Cytoplasmic LEK1 is a regulator of microtubule function through its interaction with the LIS1 pathway

Abstract

LIS1 and nuclear distribution gene E (NudE) are partner proteins in a conserved pathway regulating the function of dynein and microtubules. Here, we present data that cytoplasmic LEK1 (cytLEK1), a large protein containing a spectrin repeat and multiple leucine zippers, is a component of this pathway through its direct interaction with NudE, as determined by a yeast two-hybrid screen. We identified the binding domains in each molecule, and coimmunoprecipitation and colocalization studies confirmed the specificity of the interaction between cytLEK1 and NudE. Confocal deconvolution analysis revealed that cytLEK1 exhibits colocalization with endogenous NudE and with the known NudE binding partners, LIS1 and dynein. By localizing the NudE-binding domain of cytLEK1 to a small domain within the molecule, we were able to disrupt cytLEK1 function by using a dominant negative approach in addition to LEK1 knockdown and, thus, examine the role of the cytLEK1-NudE interaction in cells. Consistent with a defect in the LIS1 pathway, disruption of cytLEK1 function resulted in alteration of microtubule organization and cellular shape. The microtubule network of cells became tightly focused around the nucleus and resulted in a rounded cell shape. Additionally, cells exhibited a severe inability to repolymerize their microtubule networks after nocodazole challenge. Taken together, our studies revealed that cytLEK1 is essential for cellular functions regulated by the LIS1 pathway.cytoskeleton

Identification of Rare, Single-Nucleotide Mutations in NDE1 and Their Contributions to Schizophrenia Susceptibility

“…Although the contribution of R234C mutation to neurodevelopment remains unclear, the change of the axon length by R234C might relate to NDE1 structure or protein-protein interactions. In fact, several proteins besides YWHAE bind to this region of NDE1, including the S214 and R234 resides; CENPF, 52 Su48, 31 and Katanin p60 are among these. 53 Furthermore, in silico analysis with Splice Aid 2 54 indicates that the nucleotide change causing R234C may be located within an exon-splicing enhancer (ESE) motif 54 and may decrease mRNA transcription because the alternative splicing might result in exon skipping during transcription.…”

Silencing CENPF in bovine preimplantation embryo induces arrest at 8-cell stage

“…Only four of its orthologs -murine CENPF (Goodwin et al 1999, Ashe et al 2003, Dees et al 2005, Soukolis et al 2005, Evans et al 2007, avian CMF1 (Wei et al 1996, Redkar et al 2002 and worm hcp1 and hcp2 (Cheeseman et al 2005, Hajeri et al 2008) have been studied slightly more intensively. However, these proteins are expressed throughout the whole cell cycle, do not strictly localize to the nucleus or have a somewhat different function (Wei et al 1996, Goodwin et al 1999, Redkar et al 2002, Cheeseman et al 2005, Dees et al 2005, Soukolis et al 2005, Evans et al 2007, Hajeri et al 2008. This suggests that bovine CENPF does not necessarily have to be expressed in a cell-cycle-dependent manner.…”

Bves directly interacts with GEFT, and controls cell shape and movement through regulation of Rac1/Cdc42 activity

“…mGEFT was amplified from pCMVTag-2b-mGEFT and cloned in frame with GFP of pEGFP-C1 (Clontech) to generate pEGFP-mGEFT. pEGFP-mNudeL1 was generated previously by our laboratory (53). pcDNA3-N-myc-Cool1 was a gracious gift from Richard Cerione (Cornell University, Ithaca, NY), and pC.HA-Vav1 was obtained from Addgene.…”