Cep152 interacts with the cryptic Polo-box of Plk4 and is required for Plk4-induced centriole overduplication.

Primary microcephaly is a rare condition in which brain size is substantially diminished without other syndromic abnormalities. Seven autosomal loci have been genetically mapped, and the underlying causal genes have been identified for MCPH1, MCPH3, MCPH5, MCPH6, and MCPH7 but not for MCPH2 or MCPH4. The known genes play roles in mitosis and cell division. We ascertained three families from an Eastern Canadian subpopulation, each with one microcephalic child. Homozygosity analysis in two families using genome-wide dense SNP genotyping supported linkage to the published MCPH4 locus on chromosome 15q21.1. Sequencing of coding exons of candidate genes in the interval identified a nonconservative amino acid change in a highly conserved residue of the centrosomal protein CEP152. The affected children in these two families were both homozygous for this missense variant. The third affected child was compound heterozygous for the missense mutation plus a second, premature-termination mutation truncating a third of the protein and preventing its localization to centrosomes in transfected cells. CEP152 is the putative mammalian ortholog of Drosphila asterless, mutations in which affect mitosis in the fly. Published data from zebrafish are also consistent with a role of CEP152 in centrosome function. By RT-PCR, CEP152 is expressed in the embryonic mouse brain, similar to other MCPH genes. Like some other MCPH genes, CEP152 shows signatures of positive selection in the human lineage. CEP152 is a strong candidate for the causal gene underlying MCPH4 and may be an important gene in the evolution of human brain size.

The Rev protein of human immunodeficiency virus type 1 is an RNA-binding protein that is required for nuclear export of unspliced and partially spliced viral mRNAs. Nuclear import of human immunodeficiency virus type 1 Rev has been suggested to depend on the classic nuclear transport receptor importin ␤, but not on the adapter protein importin ␣. We now show that, similar to importin ␣, Rev is able to dissociate RanGTP from recycling importin ␤, a reaction that leads to the formation of a novel import complex. Besides importin ␤, the transport receptors transportin, importin 5, and importin 7 specifically interact with Rev and promote its nuclear import in digitonin-permeabilized cells. A single arginine-rich nuclear localization sequence of Rev is required for interaction with all importins tested so far. In contrast to the importin ␤-binding domain of importin ␣, Rev interacts with an N-terminal fragment of importin ␤. Transportin contains two independent binding sites for Rev. Hence, the mode of interaction of importin ␤ and transportin with Rev is clearly distinct from that with their classic import cargoes. Taken together, the viral protein takes advantage of multiple cellular transport pathways for its nuclear accumulation.The machinery for transport of macromolecules across the nuclear envelope consists of the nuclear pore complex, which is embedded between the inner and outer nuclear membrane (1), and a large variety of soluble transport factors (for reviews see Refs. 2-4). The transport cargoes are characterized by recognition sequences for soluble transport receptors: nuclear export sequences mediate the interaction of proteins with exportins, allowing transport out of the nucleus, whereas nuclear localization signals (NLSs) 2 bind to importins, promoting transport of proteins into the nucleus. Importins and exportins, collectively also referred to as karyopherins, belong to the importin ␤ superfamily of transport receptors. They interact not only with their transport cargo but also with proteins of the nuclear pore complex, as well as with the small GTP-binding protein Ran, a factor that plays an important role in the assembly or disassembly of transport complexes (3). Importin ␤, the prototype of this family, was originally identified as the transport receptor that is involved in nuclear import of proteins containing a "classic" NLS, i.e. a short stretch of amino acids enriched in basic residues, forming either a single or a bipartite transport motif. These NLSs do not bind to importin ␤ directly but via an adapter protein, importin ␣. Importin ␣ contains a characteristic importin ␤-binding-(IBB) domain. A ternary complex containing the import cargo, importin ␣, and importin ␤ assembles in the cytoplasm, translocates across the nuclear pore complex, and dissociates in the nucleus upon binding of RanGTP to importin ␤. The import receptor then recycles back to the cytoplasm in a complex with RanGTP. A dedicated exportin, CAS, is used for export of importin ␣ out of the nucleus (5). Hundreds of proteins...

SummaryCentriole duplication occurs once per cell cycle and requires Plk4, a member of the Polo-like kinase family. A key component of the centrosome is the c-tubulin ring complex (c-TuRC) that nucleates microtubules. GCP6 is a member of the c-TuRC, but its role in human cells and the regulation of its functions remain unclear. Here we report that depletion of human GCP6 prevents assembly of the c-TuRC and induces a high percentage of monopolar spindles. These spindles are characterized by a loss of centrosomal c-tubulin and reduced centriole numbers. We found that GCP6 is localized in the pericentriolar material but also at distal portions of centrioles. In addition, GCP6 is required for centriole duplication and Plk4-induced centriole overduplication. GCP6 interacts with and is phosphorylated by Plk4. Moreover, we find that Plk4-dependent phosphorylation of GCP6 regulates centriole duplication. These data suggest that GCP6 is a target of Plk4 in centriole biogenesis.

Our results suggest that She4p is a novel myosin motor domain binding protein and operates as a localized regulator of myosin function of class I and likely class V myosins.

In mammalian cells, the centrosome consists of a pair of centrioles and amorphous pericentriolar material. The centrosome duplicates once per cell cycle. Polo like kinases (Plks) perform crucial functions in cell cycle progression and during mitosis. The polo-like kinase-2, Plk2, is activated near the G(1)/S phase transition, and plays an important role in the reproduction of centrosomes. In this study, we show that the polo-box of Plk2 is required both for association to the centrosome and centriole duplication. Mutation of critical sites in the Plk2 polo-box prevents centrosomal localization and impairs centriole duplication. Plk2 is localized to centrosomes during early G(1) phase where it only associates to the mother centriole and then distributes equally to both mother and daughter centrioles at the onset of S phase. Furthermore, our results imply that Plk2 mediated centriole duplication is dependent on Plk4 function. In addition, we find that siRNA-mediated downregulation of Plk2 leads to the formation of abnormal mitotic spindles confirming that Plk2 may have a function in the reproduction of centrioles.

c-Fos, a component of the transcription factor AP-1, is rapidly imported into the nucleus after translation. We established an in vitro system using digitonin-permeabilized cells to analyze nuclear import of c-Fos in detail. Two import receptors of the importin ␤ superfamily, importin ␤ itself and transportin, promote import of c-Fos in vitro. Under conditions where importin ␤-dependent transport was blocked, c-Fos still accumulated in the nucleus in the presence of cytosol. Inhibition of the transportin-dependent pathway, in contrast, abolished import of c-Fos. Furthermore, c-Fos mutants that interact with transportin but not with importin ␤ were efficiently imported in the presence of cytosol. Hence, transportin appears to be the predominant import receptor for c-Fos. A detailed biochemical characterization revealed that the interaction of transportin with c-Fos is distinct from the interaction with its established import cargoes, the M9 sequence of heterogeneous nuclear ribonucleoprotein A1 or the nuclear localization sequence of some basic proteins. Likewise, the binding sites on importin ␤ for its classic import cargo and for c-Fos can be separated. In summary, c-Fos employs a novel mode of receptor-cargo interaction. Hence, transportin may be as versatile as importin ␤ in recognizing different nuclear import cargoes.Import of proteins into the nucleus is mediated by importins, members of the importin ␤ superfamily. The "classic" signals for nuclear import (nuclear localization signal; NLS) 2 are short basic stretches of amino acids with lysines as characteristic components, which may occur either as a single or a bipartite motif. These classic NLSs are recognized by the adapter molecule importin ␣, which forms a heterodimer with the actual transport receptor, importin ␤ (for reviews see Refs. 1-3). The importin ␣/␤-NLS-cargo complex is then translocated through the nuclear pore complex. Nucleoporins, the protein components of the nuclear pore complex, may interact with the transport receptor, facilitating the translocation of the complex. Importin ␤ interaction with RanGTP on the nuclear side of the nuclear pore complex results in dissociation of the transport complex (4). Hundreds of individual nuclear proteins or proteins that shuttle between the nucleus and the cytoplasm are thought to use this classic importin ␣/␤ nuclear import pathway. Over the last couple of years, however, a number of proteins have been described that do not depend on the adapter protein importin ␣. Some of these bind directly to the classic import receptor, importin ␤, without requiring any adapter protein. These include the proteins Rev and Rex of the human immunodeficiency virus (5, 6) and the human T-cell leukemia virus (7), respectively, the T-cell protein tyrosine phosphatase (8), the parathyroid hormone-related protein (PTHrP) (9), cyclin B1 (10), the sterol regulatory element-binding protein 2 (SREBP-2 (11)), the zinc finger protein Snail (12), and the transcription factor CREB (13). Many of these proteins also contain basi...

1 essential for initiation of centriole biogenesis, is not associated with the γ-tubulin-specific abnormal centrosomes. The amount of Plk4 at each centrosome was less in cells with abnormal centrosomes than cells without γ-tubulin-specific abnormal centrosomes. In addition, the formation of abnormal structures was abolished by expression of exogenous Plk4, but not SAS6 and Cep135/Bld10p, which are downstream regulators required for the organization of nine-triplet microtubules. These results suggest that HCT116 cells fail to organize the ninefold symmetry of centrioles due to insufficient Plk4.