Starch-binding domain-containing protein 1 (Stbd1) and glycogen metabolism: Identification of the Atg8 family interacting motif (AIM) in Stbd1 required for interaction with GABARAPL1

Glycogen, a branched polymer of glucose, acts as an intracellular carbon and energy reserve in many tissues and cell types. An important pathway for its degradation is by transport to lysosomes in an autophagy-like process. It has been proposed that starch-binding domain-containing protein 1 (Stbd1) may participate in this mechanism by anchoring glycogen to intracellular membranes. In addition, Stbd1 has been reported to interact with a known autophagy protein, GABARAPL1, a member of the Atg8 family. Here, we confirm this interaction and identify an Atg8 interacting motif (AIM) in Stbd1 necessary for GABARAPL1 binding as judged by co-immunoprecipitation from cell extracts and co-localization in cells as evidenced by immunofluorescence microscopy. The AIM sequence of Stbd1 ²⁰⁰HEEWEMV²⁰⁶ lies within a predicted disordered region of the molecule and fits the consensus of other AIM sequences in cargo-specifying proteins such as p62 and Nix. Mutation of the AIM, including single point mutations of either W203 or V206, eliminated the co-localization of Stbd1 with both over-expressed and endogenous GABARAPL1. Stbd1 may therefore function as a novel cargo binding protein that delivers glycogen to lysosomes in an autophagic pathway that could be termed “glycophagy”.     

The carbohydrate-binding domain of overexpressed STBD1 is important for its stability and protein–protein interactions

STBD1 (starch-binding domain-containing protein 1) belongs to the CBM20 (family 20 carbohydrate binding module) group of proteins, and is implicated in glycogen metabolism and autophagy. However, very little is known about its regulation or interacting partners. Here, we show that the CBM20 of STBD1 is crucial for its stability and ability to interact with glycogen-associated proteins. Mutation of a conserved tryptophan residue (W293) in this domain abolished the ability of STBD1 to bind to the carbohydrate amylose. Compared with the WT (wild-type) protein, this mutant exhibited rapid degradation that was rescued upon inhibition of the proteasome. Furthermore, STBD1 undergoes ubiquitination when expressed in COS cells, and requires the N-terminus for this process. In contrast, inhibition of autophagy did not significantly affect protein stability. In overexpression experiments, we discovered that STBD1 interacts with several glycogen-associated proteins, such as GS (glycogen synthase), GDE (glycogen debranching enzyme) and Laforin. Importantly, the W293 mutant of STBD1 was unable to do so, suggesting an additional role for the CBM20 domain in protein–protein interactions. In HepG2 hepatoma cells, overexpressed STBD1 could associate with endogenous GS. This binding increased during glycogenolysis, suggesting that glycogen is not required to bridge this interaction. Taken together, our results have uncovered new insights into the regulation and binding partners of STBD1.     

Ubiquitin ligase Kf-1 is involved in the endoplasmic reticulum-associated degradation pathway

Kf-1 was first identified as a gene showing enhanced expression in the cerebral cortex of a sporadic Alzheimer’s disease patient. To date, however, the functional properties of Kf-1 protein remain unknown. In this study, immunohistochemical analysis showed that Kf-1 immunoreactivity was detected in rat hippocampus and cerebral cortex neurons. Interestingly, it was colocalized with endoplasmic reticulum (ER) marker. To investigate the specific function of Kf-1 protein, we generated Myc tagged wild type Kf-1 (Myc-Kf-1WT) and RING finger domain deletion mutant of Kf-1 (Myc-Kf-1ΔR), and then transfected in HEK293 cells. Myc-Kf-1WT displayed a reticular pattern typical of ER localization, with large perinuclear aggregates and colocalized with ER marker, calnexin. Myc-Kf-1WT facilitated ubiquitination of endogenous proteins, whereas Myc-Kf-1ΔR did not show ubiquitin ligase activity. In addition, we found that Kf-1 interacted with components of the ER-associated degradation (ERAD) pathway, including Derlin-1 and VCP. Taken together, these properties suggest that Kf-1 is an ER ubiquitin ligase involved in the ERAD pathway.     

Human selenium binding protein-1 (hSP56) interacts with VDU1 in a selenium-dependent manner

Reduced expression of the 56-kDa human selenium binding protein-1 (hSP56) has been reported in many types of human malignancies, including prostate, lung, ovarian, thyroid and colorectal cancers. hSP56 also has been implicated in selenium-dependent cell growth inhibition. However, the molecular basis of hSP56’s function has not been elucidated. In the present study, we identified von Hippel-Lindau protein (pVHL)-interacting deubiquitinating enzyme 1 (VDU1) as a protein partner of hSP56 using a yeast two-hybrid screen. The interaction between hSP56 and VDU1 was confirmed by yeast two-hybrid analysis and in vitro binding experiments. hSP56 and VDU1 co-localized in the perinuclear region of LNCaP human prostate cancer cells. The full-length VDU1 specifically interacted with a selenium-replete form of hSP56. We also demonstrate stable incorporation of selenium into hSP56, in a mode distinct from conventional selenocysteine-containing selenoproteins. These findings suggest that hSP56 may play a role in ubiquitination/deubiquitination-mediated protein degradation pathways in a selenium-dependent manner.     

The C proteins of human parainfluenza virus type 1 block IFN signaling by binding and retaining Stat1 in perinuclear aggregates at the late endosome

Interferons (IFNs) play a crucial role in the antiviral immune response. Whereas the C proteins of wild-type human parainfluenza virus type 1 (WT HPIV1) inhibit both IFN-β induction and signaling, a HPIV1 mutant encoding a single amino acid substitution (F170S) in the C proteins is unable to block either host response. Here, signaling downstream of the type 1 IFN receptor was examined in Vero cells to define at what stage WT HPIV1 can block, and F170S HPIV1 fails to block, IFN signaling. WT HPIV1 inhibited phosphorylation of both Stat1 and Stat2, and this inhibition was only slightly reduced for F170S HPIV1. Degradation of Stat1 or Stat2 was not observed. The HPIV1 C proteins were found to accumulate in the perinuclear space, often forming large granules, and co-localized with Stat1 and the cation-independent mannose 6-phosphate receptor (M6PR) that is a marker for late endosomes. Upon stimulation with IFN-β, both the WT and F170S C proteins remained in the perinuclear space, but only the WT C proteins prevented Stat1 translocation to the nucleus. In addition, WT HPIV1 C proteins, but not F170S C proteins, co-immunoprecipitated both phosphorylated and unphosphorylated Stat1. Our findings suggest that the WT HPIV1 C proteins form a stable complex with Stat1 in perinuclear granules that co-localize with M6PR, and that this direct interaction between the WT HPIV1 C proteins and Stat1 is the basis for the ability of HPIV1 to inhibit IFN signaling. The F170S mutation in HPIV1 C did not prevent perinuclear co-localization with Stat1, but apparently weakened this interaction such that, upon IFN stimulation, Stat1 was translocated to the nucleus to induce an antiviral response.     

Immediate Translation of Formin DIAPH1 mRNA after Its Exiting the Nucleus Is Required for Its Perinuclear Localization in Fibroblasts

DIAPH1 is a formin protein which promotes actin polymerization, stabilizes microtubules and consequently is involved in cytoskeleton dynamics, cell migration and differentiation. In contrast to the relatively well-understood signaling cascades that regulate DIAPH1 activity, its spatial regulation of biogenesis is not understood. A recent report showed that synthesis of DIAPH1 is confined in the perinuclear ER compartment through translation-dependent mRNA targeting. However, the underlying mechanism of DIAPH1 local synthesis is yet to be elucidated. Here, we provide evidence to demonstrate that the 5′-cap-mediated immediate translation of DIAPH1 mRNA upon exiting nucleus is required for localizing the mRNA in the perinuclear ER compartment. This is supported by data: 1) Delayed translation of DIAPH1 mRNA resulted in loss of perinuclear localization of the mRNA; 2) Once delocalized, DIAPH1 mRNA could not be retargeted to the perinuclear region; and 3) The translation of DIAPH1 mRNA is 5′-cap dependent. These results provide new insights into the novel mechanism of DIAPH1 local synthesis. In addition, these findings have led to the development of new approaches for manipulating DIAPH1 mRNA localization and local protein synthesis in cells for functional studies. Furthermore, a correlation of DIAPH1 mRNA and DIAPH1 protein localization has been demonstrated using a new method to quantify the intracellular distribution of protein.     

Adoptive transfer of genetically engineered WT1-specific cytotoxic T lymphocytes does not induce renal injury

Because WT1 is expressed in leukemia cells, the development of cancer immunotherapy targeting WT1 has been an attractive translational research topic. However, concern of this therapy still remains, since WT1 is abundantly expressed in renal glomerular podocytes. In the present study, we clearly showed that WT1-specific cytotoxic T lymphocytes (CTLs) certainly exerted cytotoxicity against podocytes in vitro; however, they did not damage podocytes in vivo. This might be due to the anatomical localization of podocytes, being structurally separated from circulating CTLs in glomerular capillaries by an exceptionally thick basement membrane.     

The 3' untranslated region of human vimentin mRNA interacts with protein complexes containing eEF-1gamma and HAX-1

Previously, we have shown that the vimentin 3′ untranslated region (3′UTR) contains a highly conserved region, which is sufficient for the perinuclear localization of a reporter mRNA. This region was shown to specifically bind protein(s) by band shift analyses. UV-cross-linking studies suggest these proteins are 46- and 35-kDa in mass. Here, we have used this sequence as ‘bait’ to isolate RNA binding proteins using the yeast three-hybrid method. This technique relies on a functional assay detecting bona fide RNA–protein interaction in vivo. Three cDNA isolates, HAX-1, eEF-1γ and hRIP, code for proteins of a size consistent with in vitro cross- linking studies. In all cases, recombinant proteins were capable of binding RNA in vitro. Although hRIP is thought to be a general mRNA binding protein, this represents an unreported activity for eEF-1γ and HAX-1. Moreover, HAX-1 binding appears to be specific to vimentin’s 3′UTR. Both in vivo synthesized eEF-1γ and HAX-1 proteins were ‘pulled out’ of HeLa whole cell extracts by binding to a RNA affinity column comprised of vimentin’s 3′UTR. Moreover, size-fractionation of extracts results in the separation of large complexes containing either eEF-1γ or HAX-1. Thus, in addition to their known functions, both eEF-1γ and HAX-1 are RNA binding proteins, which suggests new roles in mRNA translation and/or perinuclear localization.     

Characterization of YDJ1: a yeast homologue of the bacterial dnaJ protein

The YDJ1 (yeast dnaJ) gene was isolated from a yeast expression library using antisera made against a yeast nuclear sub-fraction termed the matrix lamina pore complex. The predicted open reading frame displays a 32% identity with the sequence of the Escherichia coli heat shock protein dnaJ. Localization of YDJ1 protein (YDJ1p) by indirect immunofluorescence reveals it to be concentrated in a perinuclear ring as well as in the cytoplasm. YDJ1p cofractionates with nuclei and also microsomes, suggesting that its perinuclear localization reflects association with the ER. YDJ1p is required for normal growth and disruption of its gene results in very slow growing cells that have pleiotropic morphological defects. Haploid cells carrying the disrupted YDJ1 gene are inviable for growth in liquid media. We further show that a related yeast protein, SIS1, is a multicopy suppressor of YDJ1.     

Vascular smooth muscle cell proliferation depends on caveolin-1-regulated polyamine uptake

Much evidence highlights the importance of polyamines for VSMC (vascular smooth muscle cell) proliferation and migration. Cav-1 (caveolin-1) was recently reported to regulate polyamine uptake in intestinal epithelial cells. The aim of the present study was to assess the importance of Cav-1 for VSMC polyamine uptake and its impact on cell proliferation and migration. Cav-1 KO (knockout) mouse aortic cells showed increased polyamine uptake and elevated proliferation and migration compared with WT (wild-type) cells. Both Cav-1 KO and WT cells expressed the smooth muscle differentiation markers SM22 and calponin. Cell-cycle phase distribution analysis revealed a higher proportion of Cav-1 KO than WT cells in the S phase. Cav-1 KO cells were hyper-proliferative in the presence but not in the absence of extracellular polyamines, and, moreover, supplementation with exogenous polyamines promoted proliferation in Cav-1 KO but not in WT cells. Expression of the solute carrier transporters Slc7a1 and Slc43a1 was higher in Cav-1 KO than in WT cells. ODC (ornithine decarboxylase) protein and mRNA expression as well as ODC activity were similar in Cav-1 KO and WT cells showing unaltered synthesis of polyamines in Cav-1 KO cells. Cav-1 was reduced in migrating cells in vitro and in carotid lesions in vivo. Our data show that Cav-1 negatively regulates VSMC polyamine uptake and that the proliferative advantage of Cav-1 KO cells is critically dependent on polyamine uptake. We provide proof-of-principle for targeting Cav-1-regulated polyamine uptake as a strategy to fight unwanted VSMC proliferation as observed in restenosis.     

The t complex polypeptide 1 (TCP-1) is associated with the cytoplasmic aspect of Golgi membranes

The t complex polypeptide 1 (TCP-1) is a protein of unknown function expressed in large amounts during spermatogenesis. Rat monoclonal antibodies recognizing TCP-1 have been prepared and used to immunoprecipitate and Western blot a 57 kd protein from germ cell and tissue culture cell extracts. In tissue culture cells, indirect immunofluorescent localization of antigen indicated a perinuclear distribution similar to that of the Golgi apparatus. Analysis of the TCP-1 distribution in tissue culture cells showed that the polypeptide was associated with the cytoplasmic aspect of membranes of the trans-Golgi network (TGN). The distribution in spermatids suggested that TCP-1 was localized to structures often associated with the developing acrosome. The TCP-1 antigenic epitopes are highly conserved, allowing the protein to be identified in cells across a wide variety of vertebrate species and tissues. These experiments suggest that TCP-1 may be essential for transport of proteins through the exocytic pathway in all cells and required in large amounts for acrosome formation in developing spermatids.     

The Recruitment of AMP-activated Protein Kinase to Glycogen Is Regulated by Autophosphorylation

The mammalian AMP-activated protein kinase (AMPK) is an obligatory αβγ heterotrimeric complex carrying a carbohydrate-binding module (CBM) in the β-subunit (AMPKβ) capable of attaching AMPK to glycogen. Nonetheless, AMPK localizes at many different cellular compartments, implying the existence of mechanisms that prevent AMPK from glycogen binding. Cell-free carbohydrate binding assays revealed that AMPK autophosphorylation abolished its carbohydrate-binding capacity. X-ray structural data of the CBM displays the central positioning of threonine 148 within the binding pocket. Substitution of Thr-148 for a phospho-mimicking aspartate (T148D) prevents AMPK from binding to carbohydrate. Overexpression of isolated CBM or β1-containing AMPK in cellular models revealed that wild type (WT) localizes to glycogen particles, whereas T148D shows a diffuse pattern. Pharmacological AMPK activation and glycogen degradation by glucose deprivation but not forskolin enhanced cellular Thr-148 phosphorylation. Cellular glycogen content was higher if pharmacological AMPK activation was combined with overexpression of T148D mutant relative to WT AMPK. In summary, these data show that glycogen-binding capacity of AMPKβ is regulated by Thr-148 autophosphorylation with likely implications in the regulation of glycogen turnover. The findings further raise the possibility of regulated carbohydrate-binding function in a wider variety of CBM-containing proteins.     

In MMTV-Her-2/neu transgenic mammary tumors the absence of caveolin-1−/− alters PTEN and NHERF1 but not β-catenin expression

In a recent study, we have shown that in mammary tumors from mice lacking the Cav-1 gene, there are alterations in specific heat shock proteins as well as in tumor development. With this in mind, we have now investigated other proteins in the same mammary mouse tumor model (Her-2/neu expressing mammary tumors from Cav-1 wild type and Cav-1 null mice), to further comprehend the complex tumor-stroma mechanisms involved in regulating stress responses during tumor development. In this tumor model the cancer cells always lacked of Cav-1, so the KO influenced the Cav-1 in the stroma. By immunohistochemistry, we have found a striking co-expression of β-catenin and Her-2/neu in the tumor cells. The absence of Cav-1 in the tumor stroma had no effect on expression or localization of β-catenin and Her-2/neu. Both proteins appeared co-localized at the cell surface during tumor development and progression. Since Her-2/neu activation induces MTA1, we next evaluated MTA1 in the mouse tumors. Although this protein was found in numerous nuclei, the absence of Cav-1 did not alter its expression level. In contrast, significantly more PTEN protein was noted in the tumors lacking Cav-1 in the stroma, with the protein localized mainly in the nuclei. P-Akt levels were relatively low in tumors from both Cav-1 WT and Cav-1 KO mice. There was also an increase in nuclear NHERF1 expression levels in the tumors arising from Cav-1 KO mice. The data obtained in the MMTV-neu model are consistent with a role for Cav-1 in adjacent breast cancer stromal cells in modulating the expression and localization of important proteins implicated in tumor cell behavior.     

Hypoxia-inducible factor 1-mediated regulation of PPP1R3C promotes glycogen accumulation in human MCF-7 cells under hypoxia

Hundreds of genes can be regulated by hypoxia-inducible factor 1 (HIF1) under hypoxia. Here we demonstrated a HIF1-mediated induction of protein phosphatase 1, regulatory subunit 3C gene (PPP1R3C) in human MCF7 cells under hypoxia. By mutation analysis we confirmed the presence of a functional hypoxia response element that is located 229 bp upstream from the PPP1R3C gene. PPP1R3C induction correlates with a significant glycogen accumulation in MCF7 cells under hypoxia. Knockdown of either HIF1α or PPP1R3C attenuated hypoxia-induced glycogen accumulation significantly. Knockdown of HIF2α reduced hypoxia-induced glycogen accumulation slightly (but not significantly). Our results demonstrated that HIF1 promotes glycogen accumulation through regulating PPP1R3C expression under hypoxia, which revealed a novel metabolic adaptation of cells to hypoxia.     

The nuclear localization of 3'-phosphoinositide-dependent kinase-1 is dependent on its association with the protein tyrosine phosphatase SHP-1

3'-Phosphoinositide-dependent protein kinase-1 (PDK1), the direct upstream kinase of Akt, can localize to the nucleus during specific signalling events. The mechanism used for its import into the nucleus, however, remains unresolved as it lacks a canonical nuclear localization signal (NLS). Expression of activated Src kinase in C6 glioblastoma cells promotes the association of tyrosylphosphorylated PDK1 with the NLS-containing tyrosine phosphatase SHP-1 as well as the nuclear localization of both proteins. A constitutive nucleo-cytoplasmic SHP-1:PDK1 shuttling complex is supported by several lines of evidence including (i) the distribution of both proteins to similar subcellular compartments following manipulation of the nuclear pore complex, (ii) the nuclear retention of SHP-1 upon overexpression of a PDK1 protein bearing a disrupted nuclear export signal (NES), and (iii) the exclusion of PDK1 from the nucleus upon overexpression of SHP-1 lacking the NLS or following siRNA-mediated knock-down of SHP-1. The latter case results in a perinuclear distribution of PDK1 that corresponds with the distribution of PIP3 (phosphatidylinositol 3,4,5-triphosphate), while a PDK1 protein bearing a mutated PH domain that abrogates PIP3-binding is excluded from the nucleus. Our data suggest that the SHP-1:PDK1 complex is recruited to the nuclear membrane by binding to perinuclear PIP3, whereupon SHP-1 (and its NLS) facilitates active import. Export from the nucleus relies on PDK1 (and its NES). The intact complex contributes to Src kinase-induced, Akt-sensitive podial formation in C6 cells.     

Snail1 induced in breast cancer cells in 3D collagen I gel environment suppresses cortactin and impairs effective invadopodia formation

Although an in vitro 3D environment cannot completely mimic the in vivo tumor site, embedding tumor cells in a 3D extracellular matrix (ECM) allows for the study of cancer cell behaviors and the screening of anti-metastatic reagents with a more in vivo-like context. Here we explored the behaviors of MDA-MB-231 breast cancer cells embedded in 3D collagen I. Diverse tumor environmental conditions (including cell density, extracellular acidity, or hypoxia as mimics for a continuous tumor growth) reduced JNKs, enhanced TGFβ1/Smad signaling activity, induced Snail1, and reduced cortactin expression. The reduced JNKs activity blocked efficient formation of invadopodia labeled with actin, cortactin, or MT1-MMP. JNKs inactivation activated Smad2 and Smad4, which were required for Snail1 expression. Snail1 then repressed cortactin expression, causing reduced invadopodia formation and prominent localization of MT1-MMP at perinuclear regions. MDA-MB-231 cells thus exhibited less efficient collagen I degradation and invasion in 3D collagen I upon JNKs inhibition. These observations support a signaling network among JNKs, Smads, Snail1, and cortactin to regulate the invasion of MDA-MB-231 cells embedded in 3D collagen I, which may be targeted during screening of anti-invasion reagents.     

Characterization of a novel mutant KCNQ1 channel subunit lacking a large part of the C-terminal domain

A mutation of KCNQ1 gene encoding the alpha subunit of the channel mediating the slow delayed rectifier K⁺ current in cardiomyocytes may cause severe arrhythmic disorders. We identified KCNQ1(Y461X), a novel mutant gene encoding KCNQ1 subunit whose C-terminal domain is truncated at tyrosine 461 from a man with a mild QT interval prolongation. We made whole-cell voltage-clamp recordings from HEK-293T cells transfected with either of wild-type KCNQ1 [KCNQ1(WT)], KCNQ1(Y461X), or their mixture plus KCNE1 auxiliary subunit gene. The KCNQ1(Y461X)-transfected cells showed no delayed rectifying current. The cells transfected with both KCNQ1(WT) and KCNQ1(Y461X) showed the delayed rectifying current that is thought to be mediated largely by homomeric channel consisting of KCNQ1(WT) subunit because its voltage-dependence of activation, activation rate, and deactivation rate were similar to the current in the KCNQ1(WT)-transfected cells. The immunoblots of HEK-293T cell-derived lysates showed that KCNQ1(Y461X) subunit cannot form channel tetramers by itself or with KCNQ1(WT) subunit. Moreover, immunocytochemical analysis in HEK-293T cells showed that the surface expression level of KCNQ1(Y461X) subunit was very low with or without KCNQ1(WT) subunit. These findings suggest that the massive loss of the C-terminal domain of KCNQ1 subunit impairs the assembly, trafficking, and function of the mutant subunit-containing channels, whereas the mutant subunit does not interfere with the functional expression of the homomeric wild-type channel. Therefore, the homozygous but not heterozygous inheritance of KCNQ1(Y461X) might cause major arrhythmic disorders. This study provides a new insight into the structure–function relation of KCNQ1 channel and treatments of cardiac channelopathies.     

The Effect of PRMT1-Mediated Arginine Methylation on the Subcellular Localization,Stress Granules,and Detergent-Insoluble Aggregates of FUS/TLS

Fused in sarcoma/translocated in liposarcoma (FUS/TLS) is one of causative genes for familial amyotrophic lateral sclerosis (ALS). In order to identify binding partners for FUS/TLS, we performed a yeast two-hybrid screening and found that protein arginine methyltransferase 1 (PRMT1) is one of binding partners primarily in the nucleus. In vitro and in vivo methylation assays showed that FUS/TLS could be methylated by PRMT1. The modulation of arginine methylation levels by a general methyltransferase inhibitor or conditional over-expression of PRMT1 altered slightly the nucleus-cytoplasmic ratio of FUS/TLS in cell fractionation assays. Although co-localized primarily in the nucleus in normal condition, FUS/TLS and PRMT1 were partially recruited to the cytoplasmic granules under oxidative stress, which were merged with stress granules (SGs) markers in SH-SY5Y cell. C-terminal truncated form of FUS/TLS (FUS-dC), which lacks C-terminal nuclear localization signal (NLS), formed cytoplasmic inclusions like ALS-linked FUS mutants and was partially co-localized with PRMT1. Furthermore, conditional over-expression of PRMT1 reduced the FUS-dC-mediated SGs formation and the detergent-insoluble aggregates in HEK293 cells. These findings indicate that PRMT1-mediated arginine methylation could be implicated in the nucleus-cytoplasmic shuttling of FUS/TLS and in the SGs formation and the detergent-insoluble inclusions of ALS-linked FUS/TLS mutants.     

Human Asunder promotes dynein recruitment and centrosomal tethering to the nucleus at mitotic entry

Recruitment of dynein motors to the nuclear surface is an essential step for nucleus–centrosome coupling in prophase. In cultured human cells, this dynein pool is anchored to nuclear pore complexes through RanBP2–Bicaudal D2 (BICD2) and Nup133– centromere protein F (CENP-F) networks. We previously reported that the asunder (asun) gene is required in Drosophila spermatocytes for perinuclear dynein localization and nucleus–centrosome coupling at G2/M of male meiosis. We show here that male germline expression of mammalian Asunder (ASUN) protein rescues asun flies, demonstrating evolutionary conservation of function. In cultured human cells, we find that ASUN down-regulation causes reduction of perinuclear dynein in prophase of mitosis. Additional defects after loss of ASUN include nucleus–centrosome uncoupling, abnormal spindles, and multinucleation. Coimmunoprecipitation and overlapping localization patterns of ASUN and lissencephaly 1 (LIS1), a dynein adaptor, suggest that ASUN interacts with dynein in the cytoplasm via LIS1. Our data indicate that ASUN controls dynein localization via a mechanism distinct from that of either BICD2 or CENP-F. We present a model in which ASUN promotes perinuclear enrichment of dynein at G2/M that facilitates BICD2- and CENP-F-mediated anchoring of dynein to nuclear pore complexes.