Cytoplasmic LEK1 is a regulator of microtubule function through its interaction with the LIS1 pathway.
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.
Soukoulis
Victor
V
Stahlman Cardiovascular Research Laboratories, Program for Developmental Biology, and Department of Medicine, Vanderbilt University Medical Center, Nashville, TN 37232-6300, USA.
Reddy
Samyukta
S
Pooley
Ryan D
RD
Feng
Yuanyi
Y
Walsh
Christopher A
CA
Bader
David M
DM
D000818:Animals; D019556:COS Cells; D002352:Carrier Proteins / Q000378:metabolism*; D002522:Chlorocebus aethiops; D003593:Cytoplasm / Q000378:metabolism*; D007150:Immunohistochemistry; D008869:Microtubule-Associated Proteins / Q000378:metabolism*; D008870:Microtubules / Q000378:metabolism / Q000502:physiology*; D015739:Nocodazole; D016376:Oligonucleotides, Antisense; D020798:Two-Hybrid System Techniques
D002352:Carrier Proteins; D008869:Microtubule-Associated Proteins; D016376:Oligonucleotides, Antisense; D015739:Nocodazole