CEP120 interacts with CPAP and positively regulates centriole elongation

Centriole duplication begins with the formation of a single procentriole next to a preexisting centriole. CPAP (centrosomal protein 4.1–associated protein) was previously reported to participate in centriole elongation. Here, we show that CEP120 is a cell cycle–regulated protein that directly interacts with CPAP and is required for centriole duplication. CEP120 levels increased gradually from early S to G2/M and decreased significantly after mitosis. Forced overexpression of either CEP120 or CPAP not only induced the assembly of overly long centrioles but also produced atypical supernumerary centrioles that grew from these long centrioles. Depletion of CEP120 inhibited CPAP-induced centriole elongation and vice versa, implying that these proteins work together to regulate centriole elongation. Furthermore, CEP120 was found to contain an N-terminal microtubule-binding domain, a C-terminal dimerization domain, and a centriolar localization domain. Overexpression of a microtubule binding–defective CEP120-K76A mutant significantly suppressed the formation of elongated centrioles. Together, our results indicate that CEP120 is a CPAP-interacting protein that positively regulates centriole elongation.     

A novel GTPase-activating protein for ARF6 directly interacts with clathrin and regulates clathrin-dependent endocytosis

ADP-ribosylation factor 6 (Arf6) is a small-GTPase that regulates the membrane trafficking between the plasma membrane and endosome. It is also involved in the reorganization of the actin cytoskeleton. GTPase-activating protein (GAP) is a critical regulator of Arf function as it inactivates Arf. Here, we identified a novel species of GAP denoted as SMAP1 that preferentially acts on Arf6. Although overexpression of SMAP1 did not alter the subcellular distribution of the actin cytoskeleton, it did block the endocytosis of transferrin receptors. Knock down of endogenous SMAP1 also abolished transferrin internalization, which confirms that SMAP1 is needed for this endocytic process. SMAP1 overexpression had no effect on clathrin-independent endocytosis, however. Intriguingly, SMAP1 binds directly to the clathrin heavy chain via its clathrin-box and mutation studies revealed that its GAP domain and clathrin-box both contribute to the role SMAP1 plays in clathrin-dependent endocytosis. These observations suggest that SMAP1 may be an Arf6GAP that specifically regulates one of the multiple functions of Arf6, namely, clathrin-dependent endocytosis, and that it does so by binding directly to clathrin.     

Axin directly interacts with plakoglobin and regulates its stability.

Plakoglobin is homologous to beta-catenin. Axin, a Wnt signal negative regulator, enhances glycogen synthase kinase (GSK)-3beta-dependent phosphorylation of beta-catenin and stimulates the degradation of beta-catenin. Therefore, we examined the effect of Axin on plakoglobin stability. Axin formed a complex with plakoglobin in COS cells and SW480 cells. Axin directly bound to plakoglobin, and this binding was inhibited by beta-catenin. Axin promoted GSK-3beta-dependent phosphorylation of plakoglobin. Furthermore, overexpression of Axin down-regulated the level of plakoglobin in SW480 cells. These results suggest that Axin regulates the stability of plakoglobin by enhancing its phosphorylation by GSK-3beta and that Axin may act on beta-catenin and plakoglobin in similar manners.     

Phosphatidylserine directly and positively regulates fusion of myoblasts into myotubes

Cell membrane consists of various lipids such as phosphatidylserine (PS), phosphatidylcholine (PC), and phosphatidylethanolamine (PE). Among them, PS is a molecular marker of apoptosis, because it is located to the inner leaflet of plasma membrane generally but it is moved to the outer leaflet during programmed cell death. The process of apoptosis has been implicated in the fusion of muscle progenitor cells, myoblasts, into myotubes. However, it remained unclear whether PS regulates muscle cell differentiation directly. In this paper, localization of PS to the outer leaflet of plasma membrane in proliferating primary myoblasts and during fusion of these myoblasts into myotubes is validated using Annexin V. Moreover, we show the presence of PS clusters at the cell–cell contact points, suggesting the importance of membrane ruffling and PS exposure for the myogenic cell fusion. Confirming this conclusion, experimentally constructed PS, but not PC liposomes dramatically enhance the formation of myotubes from myoblasts, thus demonstrating a direct positive effect of PS on the muscle cell fusion. In contrast, myoblasts exposed to PC liposomes produce long myotubes with low numbers of myonuclei. Moreover, pharmacological masking of PS on the myoblast surface inhibits fusion of these cells into myotubes in a dose-dependent manner.     

Hepatopoietin interacts directly with COP9 signalosome and regulates AP-1 activity

Hepatopoietin (HPO)/augmenter of liver regeneration (ALR) is a specific hepatotrophic growth factor, which plays a key role in liver regeneration. Our previous study indicated that HPO executes its function by an inter-reactive network of the autocrine, paracrine and endocrine pathways. Recently, we have demonstrated that intracellular HPO interacts with Jun activation domain-binding protein 1 (JAB1) and leads to potentiation of activating protein-1 (AP-1) activity in a MAPK independent fashion. JAB1 is the fifth subunit of the COP9 signalosome (CSN), which is first identified as a suppressor of plant morphogenesis. A protein complex kinase activity associated with the CSN has been reported but not identified yet. In this report, we investigated further the association of HPO with the whole CSN. HPO exists in a complex with the eight-component CSN, both when purified from glycerol gradient centrifugation and when reciprocal immunoprecipitated from the lysates of transfected COS-7 cells. Intracellular HPO colocalizes with endogenous CSN in nucleus of hepatic cells. In addition, intracellular function of HPO that increases the phosphorylation of c-Jun leading to potentiate the AP-1 activity is inhibited by curcumin, a potent inhibitor of CSN-associated kinase. Taken together, these results elucidate a novel relationship of intracellular growth factor, HPO with large protein complex, CSN, which suggests a possible linkage between CSN and liver regeneration.     

The Rab5 effector EEA1 interacts directly with syntaxin-6.

The fusion of transport vesicles with their cognate target membranes, an essential event in intracellular membrane trafficking, is regulated by SNARE proteins and Rab GTPases. Rab GTPases are thought to act prior to SNAREs in vesicle docking, but the exact biochemical relationship between the two classes of molecules is not known. We recently identified the early endosomal autoantigen EEA1 as an effector of Rab5 in endocytic membrane fusion. Here we demonstrate that EEA1 interacts directly and specifically with syntaxin-6, a SNARE implicated in trans-Golgi network to early endosome trafficking. The binding site for syntaxin-6 overlaps with that of Rab5-GTP at the C terminus of EEA1. Syntaxin-6 and EEA1 were found to colocalize extensively on early endosomes, although syntaxin-6 is present in the trans-Golgi network as well. Our results indicate that SNAREs can interact directly with Rab effectors, and suggest that EEA1 may participate in trans-Golgi network to endosome as well as in endocytic membrane traffic.     

The mammalian Sec6/8 complex interacts with Ca(2+) signaling complexes and regulates their activity

The localization of various Ca(2+) transport and signaling proteins in secretory cells is highly restricted, resulting in polarized agonist-stimulated Ca(2+) waves. In the present work, we examined the possible roles of the Sec6/8 complex or the exocyst in polarized Ca(2+) signaling in pancreatic acinar cells. Immunolocalization by confocal microscopy showed that the Sec6/8 complex is excluded from tight junctions and secretory granules in these cells. The Sec6/8 complex was found in at least two cellular compartments, part of the complex showed similar, but not identical, localization with the Golgi apparatus and part of the complex associated with Ca(2+) signaling proteins next to the plasma membrane at the apical pole. Accordingly, immunoprecipitation (IP) of Sec8 did not coimmunoprecipitate betaCOP, Golgi 58K protein, or mannosidase II, all Golgi-resident proteins. By contrast, IP of Sec8 coimmunoprecipitates Sec6, type 3 inositol 1,4,5-trisphosphate receptors (IP(3)R3), and the Gbetagamma subunit of G proteins from pancreatic acinar cell extracts. Furthermore, the anti-Sec8 antibodies coimmunoprecipitate actin, Sec6, the plasma membrane Ca(2+) pump, the G protein subunits Galphaq and Gbetagamma, the beta1 isoform of phospholipase C, and the ER resident IP(3)R1 from brain microsomal extracts. Antibodies against the various signaling and Ca(2+) transport proteins coimmunoprecipitate Sec8 and the other signaling proteins. Dissociation of actin filaments in the immunoprecipitate had no effect on the interaction between Sec6 and Sec8, but released the actin and dissociated the interaction between the Sec6/8 complex and Ca(2+) signaling proteins. Hence, the interaction between the Sec6/8 and Ca(2+) signaling complexes is likely mediated by the actin cytoskeleton. The anti-Sec6 and anti-Sec8 antibodies inhibited Ca(2+) signaling at a step upstream of Ca(2+) release by IP(3). Disruption of the actin cytoskeleton with latrunculin B in intact cells resulted in partial translocation of Sec6 and Sec8 from membranes to the cytosol and interfered with propagation of agonist-evoked Ca(2+) waves. Our results suggest that the Sec6/8 complex has multiple roles in secretory cells including governing the polarized expression of Ca(2+) signaling complexes and regulation of their activity.     

Type Igamma661 phosphatidylinositol phosphate kinase directly interacts with AP2 and regulates endocytosis

Clathrin-coated vesicles mediate sorting and intracellular transport of membrane-bound proteins. The formation of these coats is initiated by the assembly of adaptor proteins (AP), which specifically bind to membrane cargo proteins via recognition of endocytic sorting motifs. The lipid signaling molecule phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) is critical for this process, as it serves as both a targeting and regulatory factor. PI(4,5)P(2) is synthesized by type I phosphatidylinositol phosphate kinases (PIPKI). We have discovered a direct interaction between the mu2-subunit of the AP2 complex and PIPKIgamma661 via a yeast two-hybrid screen. This interaction was confirmed using both the mu2-subunit in glutathione S-transferase pulldowns and via coimmunoprecipitation of endogenous PIPKIgamma661 with the AP2 complex from HEK293 cells. The interaction is mediated, in vivo, by a tyrosine-based motif in the 26-amino acid tail of PIPKIgamma661. Because AP2 regulates endocytosis of transferrin receptor from the plasma membrane, we also examined a role for PIPKIgamma661 using a flow cytometry endocytosis assay. We observed that stable expression of wild type PIPKIgamma661 in Madin-Darby canine kidney cells enhanced transferrin uptake, whereas stable expression of kinase-dead PIPKIgamma661 had an inhibitory effect. Neither condition affected the overall cellular level of PI(4,5)P(2). RNA interference-based knockdown of PIPKIgamma661 in HeLa cells also had an inhibitory effect on transferrin endocytosis using the same assay system. Collectively, this evidence implies an important role for PIPKIgamma661 in the AP2-mediated endocytosis of transferrin.     

G protein beta2 subunit interacts directly with neuropathy target esterase and regulates its activity

Neuropathy target esterase (NTE) was identified as the primary target of organophosphate compounds that cause a delayed neuropathy with degeneration of nerve axons. NTE is a novel phospholipase B anchored to the cytoplasmic face of endoplasmic reticulum and essential for embryonic and nervous development. However, little is known about the regulation of NTE. A human fetal brain cDNA library was screened for proteins that interact with NTE, Gbeta2 and Gbeta2-like I subunits were found to be able to bind the C-terminal of NTE in yeast. The interaction of Gbeta2 and NTE was confirmed by in vivo co-immunoprecipitation analysis in COS7 cells. Furthermore, depletion of Gbeta2 by RNA interference down regulated the activity of NTE but not its expression level. In addition, the activity of NTE was down regulated by the G protein signal pathway influencing factor, pertussis toxin, treatment in vivo. These findings suggest that Gbeta2 may play a significant role in maintaining the activity of NTE.     

Macrophage migration inhibitory factor directly interacts with hepatopoietin and regulates the proliferation of hepatoma cell

Macrophage migration inhibitory factor (MIF) is a pluripotent cytokine involved in inflammation and immune responses as well as in growth factor-dependent cell proliferation, cell cycle, angiogenesis, and tumorigenesis. Several studies have documented MIF expression in the sera following hepatic resection or in the course of liver cancer progression, but there is a paucity of information regarding the effect of MIF on hepatoma cells and relating mechanisms. In this paper, by [3H] thymidine incorporation, we found that exogenously added MIF could promote the proliferation of HepG2 in a dose-dependent manner. Hepatopoietin (HPO), as a liver-specific regeneration augmenter, could be induced by the expression of MIF in hepatoma cells. The activity of HPO promoter was increased, and its levels were enhanced after MIF was overexpressed in hepatoma cells. The similarities between HPO and MIF in structure and action led us to investigate their interaction and the inducing biological significance. Using yeast two-hybrid identification, we found that HPO interacted with MIF in yeast cells, and their binding ability was higher than that between HPO and JAB1 (Jun activation domain binding protein) or MIF and JAB1 in yeast cells. Their interaction was further verified by His pull-down assay in vitro and coimmunoprecipitation experiment in vivo. They were colocalized in the cytoplasm. Both HPO and MIF could bind to JAB1 and modulate the AP-1 pathway. When HPO and MIF were cotransfected into HepG2 cells, the binding activity of MIF to JAB1 was reduced, and the activity of AP-1 was improved. In contrast, MIF overexpressed in HepG2 was unable to interfere with the binding activity of HPO to JAB1, but its potentiation on AP-1 activity was reduced significantly. Taken together, these results indicate that MIF plays an important role in the proliferation of hepatoma cells, and the effect of MIF is in concert with HPO.     

The DNA damage response mediator MDC1 directly interacts with the anaphase-promoting complex/cyclosome

MDC1 (NFBD1), a mediator of the cellular response to DNA damage, plays an important role in checkpoint activation and DNA repair. Here we identified a cross-talk between the DNA damage response and cell cycle regulation. We discovered that MDC1 binds the anaphase-promoting complex/cyclosome (APC/C), an E3 ubiquitin ligase that controls the cell cycle. The interaction is direct and is mediated by the tandem BRCA1 C-terminal domains of MDC1 and the C terminus of the Cdc27 (APC3) subunit of the APC/C. It requires the phosphorylation of Cdc27 and is enhanced after induction of DNA damage. We show that the tandem BRCA1 C-terminal domains of MDC1, known to directly bind the phosphorylated form of histone H2AX (gamma-H2AX), also bind the APC/C by the same mechanism, as phosphopeptides that correspond to the C termini of gamma-H2AX and Cdc27 competed with each other for the binding to MDC1. Our results reveal a link between the cellular response to DNA damage and cell cycle regulation, suggesting that MDC1, known to have a role in checkpoint regulation, executes part of this role by binding the APC/C.     

The Bloom's syndrome helicase interacts directly with the human DNA mismatch repair protein hMSH6

Bloom's syndrome (BS) is a rare genetic disorder characterised by genome instability and cancer susceptibility. BLM, the BS gene product, belongs to the highly-conserved RecQ family of DNA helicases. Although the exact function of BLM in human cells remains to be defined, it seems likely that BLM eliminates some form of homologous recombination (HR) intermediate that arises during DNA replication. Similarly, the mismatch repair (MMR) system also plays a crucial role in the maintenance of genomic stability, by correcting DNA errors generated during DNA replication. Recent evidence implicates components of the MMR system also in HR repair. We now show that hMSH6, a component of the heterodimeric mismatch recognition complex hMSH2/hMSH6 (hMutS(alpha)), interacts with the BLM protein both in vivo and in vitro. In agreement with these findings, BLM and hMSH6 co-localise to discrete nuclear foci following exposure of the cells to ionising radiation. However, the purified recombinant MutS(alpha) complex does not affect the helicase activity of BLM in vitro. As BLM has previously been shown to interact with the hMLH1 component of the hMLH1/hPMS2 (hMutL(alpha)) heterodimeric MMR complex, our present findings further strengthen the link between BLM and processes involving correction of DNA mismatches, such as in the regulation of the fidelity of homologous recombination events.     

The coat protein complex II, COPII, protein Sec13 directly interacts with presenilin-1

Mutations in the human gene encoding presenilin-1, PS1, account for most cases of early-onset familial Alzheimer’s disease. PS1 has nine transmembrane domains and a large loop orientated towards the cytoplasm. PS1 locates to cellular compartments as endoplasmic reticulum (ER), Golgi apparatus, vesicular structures, and plasma membrane, and is an integral member of γ-secretase, a protein protease complex with specificity for intra-membranous cleavage of substrates such as β-amyloid precursor protein. Here, an interaction between PS1 and the Sec13 protein is described. Sec13 takes part in coat protein complex II, COPII, vesicular trafficking, nuclear pore function, and ER directed protein sequestering and degradation control. The interaction maps to the N-terminal part of the large hydrophilic PS1 loop and the first of the six WD40-repeats present in Sec13. The identified Sec13 interaction to PS1 is a new candidate interaction for linking PS1 to secretory and protein degrading vesicular circuits.     

The endosomal sorting complex required for transport complex negatively regulates Erg6 degradation under specific glucose restriction conditions

Lipid droplets (LDs) are cytosolic fat storage organelles that play roles in lipid metabolism, trafficking and signaling. Breakdown of LDs in Saccharomyces cerevisiae is mainly achieved by lipolysis and lipophagy. In this study, we found that the endosomal sorting complex required for transport (ESCRT) in S. cerevisiae negatively regulated the turnover of a LD marker, Erg6, under both simplified glucose restriction (GR) and acute glucose restriction (AGR) conditions by monitoring the localization and degradation of Erg6. Loss of Vps27, Snf7 or Vps4, representative subunits of the ESCRT machinery, facilitated the delivery of Erg6‐GFP to vacuoles and its degradation depending on the lipophagy protein Atg15 under simplified GR. Additionally, the lipolysis proteins Tgl3 and Tgl4 were also involved in the enhanced vacuolar localization and degradation of Erg6‐GFP in vps4Δ cells. Furthermore, we found that Atg14, which is required for the formation of putatively liquid‐ordered (Lo) membrane domains on the vacuole that act as preferential internalization sites for LDs, abundantly localized to vacuolar membranes in ESCRT mutants. Most importantly, the depletion or overexpression of Atg14 correspondingly abolished or promoted the observed Erg6 degradation in ESCRT mutant cells. We propose that Atg14 together with other proteins promotes Erg6 degradation in ESCRT mutant cells under specific glucose restriction conditions. These results shed new light on the regulation of ESCRT on LD turnover.     

The mitochondrial receptor complex: Mom22 is essential for cell viability and directly interacts with preproteins.

A multisubunit complex in the mitochondrial outer membrane is responsible for targeting and membrane translocation of nuclear-encoded preproteins. This receptor complex contains two import receptors, a general insertion pore and the protein Mom22. It was unknown if Mom22 directly interacts with preproteins, and two views existed about the possible functions of Mom22: a central role in transfer of preproteins from both receptors to the general insertion pore or a more limited function dependent on the presence of the receptor Mom19. For this report, we identified and cloned Saccharomyces cerevisiae MOM22 and investigated whether it plays a direct role in targeting of preproteins. A preprotein accumulated at the mitochondrial outer membrane was cross-linked to Mom22. The cross-linking depended on the import stage of the preprotein. Overexpression of Mom22 suppressed the respiratory defect of yeast cells lacking Mom19 and increased preprotein import into mom19 delta mitochondria, demonstrating that Mom22 can function independently of Mom19. Overexpression of Mom22 even suppressed the lethal phenotype of a double deletion of the two import receptors known so far (mom19 delta mom72 delta). Deletion of the MOM22 gene was lethal for yeast cells, identifying Mom22 as one of the few mitochondrial membrane proteins essential for fermentative growth. These results suggest that Mom22 plays an essential role in the mitochondrial receptor complex. It directly interacts with preproteins in transit and can perform receptor-like activities.     

Human alpha-synemin interacts directly with vinculin and metavinculin

Synemin is a very large, unique member of the IF (intermediate filament) protein superfamily. Association of synemin with the major IF proteins, desmin and/or vimentin, within muscle cells forms heteropolymeric IFs. We have previously identified interactions of avian synemin with alpha-actinin and vinculin. Avian synemin, however, is expressed as only one form, whereas human synemin is expressed as two major splice variants, namely alpha- and beta-synemins. The larger alpha-synemin contains an additional 312-amino-acid insert (termed SNTIII) located near the end of the long C-terminal tail domain. Whether alpha- and beta-synemins have different cellular functions is unclear. In the present study we show, by in vitro protein-protein interaction assays, that SNTIII interacts directly with both vinculin and metavinculin. Furthermore, SNTIII interacts with vinculin in vivo, and this association is promoted by PtdIns(4,5)P(2). SNTIII also specifically co-localizes with vinculin within focal adhesions when transiently expressed in mammalian cells. In contrast, other regions of synemin show distinct localization patterns in comparison with those of SNTIII, without labelling focal adhesions. Our results indicate that alpha-synemin, but not beta-synemin, interacts with both vinculin and metavinculin, thereby linking the heteropolymeric IFs to adhesion-type junctions, such as the costameres located within human striated muscle cells.