Cell Cycle Regulated Phosphorylation of the Telomere-Associated Protein TIN2

<div><p>The protein TIN2 is a member of telomere-binding protein complex that serves to cap and protect mammalian chromosome ends. As a number of proteins in this complex are phosphorylated in a cell cycle-dependent manner, we investigated whether TIN2 is modified by phosphorylation as well. We performed phospho-proteomic analysis of human TIN2, and identified two phosphorylated residues, serines 295 and 330. We demonstrated that both these sites were phosphorylated during mitosis in human cells, as detected by Phos-tag reagent and phosphorylation-specific antibodies. Phosphorylation of serines 295 and 330 appeared to be mediated, at least in part, by the mitotic kinase RSK2. Specifically, phosphorylation of TIN2 at both these residues was increased upon expression of RSK2 and reduced by an inhibitor of the RSK family of kinases. Moreover, RSK2 phosphorylated TIN2 <i>in vitro</i>. The identification of these specifically timed post-translational events during the cell cycle suggests a potential mitotic regulation of TIN2 by phosphorylation.</p></div

TIN2 is phosphorylated on S295 and S330 during mitosis.

<p>(<b>A</b>) Detection of S295 and S330 phosphorylation of TIN2 during mitosis by the Phos-tag reagent after release from a double thymidine block. HeLa cells stably infected with a retrovirus encoding Flag-TIN2 in the WT, S330A, or S295A configuration were collected from asynchronous populations (A), populations arrested with a double thymidine block corresponding to the G1/S phase of the cell cycle, or populations at the points corresponding to S, G2, M and early or middle G1 (EG1 or MG1) after release from the double thymidine block. Derived lysates were then either subjected to (<i>top</i>) immunoprecipitation (IP) with an anti-Flag antibody and resolved by SDS-PAGE in the presence of the Phos-tag reagent and immunoblotted (IB) with an anti-TIN2 antibody or (<i>bottom</i>) resolved by normal SDS-PAGE and immunoblotted with either an anti-Phos-HH3 antibody to monitor cell cycle progression or an anti-Tubulin antibody as a loading control. The supershifted bands corresponding to S295, S330, or S295 and S330 phosphorylation, as well as the unphosphorylated TIN2 (UP) are denoted on the left of the upper panels. Left and right panels are different exposures. Representative of two experiments. (<b>B</b>) Detection of S295 and S330 phosphorylation of TIN2 by the Phos-tag reagent in cells arrested with nocodazole. HeLa cells stably infected with a retrovirus encoding no transgene (vector, V) or Flag-TIN2 in the WT, S330A, S295A, or AA configuration were collected from asynchronous populations (Asyn) or populations arrested in G2/M by treatment with nocodazole (Noc). Derived lysates were then subjected to either (<i>top</i>) immunoprecipitation (IP) with αFlag and resolved by SDS-PAGE in the presence of the Phos-tag reagent and immunoblotted (IB) with an anti-TIN2 antibody or (<i>bottom</i>) resolved by normal SDS-PAGE and immunoblotted with either an anti-Phos-HH3 antibody to monitor cell cycle progression or an anti-Tubulin antibody as a loading control. The supershifted bands corresponding to S295, S330, or S295 and S330 phosphorylation, as well as the unphosphorylated TIN2 (UP), are denoted on the left of the upper panel. Representative of three experiments. (<b>C</b>) Detection of S295 phosphorylation of endogenous TIN2 with a phosphorylation-specific antibody in cells arrested with nocodazole. Lysates from HeLa cells were collected from asynchronous populations (Asyn) or populations arrested in G2/M by treatment with nocodazole (Noc), resolved by SDS-PAGE and immunoblotted (IB) with an anti-Phos-S295, anti-TIN2, anti-Phos-HH3, or anti-HH3 (loading control) antibody. Representative of two experiments. (<b>D</b>) Detection of S330 phosphorylation of endogenous TIN2 with a phosphorylation-specific antibody in cells arrested with nocodazole. HeLa cells were collected from asynchronous populations (Asyn) or populations arrested in G2/M by treatment with nocodazole (Noc). Derived lysates were then either subjected to (<i>top</i>) immunoprecipitation (IP) with an anti-TIN2 antibody, resolved by SDS-PAGE in the presence of the Phos-tag reagent, and immunoblotted (IB) with either an anti-Phos-S330 or anti-TIN2 antibody, or (<i>bottom</i>) resolved by normal SDS-PAGE and immunoblotted with an anti-Phos-HH3 antibody, to monitor cell cycle progression, or an anti-Tubulin antibody as a loading control. Representative of one experiment.</p

Reduced HRAS^G12V-Driven Tumorigenesis of Cell Lines Expressing KRAS^C118S

<div><p>In many different human cancers, one of the <i>HRAS</i>, <i>NRAS</i>, or <i>KRAS</i> genes in the RAS family of small GTPases acquires an oncogenic mutation that renders the encoded protein constitutively GTP-bound and thereby active, which is well established to promote tumorigenesis. In addition to oncogenic mutations, accumulating evidence suggests that the wild-type isoforms may also be activated and contribute to oncogenic RAS-driven tumorigenesis. In this regard, redox-dependent reactions with cysteine 118 (C118) have been found to promote activation of wild-type HRAS and NRAS. We sought to determine if this residue is also important for the activation of wild-type KRAS and promotion of tumorigenesis. Thus, we mutated C118 to serine (C118S) in wild-type KRAS to block redox-dependent reactions at this site. We now report that this mutation reduced the level of GTP-bound KRAS and impaired RAS signaling stimulated by the growth factor EGF. With regards to tumorigenesis, we also report that oncogenic HRAS-transformed human cells in which endogenous KRAS was knocked down and replaced with KRAS^C118S exhibited reduced xenograft tumor growth, as did oncogenic HRAS-transformed <i>Kras^C118S/C118S</i> murine cells in which the C118S mutation was knocked into the endogenous <i>Kras</i> gene. Taken together, these data suggest a role for redox-dependent activation of wild-type KRAS through C118 in oncogenic HRAS-driven tumorigenesis.</p></div

TIN2 is phosphorylated by the mitotic kinase RSK2.

<p>(<b>A</b>) Detection of S330 phosphorylation of TIN2 with a phosphorylation-specific antibody in cells arrested with nocodazole and treated with kinase inhibitors. HeLa cells stably expressing wild-type Flag-TIN2 were treated with DMSO, H-89, BI-D1870, BI 2536 or VX-680 in the presence of either nocodazole (Noc) or vehicle (DMSO). Derived lysates were immunoprecipitated (IP) with an anti-Flag antibody, resolved by SDS-PAGE, and immunoblotted (IB) with an anti-Phos-S330 antibody or, as a loading control, an anti-TIN2 antibody. Representative of two experiments. (<b>B</b>) DNA profiles of HeLa cells treated with BI-D1870. HeLa cells treated with DMSO, nocodazole (Noc), or nocodazole+ BI-D1870 were harvested, stained with propidium iodide, and subjected to fluorescence-activated cell sorting (FACS) analysis. Representative of two experiments. (<b>C</b>) Detection of S295 and S330 phosphorylation of TIN2 by the Phos-tag reagent in asynchronous or nocodazole arrested cells with or without the RSK2 inhibitor BI-D1870. 293T cells were either untreated or treated with nocodazole (Noc), BI-D1870, or both compounds. Derived lysates were then subjected to immunoprecipitation (IP) with an anti-Flag antibody and resolved by SDS-PAGE in the presence of the Phos-tag reagent and immunoblotted (IB) with an anti-TIN2 antibody. The supershifted bands corresponding to S295, S330, or S295 and S330 phosphorylation, as well as the unphosphorylated TIN2 (UP), are denoted on the left. Representative of two experiments. (<b>D</b>) Detection of S295 and S330 phosphorylation of TIN2 by the Phos-tag reagent in asynchronous cells with ectopic RSK2 and/or the RSK2 inhibitor BI-D1870. 293T cells transiently transfected with Flag-TIN2 and the Y707A constitutively active mutant form of RSK2 (Flag-RSK2^Y707A) were either left untreated or treated with RSK kinase inhibitor BI-D1870. Derived lysates were split into two portions. The first portions were subjected to immunoprecipitation (IP) with an anti-Flag antibody, resolved by SDS-PAGE in the presence of the Phos-tag reagent, and immunoblotted (IB) with an anti-TIN2 antibody. The supershifted bands corresponding to S295, S330, or S295 and S330 phosphorylation, as well as the unphosphorylated TIN2 (UP), are denoted on the left (<i>top</i>). The second portions were resolved by normal SDS-PAGE and immunoblotted with either an anti-Phospho-S6 antibody to monitor RSK2 kinase activity, or an anti-Tubulin antibody as a loading control (<i>bottom</i>). Representative of two experiments. (<b>E</b>) Detection of TIN2 phosphorylation by RSK2 <i>in vitro</i>. Recombinant maltose-binding protein (MBP) or N-terminal MBP-tagged TIN2 (MBP-TIN2) in the WT, S295A, S330A, or AA mutant configuration were captured with amylose resin and eluted with maltose. No protein (-) or equal amounts of the aforementioned purified MBP-TIN2 proteins were incubated with recombinant N-terminal 6His-tagged RSK2 (6His-RSK2) in the presence of ATP³², after which the reaction products were resolved by SDS-PAGE and either (<i>top</i>) exposed to autographic film or (<i>bottom</i>) stained with Coomassie Brilliant Blue (CBB staining). Phosphorylated (P³²) MBP-TIN2 and a non-specific band (*) are denoted on the left top panel. MBP-TIN2 and MBP are denoted on the left bottom panel. Representative of two experiments.</p

Introducing a C118S mutation into wild-type KRAS impairs HRAS^G12V-driven tumor growth.

<p>(<b>A</b>) RT-PCR amplification of <i>Flag-KRAS</i>, <i>Flag-HRAS</i>, <i>p110CAAX</i> and <i>GAPDH</i> mRNA isolated from oncogenic Flag-HRAS^G12V-transformed HEK-TtH cells infected with retroviruses encoding p110-CAAX and either scramble (scram) control shRNA or <i>KRAS</i> shRNA in the absence (-) or presence of shRNA-resistant wild-type (WT) or C118S mutant Flag-tagged KRAS. One of two replicate experiments. (<b>B</b>) Immunoblot detection of endogenous KRAS, Flag-tagged HRAS^G12V and tubulin in oncogenic Flag-HRAS^G12V-transformed HEK-TtH cells infected with retroviruses encoding p110-CAAX and either KRAS shRNA or a scramble control (scram) shRNA. One of three replicate experiments. (<b>C</b>) Mean ± SEM tumor volume over time, (<b>D</b>) photographs of excised tumors at end point and (<b>E</b>) mean ± SEM tumor weight at end point of tumors developing in immunocompromised mice (n = 5) injected with oncogenic Flag-HRAS^G12V-transformed HEK-TtH cells infected with retroviruses encoding p110-CAAX and either scramble control shRNA (pink circles, scram) or <i>KRAS</i> shRNA without (orange squares, <i>KRASi</i>) or in conjunction with shRNA-resistant and Flag-tagged wild-type (light green triangles, <i>KRASi</i>+KRAS) or C118S-mutant (dark green reverse triangles, <i>KRASi</i>+KRAS^C118S) <i>KRAS</i>. ns: non-significant, *: <i>P</i><0.05, **: <i>P</i><0.01 and ***: <i>P</i><0.001, as determined by one-way ANOVA plus post-hoc Bonferroni’s multiple comparison test using GraphPad Prism 5 Software. Full-length immunoblots and gels are shown in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0123918#pone.0123918.s001" target="_blank">S1A and S1B</a> Fig</b>.</p

Flag-Snm1B co-immunoprecipitates with Mus81 and Myc-PSF2.

<p>The indicated wild type (WT) or mutant (Δ222–363 or 1–494) Flag epitope-tagged Snm1B (Flag-Snm1B) and HA epitope-tagged Mus81 (HA-Mus81) proteins transiently expressed in 293T cells in the absence or presence of Myc epitope-tagged PSF2 (Myc-PSF2) were immunoprecipitated (IP) with anti-Flag antibody and immunoblotted (IB) with an anti-Flag antibody to visualize Flag-Snm1B (WT, Δ222–363, or 1–494), anti-Myc antibody to visualize Myc-PSF2, or an anti-Mus81 antibody to visualize Mus81. Bottom: fold increase in Mus81 co-immunoprecipitating with Flag-Snm1B. Data are representative of at least two independent experiments.</p

Introducing a C118S mutation into the endogenous wild-type <i>Kras</i> gene impairs HRAS^G12V-driven tumor growth.

<p>(<b>A</b>) PCR amplification of genomic DNA yielding a 621 bp or 517bp fragment indicative of the wild-type or C118S <i>Kras</i> alleles in three SV40-immortalized <i>Kras</i>^<i>+/+</i> or <i>Kras</i>^{<i>C118S/C118S</i>} MEF cell lines, respectively. (<b>B</b>) Immunoblot detection of endogenous Kras, HRAS and tubulin in the indicated SV40-immortalized <i>Kras</i>^<i>+/+</i> or <i>Kras</i>^{<i>C118S/C118S</i>} MEF cell lines transformed with HRAS^G12V. Relative Kras or HRAS levels normalized to tubulin are shown beneath the immunoblot. (<b>C</b>) Representative images and (<b>D</b>) the mean ± SEM number of colonies growing in soft agar by SV40-immortalized <i>Kras</i>^<i>+/+</i> versus <i>Kras</i>^{<i>C118S/C118S</i>} MEF cell lines stably infected with a retrovirus encoding no transgene (vector) or HRAS^G12V, seeded in triplicate. (<b>E</b>) Mean ± SEM tumor volume over time, (<b>F</b>) photographs of excised tumors at end point and (<b>G</b>) mean ± SEM tumor weight at end point of tumors developing in immunocompromised mice (n = 5) injected with SV40-immortalized <i>Kras</i>^<i>+/+</i> (pink boxes) versus <i>Kras</i>^{<i>C118S/C118S</i>} (orange squares) MEF cell lines transformed with HRAS^G12V. (<b>H</b>) Mean ± SEM tumor volume over time of tumors developing in immunocompromised mice (n = 5) injected with SV40-immortalized and HRAS^G12V-transformed <i>Kras</i>^<i>+/+</i> MEFs (pink circles), <i>Kras</i>^{<i>C118S/C118S</i>} MEFs (orange squares) or <i>Kras</i>^{<i>C118S/C118S</i>} MEFs stably infected with a retrovirus encoding KRAS* (light green triangles) or KRAS*^C118S (dark green reverse triangles). (<b>I</b>) Kaplan-Meier survival curves based on the time to reach end point of immunocompromised mice (n = 5) each injected with one of the three SV40-immortalized <i>Kras</i>^<i>+/+</i> (pink line) versus <i>Kras</i>^{<i>C118S/C118S</i>} (orange line) MEF cell lines transformed with HRAS^G12V. ns: non-significant, **: <i>P</i><0.01, ***: <i>P</i><0.001 and ****: <i>P</i><0.0001, as determined by one-way ANOVA plus post-hoc Bonferroni’s multiple comparison test (D, H), two-tailed unpaired <i>t</i> test (E, G) or long-rank test (I) using GraphPad Prism 5 Software. Full-length immunoblots and gels are shown in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0123918#pone.0123918.s001" target="_blank">S1F and S1G</a> Fig.</b></p

Introducing a G12V activating mutation overcomes the inability of KRAS^C118S to promote HRAS^G12V-driven tumor growth.

<p>(<b>A</b>) RT-PCR amplification of <i>Flag-KRAS</i>, <i>Flag-HRAS</i>, <i>p110CAAX</i> and <i>GAPDH</i> mRNA (note that this is the same gel as in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0123918#pone.0123918.g001" target="_blank">Fig 1A</a></b>, except with the addition of analysis for Flag-KRAS^G12V,C118S, one of two replicate experiments), (<b>B</b>) mean ± SEM tumor volume over time, (<b>C</b>) photographs of excised tumors at end point and (<b>D</b>) mean ± SEM tumor weight at end point of tumors developing in immunocompromised mice (n = 5) injected with Flag-HRAS^G12V-transformed HEK-TtH cells infected with retroviruses encoding p110CAAX and <i>KRAS</i> shRNA alone (pink circles) or with wild-type (WT, orange squares), C118S-mutant (light green triangles) or G12V,C118S-mutant (dark green reverse triangles) Flag-tagged and shRNA-resistant KRAS. ns: non-significant, *: <i>P</i><0.05, **: <i>P</i><0.01 and ***: <i>P</i><0.001, as determined by one-way ANOVA plus post-hoc Bonferroni’s multiple comparison test using GraphPad Prism 5 Software. The full-length gel is shown in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0123918#pone.0123918.s001" target="_blank">S1A Fig</a>.</b></p

Introducing a C118S mutation into wild-type KRAS impairs EGF stimulation of AKT and ERK1/2 phosphorylation.

<p>Immunoblot detection of input and GTP-bound Flag-KRAS captured by the Ras-binding domain of Raf1, as well as HA-eNOS and tubulin, in (<b>A</b>) HEK-TtH cells or (<b>B</b>) SV40-immortalized MEFs stably infected with retroviruses encoding either wild-type (WT) or C118S-mutant Flag-tagged KRAS* in conjunction with either the HA-tagged S1177D constitutively-active or the S1177A inactive mutant versions of eNOS. High molecular weigh bands were variably detected using this assay, and because they are above the size of Ras, they were considered to be non-specific. Relative mean ± SEM of GTP-bound KRAS (normalized to KRAS input) is shown beneath the immunoblot. One of three replicate experiments. (<b>C</b>) Immunoblot detection of total (T) and phosphorylated (P) AKT and ERK1/2 in HEK-TtH cells stably infected with retroviruses encoding a scramble control (scram) shRNA, a <i>KRAS</i> shRNA, or <i>KRAS</i> shRNA and wild-type (WT) or C118S-mutant Flag-tagged shRNA-resistant KRAS and serum starved overnight and treated without (-) or with (+) EGF for five minutes. Relative mean ± SEM of P-AKT (normalized to T-AKT) or P-ERK1/2 (normalized to T-ERK) is shown beneath the immunoblot. One of three replicate experiments. Full-length immunoblots are shown in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0123918#pone.0123918.s001" target="_blank">S1C–S1E Fig</a>.</b></p

TIN2 is phosphorylated on Serine 295 and Serine 330.

<p>(<b>A</b>) Identification of phosphorylation sites on TIN2 by mass spectrometry. <i>Left,</i> A lysate from HeLa cells stably infected with a retrovirus encoding N-terminal Flag epitope-tagged TIN2 (Flag-TIN2) was subjected to immunoprecipitation (IP) with an anti-Flag antibody, resolved by SDS-PAGE, and detected by Coomassie Brilliant Blue staining. M: marker lane. <i>Right,</i> The purified protein was then recovered and digested by trypsin, followed by TiO2 enrichment and mass spectrometry analysis, revealing two peptides with phosphorylated serine residues (denoted with an *). Representative of one experiment. (<b>B</b>) Detection of phosphorylation of TIN2 at S295 and S330 by the Phos-tag reagent. Lysates from HeLa cells stably infected with a retrovirus encoding Flag-TIN2 in the wild-type (WT), S330A, or S295A configuration were subjected to immunoprecipitation (IP) with an anti-Flag antibody and then either left untreated or treated with calf intestine phosphatase (CIP), followed by SDS-PAGE either in the presence (<i>top</i>) or the absence (<i>bottom</i>) of the Phos-tag reagent and immunoblotted (IB) with an anti-TIN2 antibody. The supershifted bands corresponding to S295, S330, or S295 and S330 phosphorylation, as well as the unphosphorylated TIN2 (UP), are denoted on the left. Representative of three experiments. (<b>C</b>) Detection of S295 and S330 phosphorylation of TIN2 with phosphorylation-specific antibodies. Lysates from HeLa cells stably infected with a retrovirus encoding C-terminal HA epitope-tagged TIN2 (TIN2-HA) or Flag-TIN2 in WT, S295A, or S330A configuration were subjected to immunoprecipitation (IP) with either an anti-HA or anti-Flag antibody, resolved by SDS-PAGE, and immunoblotted (IB) with either an anti-Phos-S295 or anti-Phos-S330 antibody to detected the phosphorylated TIN2, and either an anti-HA or anti-Flag antibody to detect total ectopic TIN2 as a loading control. Representative of two experiments.</p