Diacylglycerol kinase is a member of the diacylglycerol kinase family of enzymes, which generate phosphatidic acid through diacylglycerol phosphorylation. In addition to the catalytic and cysteine-rich domains found in all diacylglycerol kinases, diacylglycerol kinase has a MARCKS domain as well as a C-terminal region containing four ankyrin repeats and a PDZ-binding motif. Previous reports demonstrated that diacylglycerol kinase interaction with several proteins is an important mechanism for modulating the localization and activity of this enzyme. Here we used a proteomics approach to search for novel diacylglycerol kinase -interacting proteins and identified sorting nexin 27 (SNX27), a recently described member of a protein family involved in intracellular trafficking, which has a PDZ domain in addition to the phox homology domain characteristic of SNX proteins. Co-immunoprecipitation studies and two-hybrid analysis confirmed physical, PDZ-dependent association between SNX27 and diacylglycerol kinase . Because diacylglycerol kinase is expressed abundantly in T lymphocytes, we characterized SNX27 expression and subcellular localization in these cells. SNX27 co-localized with transferrin receptor-positive vesicles, pointing to its participation in T cell endocytic recycling. Expression of deletion mutants revealed that in addition to the phox homology domain the SNX27 PDZ domain contributed to vesicle localization of this protein, suggesting that interaction with diacylglycerol kinase regulates SNX27 localization. Analysis of cells with RNA interference-mediated knockdown of diacylglycerol kinase showed accelerated transferrin receptor exit from the lymphocyte endocytic recycling compartment back to the plasma membrane, further confirming diacylglycerol kinase -dependent control of vesicle trafficking. These data support a previously unreported role for diacylglycerol kinase in the modulation of membrane trafficking, which may also help to define SNX27 function.

Sorcin, a protein overexpressed in many multi-drug resistant cancers, dynamically localizes to distinct subcellular sites in 3T3-L1 fibroblasts during cell-cycle progression. During interphase sorcin is in the nucleus, in the plasma membrane, in endoplasmic reticulum (ER) cisternae, and in ER-derived vesicles localized along the microtubules. These vesicles are positive to RyR, SERCA, calreticulin and Rab10. At the beginning of mitosis, sorcin-containing vesicles associate with the mitotic spindle, and during telophase are concentrated in the cleavage furrow and, subsequently, in the midbody. Sorcin regulates dimensions and calcium load of the ER vesicles by inhibiting RYR and activating SERCA. Analysis of sorcin interactome reveals calcium-dependent interactions with many proteins, including Polo-like kinase 1 (PLK1), Aurora A and Aurora B kinases. Sorcin interacts physically with PLK1, is phosphorylated by PLK1 and induces PLK1 autophosphorylation, thereby regulating kinase activity. Knockdown of sorcin results in major defects in mitosis and cytokinesis, increase in the number of rounded polynucleated cells, blockage of cell progression in G2/M, apoptosis and cell death. Sorcin regulates calcium homeostasis and is necessary for the activation of mitosis and cytokinesis.

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b and P2 a a a a respectively. An interaction of P0 with P1 proteins, but not with P2 proteins, was also detected. This interaction is strongly increased with the P0 carboxyl end, which is able to form a pentameric complex with the four acidic proteins. The P1/P2 binding site has been located between residues 212 and 262 using different C-terminal P0 fragments. Immunoprecipitation shows the association of EF-2 with protein P0. However, the interaction is stronger with the P1/P2 proteins than with P0 in the two-hybrid assay. This interaction improves using the 100-aminoacid-long C-end of P0 and is even higher with the last 50 amino acids. The data indicate a specific association of P1 a a a a with P2 b b b b and of P1 b b b b with P2 a a a a rather than the dimerization of the acidic proteins found in prokaryotes. In addition, they suggest that stalk assembly begins by the interaction of the P1 proteins with P0. Moreover, as functional interactions of the complete P0 were found to increase using protein fragments, the data suggest that some active sites are exposed in the ribosome as a result of conformational changes that take place during stalk assembly and function.

In this study we have used the yeast two-hybrid system to identify proteins that interact with the carboxylcytoplasmic domain (residues 464 -509) of the insulinsensitive glucose transporter GLUT4 (C-GLUT4). Using as bait C-GLUT4, we have isolated the carboxyl domain of Daxx (C-Daxx), the adaptor protein associated with the Fas and the type II TGF-␤ (T␤RII) receptors (1, 2). The two-hybrid interaction between C-GLUT4 and CDaxx is validated by the ability of in vitro translated C-GLUT4 to interact with in vitro translated full-length Daxx and C-Daxx. C-Daxx does not interact with the C-cytoplasmic domain of GLUT1, the ubiquitous glucose transporter homologous to GLUT4. Replacement of alanine and serine for the dileucine pair (Leu 489 -Leu 490 ) critical for targeting GLUT4 from the trans-Golgi network to the perinuclear intracellular store as well as for its surface internalization by endocytosis inhibits 2-fold the interaction of C-GLUT4 with Daxx. Daxx is pulled down with GLUT4 immunoprecipitated from lysates of 3T3-L1 fibroblasts stably transfected with GLUT4 and 3T3-L1 adipocytes expressing physiological levels of the two proteins. Similarly, GLUT4 is recovered with antiDaxx immunoprecipitates. Using an established cell fractionation procedure we present evidence for the existence of two distinct intracellular Daxx pools in the nucleus and low density microsomes. Confocal immunofluorescence microscopy studies localize Daxx to promyelocytic leukemia nuclear bodies and punctate cytoplasmic structures, often organized in strings and underneath the plasma membrane. Daxx and GLUT4 are SUMOlated as shown by their reaction with an anti-SUMO1 antibody and by the ability of this antibody to pull down Daxx and GLUT4.The cytoplasmic domain of membrane proteins plays important roles in their transport, signal transduction, organization of protein scaffolds, and regulation of their turnover. Trafficking of GLUT4 in adipose and skeletal muscle cells is regulated by insulin and muscle contraction and is critical for the control of glucose levels in blood. Upon increase in insulin levels and muscle contraction the GLUT4 retained in intracellular stores is translocated to the plasma membrane, where it facilitates glucose transport (3). Trafficking of GLUT4 is mediated by motifs localized to the amino and carboxyl-cytoplasmic domains of the protein, though their characterization and the identification of the factors involved in their reading is incomplete.SUMO (also called sentrin, PIC1, and GMP1), a 101-amino acid ubiquitin-like modifier protein that is highly conserved from yeast to human, appears to control protein turnover and compartmentalization (4). Three members of the SUMO family have been described in vertebrates. They show major structural differences in the sequences of their N-extensions, which are absent in ubiquitin. It has been shown recently that Ubc9, the only E2-type SUMO1-conjugating enzyme described in vertebrates, interacts with the carboxyl-cytoplasmic domain of GLUT4 as part of a mechanism that slows its...

Sorcin is an essential penta-EF hand calcium binding protein, able to confer the multi-drug resistance phenotype to drug-sensitive cancer cells and to reduce Endoplasmic Reticulum stress and cell death. Sorcin silencing blocks cell cycle progression in mitosis and induces cell death by triggering apoptosis. Sorcin participates in the modulation of calcium homeostasis and in calcium-dependent cell signalling in normal and cancer cells. The molecular basis of Sorcin action is yet unknown. The X-ray structures of Sorcin in the apo (apoSor) and in calcium bound form (CaSor) reveal the structural basis of Sorcin action: calcium binding to the EF1-3 hands promotes a large conformational change, involving a movement of the long D-helix joining the EF1-EF2 sub-domain to EF3 and the opening of EF1. This movement promotes the exposure of a hydrophobic pocket, which can accommodate in CaSor the portion of its N-terminal domain displaying the consensus binding motif identified by phage display experiments. This domain inhibits the interaction of sorcin with PDCD6, a protein that carries the Sorcin consensus motif, co-localizes with Sorcin in the perinuclear region of the cell and in the midbody and is involved in the onset of apoptosis.

Here, we report that Cdk5 activation is stimulated by insulin and plays a key role in the regulation of GLUT4-mediated glucose uptake in 3T3-L1 adipocytes. Insulin activation of Cdk5 requires PI3K signaling. Insulin-activated Cdk5 phosphorylates E-Syt1, a 5 C2-domain protein-related to the synaptotagmins that is induced during adipocyte differentiation. Phosphorylated E-Syt1 associates with GLUT4, an event inhibited by the Cdks inhibitor roscovitine. Cdk5 silencing inhibits glucose uptake by 3T3-L1 adipocytes. These studies elucidate a previously unknown activity of Cdk5 and demonstrate the involvement of this kinase in the regulation of insulin-dependent glucose uptake in adipocytes.T he atypical Cdc2-related protein kinase Cdk5 is ubiquitously expressed in mammalian cells and tissues (1). In noncycling cells, Cdk5 phosphorylates multiple substrates to control such diverse phenomena as cell signaling, adhesion and motility, cytoskeletal organization, protein trafficking, and membrane fusion and dynamic organization (2). The membrane-bound Cdk5 effectors, p35/p39, direct and activate the kinase to specific membrane targets via mechanisms that are not yet well understood (3-6). Cdk5 plays a crucial regulatory role in glucosestimulated insulin secretion in pancreatic cells (7,8). In addition, CDK5 is involved in the loss of ␤ cell function under glucotoxic conditions, revealing the potential therapeutic value of CDK5 inhibitors in the treatment of type 2 diabetes. Recently, fine mapping and genome-wide association studies have identified SNPs that affect a p35 homolog (9, 10) and calpain 10 (11) as being associated with type 2 diabetes susceptibility. The finding that the specific and strong Cdks inhibitor roscovitine inhibited 2DOG uptake by adipocytes has led us to investigate the involvement of Cdk5 in the regulation of glucose uptake in 3T3-L1 adipocytes.Here, we report that insulin stimulates Cdk5 activity in 3T3-L1 adipocytes, and knockdown of the kinase inhibits glucose uptake in these cells. Insulin-activated Cdk5 phosphorylates the synaptotagmin homolog E-Syt1 and promotes its association with GLUT4. Both E-Syt1 phosphorylation and GLUT4 association are inhibited by roscovitine. These findings are discussed in the context of the regulation of GLUT4-mediated glucose uptake in adipocytes, Cdk5 deregulation in response to calpains, and the susceptibility of families with the calpain 10 SNP-44 polymorphism to develop type 2 diabetes. ResultsCdk5 and p35 Are Coexpressed with GLUT4. We found comparable levels of Cdk5 transcript and protein in all GLUT4-expressing tissues and cells, including skeletal muscle, cardiac tissue, epidydimal white fat, and 3T3-L1 adipocytes (Fig. 1A). Both p35 transcript and protein were also detected in all samples (Fig. 1 A). The 32-kDa Cdk5 protein was abundant and unequally distributed among cytosolic (80%) plasma membrane (9%), microsomal (5%), and nuclear (6%) fractions [supporting information (SI) Fig. S1 A]. We identified 5 species of the Cdk5 activator p35, of which the 34-35...

The transition metal copper (Cu) is an essential trace element for all biota. Its redox properties bestow Cu with capabilities that are simultaneously essential and potentially damaging to the cell. Free Cu is virtually absent in the cell. The descriptions of the structural and functional organization of the metallothioneins, Cu-chaperones and P-type ATPases as well as of the mechanisms that regulate their distribution and functioning in the cell have enormously advanced our understanding of the Cu homeostasis and metabolism in the last decade. Cu is stored by metallothioneins and distributed by specialized chaperones to specific cell targets that make use of its redox properties. Transfer of Cu to newly synthesized cuproenzymes and Cu disposal is performed by the individual or concerted actions of the P-type ATPases ATP7A and ATP7B expressed in tissues. In mammalians liver is the major captor, distributor and excreter of Cu. Mutations in the P-type ATPases that interfere with their functioning and traffic are cause of the life-threatening Wilson (ATP7B) and Menkes (ATP7A) diseases.