NudC-like protein 2 regulates the LIS1/dynein pathway by stabilizing LIS1 with Hsp90

Abstract

The type I lissencephaly gene product LIS1, a key regulator of cytoplasmic dynein, is critical for cell proliferation, survival, and neuronal migration. However, little is known about the regulation of LIS1. Here, we identify a previously uncharacterized mammalian homolog of Aspergillus NudC, NudCL2 (NudC-like protein 2), as a regulator of LIS1. NudCL2 is localized to the centrosome in interphase, and spindle poles and kinetochores during mitosis, a pattern similar to the localization of LIS1 and cytoplasmic dynein. Depletion of NudCL2 destabilized LIS1 and led to phenotypes resembling those of either dynein or LIS1 deficiency. NudCL2 complexed with and enhanced the interaction between LIS1 and Hsp90. Either disruption of the LIS1-Hsp90 interaction with the C terminus of NudCL2 or inhibition of Hsp90 chaperone function by geldanamycin decreased LIS1 stability. Thus, our results suggest that NudCL2 regulates the LIS1/dynein pathway by stabilizing LIS1 with Hsp90 chaperone. This represents a hitherto undescribed mechanism of the LIS1/dynein regulation in mammalian cells.

Fig. 1.

Characterization of NudCL2. (A) Schematic comparison of human NudCL2, NudCL, and NudC, and Aspergillus NudC. Light gray bars indicate coiled-coil domains and black filled bars show p23_NudC_like domains. (A) Aspergillus; H., Human. (B and C) Subcellular localization of NudCL2. HeLa cells grown on cover slides were subjected to immunofluorescence analyses with the indicated antibodies. DNA was visualized by DAPI. (Scale bar, 10 μm.) The centrosome and mitotic spindle poles were determined by staining with anti-γ tubulin antibody (B). The mitotic kinetochores were identified by immunofluorescence with CREST autoimmune serum (C). A mitotic chromosome spread was performed to confirm the kinetochore localization of NudCL2 (Lower). High magnifications are shown in Insets.

Fig. 2.

Down-regulation of LIS1 in cells depleted of NudCL2. HeLa cells were cotransfected with the indicated vectors and pBABE-puro. At 24 h after transfection, puromycin was added to enrich the transfection-positive cells. (A) NudCL2 depletion leads to reduced LIS1. The cells harvested at various times were subjected to immunoblotting analyses. (B) Ectopic expression of RNAi-resistant NudCL2 partially reverses the down-regulation of LIS1 caused by NudCL2 depletion. RNAi-resistant NudCL2 mutant vector (pFLAG-NudCL2-MT) was constructed by the silent mutation of three nucleic acids in the RNAi targeting region of NudCL2. At 72 h posttransfection, the cells were subjected to Western analysis. Erk2 was used as a loading control.

Fig. 3.

Cellular phenotypes induced by depletion of NudCL2. HeLa cells were transfected with either pS-con or pS-NudCL2 and pBABE-puro. At 24 h after transfection, puromycin was added to select the transfection-positive cells. The cells were subjected to immunofluorescence analyses with the indicated antibodies at 72 h posttransfection. DNA was stained with DAPI. (Scale bar, 10 μm.) (A) The cells indicated with arrows show the dispersion of Golgi apparatus visualized by staining with anti-GM130 antibody. (B) The perinuclear microtubule enrichment is detected in cells with arrows. (C) The cells with arrows exhibit a significant increase in the distance between the centrosome and nucleus (>3 μm).

Fig. 4.

The interaction among NudCL2, LIS1 and Hsp90. (A and B) HeLa cells transfected with the indicated vectors were subjected to coimmunoprecipitation analyses with anti-FLAG antibody-coupled beads at 48 h posttransfection. Immunoblot analyses were performed with the antibodies as shown. NudCL2 was found to bind to LIS1 in vivo (A). Both NudCL2 and LIS1 coimmunoprecipitated with endogenous Hsp90 in vivo (B). (C) Three aliquots of lysates from HeLa cells were subjected to immunoprecipitation experiments with anti-rabbit IgG, anti-NudCL2 antibody (anti-NudCL2) and anti-NudCL2 peptide antibody (anti-NudCL2-P), respectively. Immunoprecipitated proteins were then analyzed by Western blotting with the indicated antibodies. IgG-HC, heavy chain of IgG. (D) The association of NudCL2 fragments with Hsp90 and LIS1 in vitro. Purified GST, GST-NudCL2-N, GST-NudCL2-C, and GST-NudCL2 proteins were incubated with the lysates of HeLa cells, respectively. Either LIS1 or Hsp90 that interacts with NudCL2 fragments was detected by Western blotting with the antibodies as shown.

Fig. 5.

NudCL2 regulates the interaction between LIS1 and Hsp90 in vivo. HeLa cells were transfected with the indicated vectors and subjected to coimmunoprecipitation analysis with anti-FLAG antibody-coupled beads at 72 h posttransfection. Western analyses were performed with the antibodies as shown. (A) Depletion of NudCL2 inhibits the association of LIS1 with Hsp90 in vivo. At 24 h after transfection, puromycin was added to enrich the transfection-positive cells. (B) Overexpression of the C terminus of NudCL2 that associates with LIS1 interferes with the interaction between Hsp90 and LIS1 in vivo.

Fig. 6.

Hsp90 is involved in the regulation of LIS1 stability by NudCL2. (A) Inhibition of Hsp90 chaperone function reduces LIS1 stability. HeLa cells treated with geldanamycin were collected at the indicated times and subjected to immunoblotting analyses with the antibodies as shown. Tubulin was used as a loading control. (B) Overexpression of the C terminus of NudCL2 that inhibits the interaction between Hsp90 and LIS1 decreases the stability of LIS1. HeLa cells transfected with the indicated vectors were harvested at various times and subjected to Western analyses. Erk2 was used as a loading control.