Ras transformation uncouples the kinesin-coordinated cellular nutrient response

Abstract

The kinesin family members (KIFs) KIF2A and KIF2C depolymerize microtubules, unlike the majority of other kinesins, which transport cargo along microtubules. KIF2A regulates the localization of lysosomes in the cytoplasm, which assists in activation of the mechanistic target of rapamycin complex 1 (mTORC1) on the lysosomal surface. We find that the closely related kinesin KIF2C also influences lysosomal organization in immortalized human bronchial epithelial cells (HBECs). Expression of KIF2C and, to a lesser extent, KIF2A in untransformed and mutant K-Ras-transformed cells is regulated by ERK1/2. Prolonged inhibition of ERK1/2 activation with PD0325901 mimics nutrient deprivation by disrupting lysosome organization and decreasing mTORC1 activity in HBEC, suggesting a long-term mechanism for optimization of mTORC1 activity by ERK1/2. We tested the hypothesis that up-regulation of KIF2C and KIF2A by ERK1/2 caused aberrant lysosomal positioning and mTORC1 activity in a mutant K-Ras-dependent cancer and cancer model. In Ras-transformed cells, however, mTORC1 activity and lysosome organization appear independent of ERK1/2 and these kinesins although ERK1/2 activity and the kinesins are required for Ras-dependent proliferation and migration. We conclude that mutant K-Ras repurposes these signaling and regulatory proteins to support the transformed phenotype.

Fig. 1.

KIF2C influences lysosomal organization and mTORC1 activity in HBEC3KT cells. (A) HBEC3KT cells were treated with control or KIF2C siRNA twice 48 h apart for a total of 96 h. Cells were then starved for 90 min and restimulated with amino acids (combination of each amino acid contained in l-glutamine-free DMEM plus 0.5 mM l-glutamine) for 30 min. Cells were coimmunostained for endogenous mTOR (red) and endogenous LAMP2 (green). (B) The eight-bit grayscale images from Fig. 1A were analyzed after thresholding to measure the area of mTOR localization after restimulation with amino acids by obtaining the ratio of mTOR staining per whole-cell area. (C) Cells were starved treated as above with control, KIF2C, or KIF2A siRNA, starved, and then stimulated with amino acids (AA) or fresh medium with serum (M). Lysate proteins were resolved on gels, transferred to membranes, and immunoblotted with antibodies to the indicated proteins.

Fig. 2.

Inhibition of ERK1/2 inhibits mTORC1 in immortalized bronchial epithelial cells but not in Raf-transformed cells. (A) HBEC3KT and HBEC3KTRL53 cells were treated for 2 d with 100 nM PD0325901 and immunostained as in Fig. 1A. (B) Images were analyzed as in Fig. 1 to measure the area of lysosomal distribution (LAMP2 staining) by obtaining the ratio of the organelle area per whole-cell area. (C) HBEC3KT and HBEC3KTRL53 cells were treated for 30 min or 2 d with 100 nM PD0325901. Lysate proteins were immunoblotted with the indicated antibodies.

Fig. 3.

Cancer cells with K-Ras mutations are less sensitive to starvation than normal cells. (A) HBEC3KTRL53 and HCT116 cells were starved in EBSS for 30 min or 120 min, as indicated, and coimmunostained as in Fig. 1A. (B) HBEC3KT and HBEC3KTRL53 cells were starved in EBSS for 0 min, 30 min, or 60 min. Lysate proteins were resolved on gels followed by immunoblotting with the indicated antibodies. (C) HBEC3KT and HCT116 cells were starved in EBSS for the specified times and immunoblotted as above.

Fig. 4.

KIF2A and KIF2C overexpression in HeLa and HBEC3KT activates pERK and pS6. (A) FLAG-KIF2A were expressed in HeLa cells that were starved in EBSS or for 90 min. (B) Cells expressing FLAG-KIF2A were treated with PD0325901 for 90 min. (C) FLAG-KIF2C was expressed in HBEC3KT cells that were treated with PD0325901 for 90 min. Lysate proteins were resolved on gels, followed by immunoblotting with the indicated antibodies.

Fig. 5.

Loss of KIF2C induces autophagy. (A) LC3-II was immunoblotted in equal amounts of lysates from HBEC3KT and HBEC3KTRL53. (B) Ras was depleted from HBEC3KTRL53 with siRNA. Cells were then treated for 3 d with 100 nM PD0325901, followed by immunoblotting of lysates as indicated. (C) KIF2C was depleted with siRNA from HBEC3KTRL53. Cells were starved for 12 h in EBSS or treated with 10 nM Baflomycin A and immunoblotted with antibodies to p62. ERK1/2 was used as the loading control.