Ras Transformation Uncouples the Kinesin-Coordinated Cellular Nutrient Response
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Ras transformation uncouples the kinesin-coordinated cellular nutrient response Elma Zaganjora,1, Lauren M. Weila, Joshua X. Gonzalesa, John D. Minnaa,b, and Melanie H. Cobba,2 aDepartment of Pharmacology and bHamon Cancer Center for Therapeutic Oncology Research, University of Texas Southwestern Medical Center, Dallas, TX 75390-9041 Contributed by Melanie H. Cobb, June 12, 2014 (sent for review March 13, 2014; reviewed by George S. Bloom and Anning Lin) The kinesin family members (KIFs) KIF2A and KIF2C depolymerize We recently determined that expression of KIF2C and KIF2A microtubules, unlike the majority of other kinesins, which trans- is up-regulated by the Ras-ERK pathway (19). Therefore, we port cargo along microtubules. KIF2A regulates the localization of asked whether ERK1/2 and these kinesins are required to main- lysosomes in the cytoplasm, which assists in activation of the mech- tain lysosomal organization in immortalized human bronchial anistic target of rapamycin complex 1 (mTORC1) on the lysosomal epithelial cells (HBECs) and also whether the elevated expres- surface. We find that the closely related kinesin KIF2C also influen- sion of KIF2C and KIF2A accounts for the nutrient insensitivity ces lysosomal organization in immortalized human bronchial epi- of lysosomal positioning and mTORC1 activity in a mutant thelial cells (HBECs). Expression of KIF2C and, to a lesser extent, K-Ras–dependent cancer and cancer model. KIF2A in untransformed and mutant K-Ras–transformed cells is regulated by ERK1/2. Prolonged inhibition of ERK1/2 activation Results with PD0325901 mimics nutrient deprivation by disrupting lyso- KIF2A and KIF2C Are Required for the Amino Acid-Dependent some organization and decreasing mTORC1 activity in HBEC, sug- Localization of mTORC1 to Lysosomes in Immortalized HBEC. To ex- gesting a long-term mechanism for optimization of mTORC1 plore the relationships among KIF2A and KIF2C expression, activity by ERK1/2. We tested the hypothesis that up-regulation ERK1/2, and lysosomal organization, we used a model system to of KIF2C and KIF2A by ERK1/2 caused aberrant lysosomal position- assess signaling differences between immortalized cells before ing and mTORC1 activity in a mutant K-Ras–dependent cancer and and after transformation under relatively well-defined conditions. cancer model. In Ras-transformed cells, however, mTORC1 activity We examined the characteristics of a nontumor human bronchial and lysosome organization appear independent of ERK1/2 and epithelial cell line (HBEC) that was immortalized by expression these kinesins although ERK1/2 activity and the kinesins are re- of cyclin-dependent kinase 4 (CDK4) and telomerase (hTERT), quired for Ras-dependent proliferation and migration. We conclude yielding HBEC3KT (number distinguishes different cell donors) that mutant K-Ras repurposes these signaling and regulatory pro- as described (20). These cells maintain epithelial morphology and teins to support the transformed phenotype. express E-cadherin but not N-cadherin (19). We compared the behavior of this immortalized untransformed cell line to an iso- omplex signaling pathways are engaged to coordinate cellu- genic non-small cell lung cancer (NSCLC) model that was gen- Clar growth and proliferation as dictated by stimulatory signals, erated by introducing mutant K-Ras and a stable knockdown of such as abundant nutrients and growth factors, or inhibitory sig- p53 to yield HBEC3KTRL53 (21, 22). We also examined the nals, such as limiting nutrients or other cell stresses. Disturbances patient-derived colorectal cancer cell HCT116, which has an in coordination of signaling events are often observed in pathol- oncogenic K-Ras mutation. ogies such as cancer (1). Some cancers use oncogenic mutations of To examine the effects of nutrient restriction, cells were placed ’ the small GTPase K-Ras to promote their survival and pro- in Earl s balanced salt solution (EBSS), which contains glucose liferation (2–6). Mutant K-Ras activates central signaling cascades, but lacks serum and amino acids. Because mTORC1 is, at least in among them the phosphatidylinositol 3-kinase (Ras-PI3K) and the extracellular signal-regulated kinase ERK1/2 (Ras-ERK) path- Significance ways, to facilitate continued proliferation under suboptimal con- ditions and bypass inhibitory signals from energy stress pathways. The kinesin family member 2C (KIF2C) that regulates microtu- Mechanistic target of rapamycin (mTOR), a serine/threonine bule depolymerization, is up-regulated in a number of human protein kinase in a PI3K-related family, is a central regulator of cancer cells in a Ras- and ERK1/2-dependent manner. In this events leading to cell growth and proliferation (7–12). It carries out study, we find that KIF2C expression positively regulates sig- this role primarily as part of a complex [mTOR complex 1 naling pathways downstream of Ras, ERK1/2, and mechanistic (mTORC1)], and, in this capacity, it is exquisitely responsive to target of rapamycin complex 1 in nontransformed cells to co- nutrient availability. In certain cancers, positive regulatory signals to ordinate a cellular nutrient response. Transformation by on- mTORC1 can originate from mutant K-Ras, even in nutrient-poor cogenic Ras overrides the need for this level of organization settings (13, 14). Because cancers often use mTORC1 to optimize and repurposes ERK1/2 and KIF2C signaling events. Therefore, cellular functions to promote growth and survival, we have com- this study provides a link for how cytoskeletal transformation pared how the Ras-ERK pathway influences nutrient sensing and may interconnect with cancer metabolism. mTORC1 in untransformed immortalized cells and in cancer cells. Kinesins are motor proteins that transport cargo along micro- Author contributions: E.Z. and M.H.C. designed research; E.Z., L.M.W., and J.X.G. per- formed research; J.D.M. contributed new reagents/analytic tools; E.Z., L.M.W., J.D.M., tubules. Currently, more than 40 kinesins have been found in and M.H.C. analyzed data; and E.Z. and M.H.C. wrote the paper. mammals, most of which have primary functions as cargo carriers. Reviewers: G.S.B., University of Virginia; and A.L., University of Chicago and Shanghai Kinesin family member (KIF) 2A is one of a handful of kinesins Institute of Biochemistry and Cell Biology. that apparently do not transport cargo, but instead depolymerize The authors declare no conflict of interest. microtubules. In HeLa cells, KIF2A was shown to regulate the 1Present address: Department of Cell Biology, Harvard Medical School, Boston, MA 02115. location of lysosomes in the cytoplasm, which is thought to be es- 2To whom correspondence should be addressed. Email: melanie.cobb@utsouthwestern. sential for mTORC1 activation (15–18). We find that KIF2A and edu. the closely related kinesin KIF2C (also known as MCAK) also This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. control lysosomal organization in human bronchial epithelial cells. 1073/pnas.1411016111/-/DCSupplemental. 10568–10573 | PNAS | July 22, 2014 | vol. 111 | no. 29 www.pnas.org/cgi/doi/10.1073/pnas.1411016111 Downloaded by guest on September 27, 2021 part, activated through interaction with Rag small GTPases and and less well-localized with lysosomes (Fig. S1 A, B,andF). Because the Ragulator complex on lysosomes (16), we examined how re- KIF2C has many functions that overlap with KIF2A (19), we tested moval of amino acids and serum affected lysosomes and mTOR its effects as well and found that depletion of KIF2C also prevented localization. After 60–90 min of starvation in EBSS, lysosomes in restoration of lysosomal organization or mTOR activity by amino HBEC3KT lost the typical perinuclear concentrated pattern and acids (Fig. 1 A–C and Fig. S1 D and G), suggesting that more than instead gained a more diffuse and symmetric distribution, similar one kinesin participates in lysosomal positioning. Although KIF2A to what has been observed in HEK293 and HeLa cells following siRNA and KIF2C siRNA caused a reduction in mTORC1 activity removal of amino acids (15, 16, 23) (Fig. 1 A and B). mTOR was in amino acid-replete cells, knockdown of either or both had little or no effect on mTORC1 activity in cells in serum-containing also dispersed and less concentrated on lysosomes in HBEC3KT C F G cells, and mTORC1 activity was also reduced (Fig. 1C), as mea- medium (Fig. 1 and Fig. S1 and ). ERK1/2 activation in sured by a decrease in phosphorylation of ribosomal protein S6. HBEC3KT was also reduced following KIF2A or KIF2C siRNA. ERK1/2 were also less active after an hour in EBSS. Readdition of Inhibition of the ERK1/2 Pathway Alters Lysosome Positioning and amino acids or complete medium with amino acids and serum Reduces mTORC1 Activity in Immortalized Cells. mRNAs encoding C reactivated both ERK1/2 and mTORC1 (Fig. 1 ). KIF2C and, to a lesser extent, KIF2A are decreased in both Knockdown studies revealed that the microtubule-depolymerizing HBEC3KT and HBEC3KTRL53 by extended exposure to kinesin KIF2A is required for lysosomal organization and mTORC1 PD0325901, which inhibits the enzymes MEK1 and MEK2 that positioning on lysosomes (15). We recently found that KIF2A activate ERK1/2 (19). These and other studies indicated that and the closely related kinesin KIF2C are both up-regulated in ERK1/2 stimulate KIF2A/C expression. Therefore, we explored many cancers and that their persistent expression is supported by the possibility that ERK1/2 may also influence organelle orga- oncogenic K-Ras and ERK1/2 (19). As previously found in HeLa nization in immortalized cells through their ability to control the and 293 cells, depletion of KIF2A from HBEC3KT prevented expression of proteins that alter microtubule dynamics including typical lysosomal organization, and mTOR also remained diffuse these kinesins. We inhibited ERK1/2 activation by exposure of cells to the MEK inhibitor PD0325901 briefly or for up to 2 d. We showed previously that serum can stimulate ERK1/2 im- mediately after washing out PD0325901 even if cells have been A HBEC3KT B mTOR LAMP2 MERGE treated with the inhibitor for 2 d (19).