Ruk is a recently identified gene with a complex pattern of expression in mammalian cells and tissues. Multiple Ruk transcripts and several protein isoforms have been detected in various types of cells. Ruk proteins have multidomain organization characteristic of adapter proteins involved in regulation of signal transduction. Interaction of some Ruk isoforms with several signalling proteins, including the p85 regulatory subunit of the Class IA PI 3-kinase, c-Cbl and Grb2, has been demonstrated. Ruk(l), an isoform with three SH3 domains, inhibits lipid kinase activity of the PI 3-kinase in vitro; overexpression of this protein induces apoptotic cell death of primary neurons in culture and changes in membrane trafficking in other cultured cells. However, shorter isoforms of Ruk block pro-apoptotic effect of Ruk(l), suggesting that expression of different combinations of Ruk proteins in cells could be involved in the regulation of their survival and other intracellular processes. To understand the mechanism of differential expression of Ruk proteins we studied organization of the mouse Ruk gene and its transcripts. Twenty-four exons of the Ruk gene span over 320 kb of the mouse chromosome X. Analysis of cDNA clones, ESTs and products of RT-PCR amplifications with different combinations of primers revealed how alternative splicing and promoter usage generate a variety of Ruk transcripts and encoded protein isoforms in different mouse tissues.
"Previously described protocols were used for Western blotting (Buchman et al., 2002). Protein levels were quantified using Cy3-or Cy5-conjugated secondary antibodies (Invitrogen, CA) and FluorChem Q MultiImage III system (Cell Biosciences, CA). "
[Show abstract][Hide abstract] ABSTRACT: Amyotrophic lateral sclerosis (ALS) is characterised by substantial loss of both upper and lower motor neuron function, with sensory and cognitive systems less affected. Though heritable forms of the disease have been described, the vast majority of cases are sporadic with poorly defined underlying pathogenic mechanisms. Here we demonstrate that the neurological pathology induced in transgenic mice by overexpression of γ-synuclein, a protein not previously associated with ALS, recapitulates key features of the disease, namely selective damage and loss of discrete populations of upper and lower motor neurons and their axons, contrasted by limited effects upon the sensory system.
Neurobiology of Disease 06/2012; 48(1):124-31. DOI:10.1016/j.nbd.2012.06.016 · 5.08 Impact Factor
"Proteins were resolved in SDS-PAGE and transferred to Hybond P membrane (GE Healthcare, Little Chalfont, UK). Membranes were incubated with primary rabbit polyclonal anti-FLAG (Sigma, USA), anti-Nedd4 (Abcam, UK), anti-Nedd4-2 , or anti-neomycin phosphotransferase II (Millipore, USA), mouse monoclonal anti-α-tubulin, clone DM1A antibodies (Sigma, USA) or affinity purified rat anti-MAK-V antibodies  followed by secondary anti-rabbit, anti-mouse or anti-rat horseradish peroxidase-conjugated antibodies (HRP; GE Healthcare, USA) as described previously . To detect GST, anti-GST HPR conjugate (GE Healthcare, USA) was used. "
[Show abstract][Hide abstract] ABSTRACT: MAK-V/Hunk is a scantily characterized AMPK-like protein kinase. Recent findings identified MAK-V as a pro-survival and anti-apoptotic protein and revealed its role in embryonic development as well as in tumorigenesis and metastasis. However molecular mechanisms of MAK-V action and regulation of its activity remain largely unknown. We identified Nedd4 as an interaction partner for MAK-V protein kinase. However, this HECT-type E3 ubiquitin ligase is not involved in the control of MAK-V degradation by the ubiquitin-proteasome system that regulates MAK-V abundance in cells. However, Nedd4 in an ubiquitin ligase-independent manner rescued developmental defects in Xenopus embryos induced by MAK-V overexpression, suggesting physiological relevance of interaction between MAK-V and Nedd4. This identifies Nedd4 as the first known regulator of MAK-V function.
PLoS ONE 06/2012; 7(6):e39505. DOI:10.1371/journal.pone.0039505 · 3.23 Impact Factor
"Previous expression assays have shown that at least one of the three major isoforms is expressed in every tissue type (Buchman et al., 2002; Mayevska et al., 2006). However, to date, mRNA-3 (Fig. 1A) has only been detected in Schwann cells (Fig. 2B), heart, skin, and kidney (Buchman et al., 2002). "
[Show abstract][Hide abstract] ABSTRACT: The transcription factor SOX10 has essential roles in neural crest-derived cell populations, including myelinating Schwann cells-specialized glial cells responsible for ensheathing axons in the peripheral nervous system. Importantly, SOX10 directly regulates the expression of genes essential for proper myelin function. To date, only a handful of SOX10 target loci have been characterized in Schwann cells. Addressing this lack of knowledge will provide a better understanding of Schwann cell biology and candidate loci for relevant diseases such as demyelinating peripheral neuropathies. We have identified a highly-conserved SOX10 binding site within an alternative promoter at the SH3-domain kinase binding protein 1 (Sh3kbp1) locus. The genomic segment identified at Sh3kbp1 binds to SOX10 and displays strong promoter activity in Schwann cells in vitro and in vivo. Mutation of the SOX10 binding site ablates promoter activity, and ectopic expression of SOX10 in SOX10-negative cells promotes the expression of endogenous Sh3kbp1. Combined, these data reveal Sh3kbp1 as a novel target of SOX10 and raise important questions regarding the function of SH3KBP1 isoforms in Schwann cells.
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