-associated protein 20), a human counterpart of yeast Vps25 and component of ESCRT-II, were observed by co-immunoprecipitation of epitope-tagged proteins expressed in HEK-293 cells. The in vitro pull-down assays using their recombinant proteins purified from Escherichia coli demonstrated direct physical interactions which were mediated by the N-terminal basic half of CHMP6. Overexpressed CHMP6-GFP in HeLa cells exhibited a punctate distribution throughout the cytoplasm especially in the perinuclear area, as revealed by fluorescence microscopic analysis. Accumulation of LBPA (lysobisphosphatidic acid), a major phospholipid in internal vesicles of an MVB (multivesicular body), was observed in the CHMP6–GFP-localizing area. FLAG-tagged EAP20 distributed diffusely, but exhibited a punctate distribution on co-expression with CHMP6–GFP. Overexpression of CHMP6–GFP caused reduction of transferrin receptors on the plasma membrane surface, but caused their accumulation in the cytoplasm. Ubiquitinated proteins and endocytosed EGF continuously accumulated in CHMP6–GFP-expressing cells. These results suggest that CHMP6 acts as an acceptor for ESCRT-II on endosomal membranes and regulates cargo sorting

ALG-2 belongs to the penta-EF-hand (PEF) protein family and interacts with various intracellular proteins, such as Alix and TSG101, that are involved in endosomal sorting and HIV budding. Through X-ray crystallography, we solved the structures of Ca(2+)-free and -bound forms of N-terminally truncated human ALG-2 (des3-20ALG-2), Zn(2+)-bound form of full-length ALG-2, and the structure of the complex between des3-23ALG-2 and the peptide corresponding to Alix799-814 in Zn(2+)-bound form. Binding of Ca(2+) to EF3 enables the side chain of Arg125, present in the loop connecting EF3 and EF4, to move enough to make a primary hydrophobic pocket accessible to the critical PPYP motif, which partially overlaps with the GPP motif for the binding of Cep55 (centrosome protein 55 kDa). Based on these results, together with the results of in vitro binding assay with mutant ALG-2 and Alix proteins, we propose a Ca(2+)/EF3-driven arginine switch mechanism for ALG-2 binding to Alix.