LKB1 associates with Brg1 and is necessary for Brg1-induced growth arrest.

Inactivating mutations in the serine-threonine kinase LKB1 (STK11) are found in most patients with Peutz-Jeghers syndrome; however the function of LKB1 is unknown. We found that LKB1 binds to and regulates brahma-related gene 1 (Brg1), an essential component of chromatin remodeling complexes. The association requires the N terminus of LKB1 and the helicase domain of Brg1 and LKB1 stimulates the ATPase activity of Brg1. Brg1 expression in SW13 cells induces the formation of flat cells indicative of cell cycle arrest and senescence. Expression of a kinase-dead mutant of LKB1, SL26, in SW13 cells blocks the formation of Brg1-induced flat cells, indicating that LKB1 is required for Brg1-dependent growth arrest. The inability of mutants of LKB1 to mediate Brg1-dependent growth arrest may explain the manifestations of Peutz-Jeghers syndrome.     

Active Nercc1 protein kinase concentrates at centrosomes early in mitosis and is necessary for proper spindle assembly

The Nercc1 protein kinase autoactivates in vitro and is activated in vivo during mitosis. Autoactivation in vitro requires phosphorylation of the activation loop at threonine 210. Mitotic activation of Nercc1 in mammalian cells is accompanied by Thr210 phosphorylation and involves a small fraction of total Nercc1. Mammalian Nercc1 coimmunoprecipitates gamma-tubulin and the activated Nercc1 polypeptides localize to the centrosomes and spindle poles during early mitosis, suggesting that active Nercc has important functions at the microtubular organizing center during cell division. To test this hypothesis, we characterized the Xenopus Nercc1 orthologue (XNercc). XNercc endogenous to meiotic egg extracts coprecipitates a multiprotein complex that contains gamma-tubulin and several components of the gamma-tubulin ring complex and localizes to the poles of spindles formed in vitro. Reciprocally, immunoprecipitates of the gamma-tubulin ring complex polypeptide Xgrip109 contain XNercc. Immunodepletion of XNercc from egg extracts results in delayed spindle assembly, fewer bipolar spindles, and the appearance of aberrant microtubule structures, aberrations corrected by addition of purified recombinant XNercc. XNercc immunodepletion also slows aster assembly induced by Ran-GTP, producing Ran-asters of abnormal size and morphology. Thus, Nercc1 contributes to both the centrosomal and the chromatin/Ran pathways that collaborate in the organization of a bipolar spindle.     

The alm1+ gene from Schizosaccharomyces pombe encodes a coiled-coil protein that associates with the medial region during mitosis

We have isolated a cDNA that encodes a 142-kDa protein by immunoscreening of a Schizosaccharomyces pombe expression library with a new antibody, mAb8, that reveals spindle poles and equatorial ring-like structures in several organisms. This cDNA encodes a putative protein which we termed Alm (for abnormal long morphology). The protein is predicted to be a coiled-coil protein, containing a central α-helical domain flanked by non-helical terminal domains. Immunofluorescence analysis showed that Alm1 is localized in the medial region of the cell from anaphase to the end of cytokinesis. Cells carrying an alm1::ura4 ⁺ disruption are viable and exhibit an elongated morphology. Homozygous alm1::ura4 ⁺ diploids sporulated normally but the spores did not germinate. Spores that have inherited the disruption allele from a heterozygous alm1 ⁺ / alm1::ura4 ⁺ diploid germinated but generated smaller colonies. We propose that Alm1 participates in the structural organization of the medial region in S. pombe. Received: 9 April 1999 / Accepted: 22 October 1999     

The ATP-dependent helicase RUVBL1/TIP49a associates with tubulin during mitosis

RUVBL1/TIP49a/Pontin52 is a recently identified multi-functional protein with 2 ATP binding (WALKER) sites, which is essential for cell proliferation. We recovered and identified RUVBL1/TIP49a as a tubulin-binding protein from Triton X-100 lysates of U937 promonocytic cells by protein affinity chromatography and tryptic peptide microsequencing. Performing co-immunoprecipitation using newly generated RUVBL1/TIP49a-specific antibodies (mAb and rabbit polyclonal Ab) and RUVBL1/TIP49a-GST fusion protein-pull down assays we demonstrate co-precipitation of alpha- and gamma tubulin with RUVBL1/TIP49a. Confocal immunoflourescence microscopy reveals that RUVBL1/TIP49a was present not only in the nucleus, as expected, but was also concentrated at the centrosome and at the mitotic spindle in colocalization with tubulin. The topology of RUVBL1/TIP49a at the mitotic spindle varied, depending on the mitotic stage. The protein was localized at the centrosome and at the polar and astral microtubules in metaphase, and was detectable at the zone of polar tubule interdigitation in anaphase B and telophase. During cytokinesis the protein reappeared at the area of decondensing chromosomes. Whereas preincubation of U937 cells with colcemid resulted in inhibition of mitotic spindle formation with subsequent loss of RUVBL1/TIP49a mitotic spindle staining, no relevant influence of colcemid on RUVBL1/TIP49a-tubulin binding was observed. An agonistic effect of RUVBL1/TIP49a on in vitro tubulin assembly is demonstrated. Our results reveal a new functional aspect of RUVBL1/TIP49a.     

Discovery of novel 3,5-disubstituted indole derivatives as potent inhibitors of Pim-1, Pim-2, and Pim-3 protein kinases

A series of novel 3,5-disubstituted indole derivatives as potent and selective inhibitors of all three members of the Pim kinase family is described. High throughput screen identified a pan-Pim kinase inhibitor with a promiscuous scaffold. Guided by structure-based drug design, SAR of the series afforded a highly selective indole chemotype that was further developed into a potent set of compounds against Pim-1, 2, and 3 (Pim-1 and Pim-3: IC(50)≤2nM and Pim-2: IC(50)≤100nM).     

Leucine carboxyl methyltransferase-1 is necessary for normal progression through mitosis in mammalian cells

Protein phosphatase 2A (PP2A) is a multifunctional phosphatase that plays important roles in many cellular processes including regulation of cell cycle and apoptosis. Because PP2A is involved in so many diverse processes, it is highly regulated by both non-covalent and covalent mechanisms that are still being defined. In this study we have investigated the importance of leucine carboxyl methyltransferase-1 (LCMT-1) for PP2A methylation and cell function. We show that reduction of LCMT-1 protein levels by small hairpin RNAs causes up to a 70% reduction in PP2A methylation in HeLa cells, indicating that LCMT-1 is the major mammalian PP2A methyltransferase. In addition, LCMT-1 knockdown reduced the formation of PP2A heterotrimers containing the Balpha regulatory subunit and, in a subset of the cells, induced apoptosis, characterized by caspase activation, nuclear condensation/fragmentation, and membrane blebbing. Knockdown of the PP2A Balpha regulatory subunit induced a similar amount of apoptosis, suggesting that LCMT-1 induces apoptosis in part by disrupting the formation of PP2A(BalphaAC) heterotrimers. Treatment with a pan-caspase inhibitor partially rescued cells from apoptosis induced by LCMT-1 or Balpha knockdown. LCMT-1 knockdown cells and Balpha knockdown cells were more sensitive to the spindle-targeting drug nocodazole, suggesting that LCMT-1 and Balpha are important for spindle checkpoint. Treatment of LCMT-1 and Balpha knockdown cells with thymidine dramatically reduced cell death, presumably by blocking progression through mitosis. Consistent with these results, homozygous gene trap knock-out of LCMT-1 in mice resulted in embryonic lethality. Collectively, our results indicate that LCMT-1 is important for normal progression through mitosis and cell survival and is essential for embryonic development in mice.     

N-terminal alpha-methylation of RCC1 is necessary for stable chromatin association and normal mitosis

Regulator of chromatin condensation 1 (RCC1) is the only known guanine nucleotide-exchange factor for the Ran GTPase and has pivotal roles in nucleo-cytoplasmic transport, mitosis, and nuclear-envelope assembly. RCC1 associates dynamically with chromatin through binding to histones H2A and/or H2B in a Ran-regulated manner. Here, we report that, unexpectedly, the amino-terminal serine or proline residue of RCC1 is uniquely methylated on its alpha-amino group. Methylation requires removal of the initiating methionine, and the presence of proline and lysine at positions 3 and 4, respectively. Methylation-defective mutants of RCC1 bind less effectively than wild-type protein to chromatin during mitosis, which causes spindle-pole defects. We propose a bimodal attachment mechanism for RCC1 in which the tail promotes stable RCC1 association with chromatin through DNA binding in an alpha-N-methylation-dependent manner. These data provide the first known function for N-terminal protein methylation.     

The Golgi protein p115 associates with gamma-tubulin and plays a role in Golgi structure and mitosis progression

The Golgi apparatus is a network of polarized cisternae localized to the perinuclear region in mammalian cells. It undergoes extensive vesiculation at the onset of mitosis and its reassembly requires factors that are in part segregated via the mitotic spindle. Here we show that unlike typical Golgi markers, the Golgi-protein p115 partitioned with the spindle poles throughout mitosis. An armadillo-fold in its N terminus mediated a novel interaction between p115 and γ-tubulin and functioned in its centrosomal targeting. Both the N- and C-terminal regions of p115 were required to maintain Golgi structure. Strikingly, p115 was essential for mitotic spindle function and the resolution of the cytokinetic bridge because its depletion resulted in spindle collapse, chromosome missegregation, and failed cytokinesis. We demonstrate that p115 plays a critical role in mitosis progression, implicating it as the only known golgin to regulate both mitosis and apoptosis.     

Dynamin 2 associates with microtubules at mitosis and regulates cell cycle progression

Dynamin, a ~100 kDa large GTPase, is known as a key player for membrane traffic. Recent evidence shows that dynamin also regulates the dynamic instability of microtubules by a mechanism independent of membrane traffic. As microtubules are highly dynamic during mitosis, we investigated whether the regulation of microtubules by dynamin is essential for cell cycle progression. Dynamin 2 intensely localized at the mitotic spindle, and the localization depended on its proline-rich domain (PRD), which is required for microtubule association. The deletion of PRD resulted in the impairment of cytokinesis, whereby the mutant had less effect on endocytosis. Interestingly, dominant-negative dynamin (K44A), which blocks membrane traffic but has no effect on microtubules, also blocked cytokinesis. On the other hand, the deletion of the middle domain, which binds to γ-tubulin, impaired the entry into mitosis. As both deletion mutants had no significant effect on endocytosis, dynamin 2 may participate in cell cycle progression by regulating the microtubules. These data suggest that dynamin may play a key role for cell cycle progression by two distinct pathways, membrane traffic and cytoskeleton.     

Identification and partial characterization of mitotic centromere- associated kinesin, a kinesin-related protein that associates with centromeres during mitosis

Using antipeptide antibodies to conserved regions of the kinesin motor domain, we cloned a kinesin-related protein that associates with the centromere region of mitotic chromosomes. We call the protein MCAK, for mitotic centromere-associated kinesin. MCAK appears concentrated on centromeres at prophase and persists until telophase, after which time the localization disperses. It is found throughout the centromere region and between the kinetochore plates of isolated mitotic CHO chromosomes, in contrast to two other kinetochore-associated microtubule motors: cytoplasmic dynein and CENP-E (Yen et al., 1992), which are closer to the outer surface of the kinetochore plates. Sequence analysis shows MCAK to be a kinesin-related protein with the motor domain located in the center of the protein. It is 60-70% similar to kif2, a kinesin-related protein originally cloned from mouse brain with a centrally located motor domain (Aizawa et al., 1992). MCAK protein is present in interphase and mitotic CHO cells and is transcribed as a single 3.4-kb message.     

Marlin-1, a novel RNA-binding protein associates with GABA receptors

GABA(B) receptors are heterodimeric G protein-coupled receptors that mediate slow synaptic inhibition in the central nervous system. Whereas heterodimerization between GABA(B) receptor GABA(B)R1 and GABA(B)R2 subunits is essential for functional expression, how neurons coordinate the assembly of these critical receptors remains to be established. Here we have identified Marlin-1, a novel GABA(B) receptor-binding protein that associates specifically with the GABA(B)R1 subunit in yeast, tissue culture cells, and neurons. Marlin-1 is expressed in the brain and exhibits a granular distribution in cultured hippocampal neurons. Marlin-1 binds different RNA species including the 3'-untranslated regions of both the GABA(B)R1 and GABA(B)R2 mRNAs in vitro and also associates with RNA in cultured neurons. Inhibition of Marlin-1 expression via small RNA interference technology results in enhanced intracellular levels of the GABA(B)R2 receptor subunit without affecting the level of GABA(B)R1. Together our results suggest that Marlin-1 functions to regulate the cellular levels of GABA(B) R2 subunits, which may have significant effects on the production of functional GABA(B) receptor heterodimers. Therefore, our observations provide an added level of regulation for the control of GABA(B) receptor expression and for the efficacy of inhibitory synaptic transmission.     

Hematopoietic lineage cell specific protein 1 associates with and down-regulates protein kinase CK2

The catalytic (alpha) subunit of protein kinase CK2 and the hematopoietic specific protein 1 (HS1) display opposite effects on Ha-ras induced fibroblast transformation, by enhancing and counteracting it, respectively. Here we show the occurrence of physical association between HS1 and CK2alpha as judged from both far Western blot and plasmon resonance (BIAcore) analysis. Association of HS1 with CK2alpha is drastically reduced by the deletion of the HS1 C-terminal region (403-486) containing an SH3 domain. HS1, but not its deletion mutant HS1 Delta324-393, lacking a sequence similar to an acidic stretch of the regulatory beta-subunit of CK2, inhibits calmodulin phosphorylation by CK2alpha. These data indicate that HS1 physically interacts with CK2alpha and down-regulates its activity by a mechanism similar to the beta-subunit.     

Formation of protein complexes containing plant virus movement protein TGBp3 is necessary for its intracellular trafficking

Cell-to-cell movement of Poa semilatent virus (genus Hordeivirus) in infected plants is mediated by three viral ‘triple gene block’ (TGB) proteins. One of those termed TGBp3 is an integral membrane protein essential for intracellular transport of other TGB proteins and viral genomic RNA to plasmodesmata. TGBp3 targeting to plasmodesmata-associated sites is believed to involve an unconventional mechanism which does not employ endoplasmic reticulum-derived transport vesicles. Previously TGBp3 has been shown to contain a composite transport signal consisting of the central hydrophilic protein region which includes a conserved pentapeptide YQDLN and the C-terminal transmembrane segment. This study demonstrates that these TGBp3 structural elements have distinct functions in protein transport. The YQDLN-containing region is essential for TGBp3 incorporation into high-molecular-mass protein complexes. In transient expression assay formation of such complexes is necessary for entering the TGBp3-specific pathway of intracellular transport and protein delivery to plasmodesmata-associated sites. In virus-infected plants TGBp3 is also found predominantly in the form of high-molecular-mass complexes. When the complex-formation function of YQDLN-containing region is disabled by a mutation, targeting to plasmodesmata-associated sites can be complemented by a heterologous peptide capable of formation multimeric complexes. The C-terminal transmembrane segment is found to be an essential signal of TGBp3 intracellular transport to peripheral sites.     

CLP-36 PDZ-LIM protein associates with nonmuscle alpha-actinin-1 and alpha-actinin-4

The PDZ-LIM family of proteins (Enigma/LMP-1, ENH, ZASP/Cypher, RIL, ALP, and CLP-36) has been suggested to act as adapters that direct LIM-binding proteins to the cytoskeleton. Most interactions of PDZ-LIM proteins with the cytoskeleton have been identified in striated muscle, where several PDZ-LIM proteins are predominantly expressed. By contrast, CLP-36 mRNA is expressed in several nonmuscle tissues, and here we demonstrate high expression of CLP-36 in epithelial cells by in situ hybridization analysis. Our subcellular localization studies indicate that in nonmuscle cells, CLP-36 protein localizes to actin stress fibers. This localization is mediated via the PDZ domain of CLP-36 that associates with the spectrin-like repeats of alpha-actinin. Interestingly, immunoprecipitation and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis indicate that both nonmuscle alpha-actinin-1 and alpha-actinin-4 form complexes with CLP-36. The high expression of alpha-actinin-4 in the colon, together with these results, suggests a specific function for the alpha-actinin-4-CLP-36 complex in the colonic epithelium. More generally, results presented here demonstrate that the association of PDZ-LIM proteins with the cytoskeleton extends to the actin stress fibers of nonmuscle cells.     

Phospholipase Cdelta4 associates with glutamate receptor interacting protein 1 in testis

We reported previously that phospholipase C (PLC) delta4 is required for calcium mobilization in the zona pellucida-induced acrosome reaction in sperm. Here we focused on the function of the C2 domain of PLCdelta4 and report that glutamate receptor-interacting protein1 (GRIP1) was identified as a binding protein of the PLCdelta4-C2 domain on yeast two-hybrid screening. Physiological interaction of GRIP1 with PLCdelta4 in mouse testis was confirmed by immunoprecipitation with anti-PLCdelta4 antibodies and the association seemed to correlate with the maturation stage of sperm. We also determined that a PDZ-binding motif at the C-terminus of the PLCdelta4-C2 domain is responsible for GRIP1 binding, whereas the sixth or seventh PDZ domain of GRIP1 is essential and sufficient for association with the PLCdelta4-C2 domain. These results indicate that PLCdelta4 binds via its C2 domain to the PDZ6 or PDZ7 domain of GRIP1, and that this association may play a role in spermatogenesis.