De Novo Mutations in SIK1 Cause a Spectrum of Developmental Epilepsies

Developmental epilepsies are age-dependent seizure disorders for which genetic causes have been increasingly identified. Here we report six unrelated individuals with mutations in salt-inducible kinase 1 (SIK1) in a series of 101 persons with early myoclonic encephalopathy, Ohtahara syndrome, and infantile spasms. Individuals with SIK1 mutations had short survival in cases with neonatal epilepsy onset, and an autism plus developmental syndrome after infantile spasms in others. All six mutations occurred outside the kinase domain of SIK1 and each of the mutants displayed autophosphorylation and kinase activity toward HDAC5. Three mutations generated truncated forms of SIK1 that were resistant to degradation and also showed changes in sub-cellular localization compared to wild-type SIK1. We also report the human neuropathologic examination of SIK1-related developmental epilepsy, with normal neuronal morphology and lamination but abnormal SIK1 protein cellular localization. Therefore, these results expand the genetic etiologies of developmental epilepsies by demonstrating SIK1 mutations as a cause of severe developmental epilepsy

Autosomal recessive mutations in nuclear transport factor KPNA7 are associated with infantile spasms and cerebellar malformation

Nuclear import receptors of the KPNA family recognize the nuclear localization signal in proteins and together with importin-β mediate translocation into the nucleus. Accordingly, KPNA family members have a highly conserved architecture with domains that contact the nuclear localization signal and bind to importin-β. Here, we describe autosomal recessive mutations in KPNA7 found by whole exome sequencing in a sibling pair with severe developmental disability, infantile spasms, subsequent intractable epilepsy consistent with Lennox–Gastaut syndrome, partial agenesis of the corpus callosum, and cerebellar vermis hypoplasia. The mutations mapped to exon 7 in KPNA7 result in two amino-acid substitutions, Pro339Ala and Glu344Gln. On the basis of the crystal structure of the paralog KPNA2 bound to a bipartite nuclear localization signal from the retinoblastoma protein, the amino-acid substitutions in the affected subjects were predicted to occur within the seventh armadillo repeat that forms one of the two nuclear localization signal-binding sites in KPNA family members. Glu344 is conserved in all seven KPNA proteins, and we found that the Glu354Gln mutation in KPNA2 is sufficient to reduce binding to the retinoblastoma nuclear localization signal to approximately one-half that of wild-type protein. Our data show that compound heterozygous mutations in KPNA7 are associated with a human neurodevelopmental disease, and provide the first example of a human disease associated with mutation of a nuclear transport receptor

MAP 1C is a microtubule-activated ATPase which translocates microtubules in vitro and has dynein-like properties

Abstract. We observe that one of the high molecular mass microtubule-associated proteins (MAPs) from brain exhibits nucleotide-dependent binding to microtubules. We identify the protein as MAP 1C, which was previously described in this laboratory as a minor component of standard microtubule preparations (Bloom, G. S., T. Schoenfeld, and R. B. Vallee, 1984, J. Cell Biol., 98:320-330). We find that MAP 1C is enriched in microtubules prepared in the absence of nucleotide. Kinesin is also found in these preparations, but can be specifically extracted with GTP. A fraction highly enriched in MAP 1C can be prepared by subsequent ex-traction of the microtubules with ATE Two activities cofractionate with MAP 1C upon further purification, a microtubule-activated ATPase activity and a microtubule-translocating activity. These activities indicate a role for the protein in cytoplasmic motility. MAP 1C coelectrophoreses with the beta heavy chain of Chlamydomonas flagellar dynein, and has a sedimentation coefficient of 20S. Exposure to ultraviolet light in the presence of vanadate and ATP results in the production of two large fragments of MAP 1C. These characteristics suggest that MAP 1C may be a cytoplasmic analogue of axonemal dynein. C YTOPLASMIC microtubules are composed of tubulin and a number of microtubule-associated proteins (MAPs) ~ thought to be involved in microtubule assembly regulation and microtubule function. In brain tissue the most prominent MAPs are a class of five high molecular mass proteins, which we have termed MAP 1A, MAP 1B

Homology of the 74-kD cytoplasmic dynein subunit with a flagellar dynein polypeptide suggests an intracelhilar targeting function

Abstract. In previous work we found cytoplasmic dynein to be a complex of two catalytic heavy chains and at least seven co-purifying polypeptides of unknown function. The most prominent of these is a 74kD electrophoretic species which can be resolved as two to three bands by SDS-PAGE. We have now selected a series of overlapping rat brain cDNAs encoding the 74-kD species. The deduced sequence of a full-length eDNA predicts a 72,753 D polypeptide which includes the amino acid sequences of nine peptides determined by NH2-terminal microsequencing. PCR performed on first strand rat brain eDNA together with the sequence of a partially matching tryptic peptide indicated the existence of at least three isoforms of the 74-kD cytoplasmic dynein subunit

Karyopherin α7 (KPNA7), a divergent member of the importin α family of nuclear import receptors

<p>Abstract</p> <p>Background</p> <p>Classical nuclear localization signal (NLS) dependent nuclear import is carried out by a heterodimer of importin α and importin β. NLS cargo is recognized by importin α, which is bound by importin β. Importin β mediates translocation of the complex through the central channel of the nuclear pore, and upon reaching the nucleus, RanGTP binding to importin β triggers disassembly of the complex. To date, six importin α family members, encoded by separate genes, have been described in humans.</p> <p>Results</p> <p>We sequenced and characterized a seventh member of the importin α family of transport factors, karyopherin α 7 (KPNA7), which is most closely related to KPNA2. The domain of KPNA7 that binds Importin β (IBB) is divergent, and shows stronger binding to importin β than the IBB domains from of other importin α family members. With regard to NLS recognition, KPNA7 binds to the retinoblastoma (RB) NLS to a similar degree as KPNA2, but it fails to bind the SV40-NLS and the human nucleoplasmin (NPM) NLS. KPNA7 shows a predominantly nuclear distribution under steady state conditions, which contrasts with KPNA2 which is primarily cytoplasmic.</p> <p>Conclusion</p> <p>KPNA7 is a novel importin α family member in humans that belongs to the importin α2 subfamily. KPNA7 shows different subcellular localization and NLS binding characteristics compared to other members of the importin α family. These properties suggest that KPNA7 could be specialized for interactions with select NLS-containing proteins, potentially impacting developmental regulation.</p

Molecular characterization of the 50-kD subunit of dynactin reveals function for the complex in chromosome alignment and spindle organization during mitosis

Abstract. Dynactin is a multi-subunit complex which has been implicated in cytoplasmic dynein function, though its mechanism of action is unknown. In this study, we have characterized the 50-kD subunit of dynactin, and analyzed the effects of its overexpression on mitosis in living cells. Rat and human cDNA clones revealed p50 to be novel and highly conserved, containing three predicted coiled-coil domains. Immunofluorescence staining of dynactin and cytoplasmic dynein components in cultured vertebrate cells showed that both complexes are recruited to kinetochores during prometaphase, and concentrate near spindle poles thereafter. Overexpression of p50 in COS-7 cells disrupted mitosis, causing cells to accumulate in a prometaphase-like state. Chromosomes were con-densed but unaligned, and spindles, while still bipolar, were dramatically distorted. Sedimentation analysis revealed the dynactin complex to be dissociated in the transfected cultures. Furthermore, both dynactin and cytoplasmic dynein staining at prometaphase kinetochores was markedly diminished in cells expressing high levels of p50. These findings represent clear evidence for dynactin and cytoplasmic dynein codistribution within cells, and for the presence of dynactin at kinetochores. The data also provide direct in vivo evidence for a role for vertebrate dynactin in modulating cytoplasmic dynein binding to an organelle, and implicate both dynactin and dynein in chromosome alignment and spindle organization. C YTOPLASMIC dynein is a ubiquitous, multi-subunit ATPase responsible for minus end-directed microtubule-based organelle transport (Paschal an

Isolated flagellar outer arm dynein translocates brain microtubules in vitro

The inner and outer arms of the flagellar axoneme generate the forces needed for flagellar movement; these arms contain ATPases called dyneins. To date, there has been no method for studying the mechanochemical transducing activity of isolated dyneins. Recently, it was found that the brain microtubule-associated protein (MAP) 1C is a microtubule-activated ATPase with the structural and force-producing properties of dynein. MAP 1C translocates microtubules in an in vitro gliding assay, suggesting that such an assay could also be used with axonemal dyneins. Here, we demonstrate that outer-arm dynein isolated from sea urchin (Strongylocentrotus purpuratus) sperm and adsorbed to a glass coverslip can translocate calf-brain microtubules along the surface of the coverslip. Our results conclusively demonstrate that outer-arm dynein by itself is capable of generating shearing forces. The ability to examine the force-generating properties of flagellar dynein in vitro should greatly facilitate studies of the mechanism of action of this important mechanochemical transducer