Raptor is Phosphorylated by cdc2 during Mitosis

Cell Reports, 2020

Highlights d mTORC1 activity is decreased during mitotic arrest d Mutations of raptor phosphorylation sites activate mTORC1 during mitotic arrest d Active mTORC1/S6K/PDCD4/eIF4A axis promotes survival during mitotic arrest d Inhibition of S6K or eIF4A accelerates cell death in response to mitotic poisons

mTOR is a serine/threonine kinase which acts a master regulator of cell growth and proliferation. Raptor, a scaffolding protein that recruits substrates to mTOR complex 1 (mTORC1), is known to be phosphorylated during mitosis, but the significance of this phosphorylation remains largely unknown. Here we show that raptor expression and mTORC1 activity are dramatically reduced in mitotic arrested cells across a variety of cancer and normal cell lines. Prevention of raptor phosphorylation during mitosis resulted in reactivation of mTORC1 in a rapamycin-sensitive manner. Importantly, expression of a non-phosphorylatable raptor mutant caused a dramatic reduction in cytotoxicity of the spindle poison Taxol. This effect was mediated via degradation of Programmed Cell Death Protein 4 (PDCD4), a tumor suppressor protein that inhibits eIF4A activity and is negatively regulated by the mTORC1/S6K pathway. Moreover, pharmacological inhibition of eIF4A was able to enhance the effects of taxol and...

Cell Cycle, 2011

substrate-binding element of mTORC1. Raptor's ability to properly present substrates, such as the translational regulators 4E-BP and p70 S6 kinase, to the mTOR catalytic domain is essential for their mTOR-catalyzed phosphorylation. mTOR is a key protein kinase controlling cell cycle progression, cellular proliferation and growth, autophagy and angiogenesis. In addition, new experimental evidence begins to suggest that mTOR is also directly involved in the mitotic process. In this regard, we have previously described the spatiotemporal subcellular distribution of the Serine 2481 -autophosphorylated form of mTOR (P-mTOR Ser2481 ) during the G 1 /S-to-M-phase transition, both in cultured cancer cells and in cancer tissue specimens. We have shown that P-mTOR Ser2481 is associated near and between separating chromosomes, not only during early mitotic stages, but also to the midzone and to midbody at ana/telophase through cytokinesis. 4 Indeed, we have validated P-mTOR Ser2481 as a novel high-contrast mitosis marker in breast cancer core ©2 0 1 1 L a n d e s B i o s c i e n c e . D o n o t d i s t r i b u t e .

Frontiers in Cell and Developmental Biology

Current Biology, 2004

Peptide mass fingerprinting analysis using mass spectrometry and mass spectrometric sequencing revealed that p200 is novel and not in the databases of full-length human proteins. We named the 200 kDa protein rictor for rapamycin-insensitive companion of mTOR. Starting from a truncated cDNA that encodes part of rictor (accession # KIAA1999), we used EST mining and Massachusetts Institute of Technology Nine Cambridge Center RT-PCR to assemble a full-length open reading frame that predicts a protein of 1708 amino acids and 192 kDa. We could not identify any domains of known function in rictor and, compared to mTOR, raptor, and G␤L, the Memorial-Sloan-Kettering Cancer Center 1275 York Avenue protein is not well conserved among eukaryotes. Rictor shares regions of homology with several poorly charac-New York, New York 10021 terized proteins, including pianissimo from D. discoidieum [4], STE20p from S. pombe [5], and AVO3p from S. cerevisiae [6, 7]. Proteins of similar domain structure

This review article focuses on protein kinases regulating the onset and transition through mitosis. The essay begins by introducing the structural features of the protein kinase catalytic domain and emphasizing the mechanism of enzymatic activation of this class of proteins. Next follows a short historical perspective on cell division and a description of our current understanding of mitosis. In the central part of the review I examine the four major kinases that set the stage for mitosis, which consist of Cdk1, Polo-like 1, Nek2 and Aurora kinases. For each entry dealt with, I focus particularly on studies that have linked DNA damage response pathways to inhibition of kinase activity, and I evaluate the conclusions drawn. Finally, I examine protein kinases initially described in the context of different cell cycle transitions and only later proposed to be involved in the control of mitosis.

Journal of Biological Chemistry, 2010

The rapamycin-sensitive mTOR complex 1 (mTORC1) promotes protein synthesis, cell growth, and cell proliferation in response to growth factors and nutritional cues. To elucidate the poorly defined mechanisms underlying mTORC1 regulation, we have studied the phosphorylation of raptor, an mTOR-interacting partner. We have identified six raptor phosphorylation sites that lie in two centrally localized clusters (cluster 1, Ser(696)/Thr(706) and cluster 2, Ser(855)/Ser(859)/Ser(863)/Ser(877)) using tandem mass spectrometry and generated phosphospecific antibodies for each of these sites. Here we focus primarily although not exclusively on raptor Ser(863) phosphorylation. We report that insulin promotes mTORC1-associated phosphorylation of raptor Ser(863) via the canonical PI3K/TSC/Rheb pathway in a rapamycin-sensitive manner. mTORC1 activation by other stimuli (e.g. amino acids, epidermal growth factor/MAPK signaling, and cellular energy) also promote raptor Ser(863) phosphorylation. Rheb overexpression increases phosphorylation on raptor Ser(863) as well as on the five other identified sites (e.g. Ser(859), Ser(855), Ser(877), Ser(696), and Thr(706)). Strikingly, raptor Ser(863) phosphorylation is absolutely required for raptor Ser(859) and Ser(855) phosphorylation. These data suggest that mTORC1 activation leads to raptor multisite phosphorylation and that raptor Ser(863) phosphorylation functions as a master biochemical switch that modulates hierarchical raptor phosphorylation (e.g. on Ser(859) and Ser(855)). Importantly, mTORC1 containing phosphorylation site-defective raptor exhibits reduced in vitro kinase activity toward the substrate 4EBP1, with a multisite raptor 6A mutant more strongly defective that single-site raptor S863A. Taken together, these data suggest that complex raptor phosphorylation functions as a biochemical rheostat that modulates mTORC1 signaling in accordance with environmental cues.

Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 2009

Activation of cyclin-dependent kinase complexes (CDK) at key cell cycle transitions is dependent on their dephosphorylation by CDC25 dual-specificity phosphatases (CDC25A, B and C in human). The CDC25B phosphatase plays an essential role in controlling the activity of CDK1-cyclin B complexes at the entry into mitosis and together with polo-like kinase 1 (PLK1) in regulating the resumption of cell cycle progression after DNA damage-dependent checkpoint arrest in G2. In this study, we analysed the regulation of CDC25Bdependent mitosis entry by PLK1. We demonstrate that PLK1 activity is essential for the relocation of CDC25B from the cytoplasm to the nucleus. By gain and loss of function analyses, we show that PLK1 stimulates CDC25B-induced mitotic entry in both normal conditions and after DNA-damage induced G2/M arrest. Our results support a model in which the relocalisation of CDC25B to the nucleus at the G2-M transition by PLK1 regulates its mitotic inducing activity.

Cell Cycle, 2012

Cell, 2002

gras et al., 2001). By interfering with the function of mam-Nine Cambridge Center malian TOR, rapamycin inhibits progression through the Cambridge, Massachusetts 02142 G1 phase of the cell cycle in various cell types. Because 2 Molecular Biology Program of these antiproliferative effects, rapamycin is a clinically Memorial Sloan-Kettering Cancer Center valuable drug that is currently used to block immune rejec-New York, New York 10021 tion of transplanted organs (Saunders et al., 2001) and in trials for the treatment of cancer (Dudkin et al., 2001; Hidalgo and Rowinsky, 2000) and for the prevention of restenosis after angioplasty (Sousa et al., 2001). Summary Mammalian TOR, mTOR (also known as RAFT1 or FRAP), phosphorylates at least two regulators of protein mTOR/RAFT1/FRAP is the target of the immunosupsynthesis: S6K1 (formerly called p70 ribosomal S6 kipressive drug rapamycin and the central component of nase) and an inhibitor of translation initiation, the eIFa nutrient-and hormone-sensitive signaling pathway 4E binding protein 1 (4E-BP1) (Brunn et al., 1997; Burnett that regulates cell growth. We report that mTOR forms et al., 1998; Hara et al., 1997; Isotani et al., 1999). In a stoichiometric complex with raptor, an evolutionarily mammalian cells, amino acid deprivation leads to the conserved protein with at least two roles in the mTOR dephosphorylation of both S6K1 and 4E-BP1 and to pathway. Raptor has a positive role in nutrient-stimudecreased rates of protein synthesis, effects that are lated signaling to the downstream effector S6K1, rapidly reversed by the readdition of amino acids (Fox et maintenance of cell size, and mTOR protein expresal., 1998; Hara et al., 1998). Among amino acids, changes in sion. The association of raptor with mTOR also negaleucine levels alone are sufficient to regulate the phostively regulates the mTOR kinase activity. Conditions phorylation state and activity of both downstream comthat repress the pathway, such as nutrient deprivation ponents of the mTOR pathway (Hara et al., 1998; Lynch and mitochondrial uncoupling, stabilize the mTORet al., 2000). In addition to amino acid level in the media, raptor association and inhibit mTOR kinase activity. mitochondrial function (Xu et al., 2001), glycolysis (Den-We propose that raptor is a missing component of nis et al., 2001), and cell stress (Parrott and Templeton, the mTOR pathway that through its association with 1999) regulate S6K1, as do growth factors such as insu-mTOR regulates cell size in response to nutrient levels. lin (Lawrence and Brunn, 2001). Despite extensive efforts, how nutrients regulate the Introduction mTOR signaling pathway remains poorly understood. In particular, stimuli that activate (e.g., amino acids) or Increasing evidence indicates that in eukaryotes, cell inhibit (e.g., mitochondrial uncouplers) downstream efgrowth (mass accumulation) is finely regulated in refectors of mTOR, such as S6K1 and 4E-BP1, fail to sponse to environmental and developmental conditions change the in vitro kinase activity of mTOR (Dennis et and can be deranged in human diseases such as cancer al., 2001; Hara et al., 1997). This discrepancy has led us and diabetes (reviewed by Dixon and Fordham-Skelton, to hypothesize that in vivo mTOR exists as a complex 1998; Johnston and Gallant, 2002; Katso et al., 2001; with one or more proteins that are lost during isolation Kozma and Thomas, 2002; Schmelzle and Hall, 2000).