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P27 Controls H-Ras/MAPK Activation and Cell Cycle Entry Via Modulation

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P27 Controls H-Ras/MAPK Activation and Cell Cycle Entry Via Modulation p27kip1 controls H-Ras/MAPK activation and cell cycle entry via modulation of MT stability Linda Fabrisa,1, Stefania Bertona,1, Ilenia Pellizzaria,1, Ilenia Segattoa, Sara D’Andreaa, Joshua Armeniaa, Riccardo Bombenb, Monica Schiappacassia, Valter Gatteib, Mark R. Philipsc, Andrea Vecchioned, Barbara Bellettia,2, and Gustavo Baldassarrea,2 aDivision of Experimental Oncology 2, Department of Translational Research, Centro di Riferimento Oncologico (CRO Aviano), National Cancer Institute, 33081 Aviano, Italy; bClinical and Experimental Onco-Hematology Unit, Department of Translational Research, Centro di Riferimento Oncologico (CRO Aviano), National Cancer Institute, 33081 Aviano, Italy; cPerlmutter Cancer Institute, New York University School of Medicine, New York, NY 10016; and dDivision of Pathology, II University of Rome “La Sapienza,” Santo Andrea Hospital, 00100 Rome, Italy Edited by Melanie H. Cobb, University of Texas Southwestern Medical Center, Dallas, TX, and approved October 1, 2015 (received for review May 4, 2015) The cyclin-dependent kinase (CDK) inhibitor p27kip1 is a critical stability and subcellular localization, and has been proposed to play regulator of the G1/S-phase transition of the cell cycle and also a central role in Ras-driven tumorigenesis (11, 12). regulates microtubule (MT) stability. This latter function is exerted We have recently demonstrated that p27 and stathmin genet- by modulating the activity of stathmin, an MT-destabilizing protein, ically interact in vivo and that this interaction impinges on cell and by direct binding to MTs. We recently demonstrated that in- proliferation, eventually leading to organ size determination (13). creased proliferation in p27kip1-null mice is reverted by concomitant Interestingly, both p27 and its interacting partner stathmin are im- deletion of stathmin in p27kip1/stathmin double-KO mice, suggesting plicated in the regulation of MT stability, with p27 acting as an MT – that a CDK-independent function of p27kip1 contributes to the control stabilizer and stathmin acting as an MT destabilizer (14 17). of cell proliferation. Whether the regulation of MT stability by p27kip1 Here,weinvestigatedinvitroandinvivotheroleofp27/stathmin impinges on signaling pathway activation and contributes to the de- interaction in cell proliferation. Our findings unveil a regulatory axis cision to enter the cell cycle is largely unknown. Here, we report that controlling the G1/S-phase transition in mammalian cells, relying faster cell cycle entry of p27kip1-null cells was impaired by the con- on the regulation of MT stability by p27 and eventually mod- comitant deletion of stathmin. Using gene expression profiling cou- ulating the spatiotemporal activation of the Ras-MAPK signal- ing pathway. pled with bioinformatic analyses, we show that p27kip1 and stathmin conjunctly control activation of the MAPK pathway. From a molecular Results point of view, we observed that p27kip1, by controlling MT stability, Stathmin Is Necessary for the Hyperproliferative Phenotype of p27 impinges on H-Ras trafficking and ubiquitination levels, eventually Null Primary Fibroblasts. restraining its full activation. Our study identifies a regulatory axis In vivo, the proliferation-related pheno- types of p27-null animals are partially reverted by concomitant controlling the G1/S-phase transition, relying on the regulation of KO of stathmin (13). To understand the molecular mechanisms MT stability by p27kip1 and finely controlling the spatiotemporal underlying this reversion, we generated primary mouse embry- activation of the Ras-MAPK signaling pathway. onic fibroblasts (MEFs) from WT, p27KO, stathmin KO (StmKO), p27kip1 | stathmin | Ras | cell cycle | microtubules Significance n multicellular organisms, each cell responds to external stimuli Different functions have been ascribed to p27kip1, originally by finely modulating, in time and space, the activation of in- I identified as a universal cyclin-dependent kinase (CDK) in- tracellular signal transduction pathways (1). Microtubules (MTs) hibitor, fundamental for the control of cell proliferation and represent one of the preferential networks involved in the or- tumor progression. Yet, not all p27 functions can be explained dered propagation of this information within the cell. MTs are by its ability to bind and inhibit CDKs. Here, we demonstrate organized into a polarized array that defines cellular polarity and that p27kip1 controls cell cycle entry also through a CDK- serves to directionally transmit extra- to intracellular signals, and independent function, by regulating microtubule stability. vice versa (2). MT functions are strictlylinkedtoMTstability Following growth factor stimulation, p27kip1 prevents full activa- and dynamics, which govern signaling compartmentalization; ac- tion of H-Ras, acting on its subcellular compartmentalization, tivation levels of signal transduction pathways; and, consequently, eventually restraining the activation of the MAPK pathway. Our several cellular processes, including cell cycle progression (2, 3). work provides additional understanding of the mechanisms reg- In particular, passage through the restriction point represents the ulating the cell cycle and anticipates potential implications in dis- prototype of a cellular process in which ordered integration of eases characterized by deregulated proliferation, such as cancer. external and internal stimuli is strictly required (4). A prominent role in this integrated system of growth control Author contributions: B.B. and G.B. designed research; L.F., S.B., I.P., I.S., S.D., J.A., and R.B. has been ascribed to the Ras-MAPK pathway (5). Ras is a small performed research; M.S., V.G., M.R.P., and A.V. contributed new reagents/analytic tools; guanosine triphosphatase (GTPase) that localizes to the membrane L.F., S.B., I.P., I.S., B.B., and G.B. analyzed data; R.B. performed and analyzed the micro- and requires recycling in the vesicle compartment to be fully acti- array data; and B.B. and G.B. wrote the paper. vated (6, 7). Full activation of the Ras-MAPK pathway results in The authors declare no conflict of interest. ERK1/2 nuclear translocation and expression of specific transcrip- This article is a PNAS Direct Submission. tion factors, such as EGR-1, Jun-B, and c-Fos (8). The regulation of Freely available online through the PNAS open access option. this pathway by MTs has been proposed, but the molecular mech- Data deposition: The microarray data reported in this paper have been deposited in the anisms have never been elucidated (2). Gene Expression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. The Ras-MAPK cascade directly signals on two key regulators GSE31533). kip1 of cell cycle entry, namely, cyclin D1 and p27 (9). Cyclin D1 is 1L.F., S.B., and I.P. contributed equally to this work. transcribed following Ras-MAPK pathway activation, and the ab- 2To whom correspondence may be addressed. Email: [email protected] or gbaldassarre@ erration of this molecular event underlies Ras-driven carci- cro.it. kip1 nogenesis (9, 10). Phosphorylation of p27 (hereafter p27) This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. on Ser-10, following Ras-MAPK pathway activation, impinges on its 1073/pnas.1508514112/-/DCSupplemental. 13916–13921 | PNAS | November 10, 2015 | vol. 112 | no. 45 www.pnas.org/cgi/doi/10.1073/pnas.1508514112 Downloaded by guest on October 4, 2021 and p27KO-StmKO [hereafter called double-KO (DKO)] mice and to p27KO cells (SI Appendix,Fig.S1D). Although stathmin is a studied their proliferative behavior in vitro. Growth curve analyses substrate for both CDK2 and CDK1 (15, 21, 22), increasing doses of demonstrated that DKO MEFs grew at an intermediate rate be- recombinant stathmin did not have an impact on the kinase activity tween WT and p27-null cells (Fig. 1A). BrdU incorporation (Fig. 1B) of either endogenous or recombinant protein complexes (SI Ap- and TUNEL assays (Fig. 1C) indicated that the differences between pendix,Fig.S2A and B). p27KOandDKOMEFswereduetoincreased proliferation, rather Overall, our data confirm that the higher CDK2 activity observed than decreased apoptosis. No differences were observed between in p27-null cells is not sufficient to explain their increased pro- WT and StmKO MEFs (Fig. 1 A–C), which, accordingly, increased liferation rate, as also proposed by others (19, 20). their number twofold in 4 days of culture (n = 5; WT = 1.96 ± 0.18, StmKO = 1.86 ± 0.20) (Fig. 1D).Inthesametime,p27KOMEFs p27/Stathmin Interaction Regulates Cell Cycle Entry. The above data increased about fourfold and DKO MEFs displayed an intermediate suggested that G1/S-phase progression could be affected by stath- phenotype, increasing 2.8-fold (Fig. 1D). The same differences were min loss in the absence of p27. Consistently, following serum ∼ also observed when MEFs were generated from different genetic stimulation, p27KO cells entered into S phase 4 h earlier than backgrounds (SI Appendix,Fig.S1A). Overexpression of stathmin in WT and DKO cells, showing an anticipated increase in cyclin A WT, but not in p27KO, 3T3 fibroblasts led to an increase of their expression (S-phase marker) and CDK2, as well as cyclin E-, cyclin A-, and cyclin B1-associated kinase activities, with respect to both proliferation (SI Appendix,Fig.S1B), supporting the possibility that – stathmin actively participated in the control of cell proliferation in a WT and DKO MEFs (SI Appendix,Fig.S1F I). Accordingly, p27-dependent
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