Identification of Phosphoproteins and Their Phosphorylation Sites in the WEHI-231 B Lymphoma Cell Line

Shu, Hongjun; Chen, She; Bi, Qun; Mumby, Marc C.; Brekken, Deirdre L.

doi:10.1074/mcp.d300003-mcp200

Cited by 81 publications

(91 citation statements)

References 14 publications

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“…Treatment-The use of IMAC for phosphopeptide enrichment is well documented (5,31). However, there is only one published report on the use of IMAC application for the enrichment of intact phosphoproteins (32).…”

Section: Identification Of Proteins Phosphorylated At Tyr Residues (Amentioning

confidence: 99%

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Quantification of Gel-separated Proteins and Their Phosphorylation Sites by LC-MS Using Unlabeled Internal Standards

Cutillas

Geering

Waterfield

et al. 2005

Molecular & Cellular Proteomics

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Protein phosphorylation plays a critical role in normal cellular function and is often subverted in disease. Although major advances have recently been made in identification and quantitation of protein phosphorylation sites by MS, current methodological limitations still preclude routine, easily usable, and comprehensive quantitative analysis of protein phosphorylation. Here we report a simple LC-MS method to quantify gel-separated proteins and their sites of phosphorylation; in this approach, integrated chromatographic peak areas of peptide analytes from proteins under study are normalized to those of a nonisotopically labeled internal standard protein spiked into the excised gel samples just prior to in-gel digestion. The internal standard intensities correct for differences in enzymatic activities and sample losses that may occur during the processes of in-gel digestion and peptide extraction from the gel pieces. We used this method of peak area measurement with an internal standard to investigate the effects of pervanadate on protein phosphorylation in the WEHI-231 B cell lymphoma cell line and to assess the role of phosphoinositide 3-kinase (PI3K) in these phosphorylation events. Phosphoproteins, isolated from total cell lysates using IMAC or by immunoprecipitation using Tyr(P) antibodies, were analyzed using this method, leading to identification of >400 proteins, several of which were found at higher levels in phosphoprotein fractions after pervanadate treatment. Pretreatment of cells with the PI3K inhibitor wortmannin reduced the phosphorylation level of certain proteins (e.g. STAT1 and phospholipase C␥2) while increasing the phosphorylation of several others. Peak area measurement with an internal standard was also used to follow the dynamics of PI3K-dependent and -independent changes in the post-translational modification of both known and novel phospho- Reversible covalent modification of proteins is a key molecular mechanism by which membrane receptor activation is converted into intracellular signaling cascades that mediate physiological change. Engagement of ligands with their receptors results in rapid and transient phosphorylation of downstream protein targets, which as a consequence modify their catalytic properties, cellular localization, and/or binding partners. As a consequence, intracellular signaling pathways can control critical aspects of metabolism, cell cycle progression, migration, differentiation, and protein synthesis. Many diseases are a direct consequence of deregulation in these processes (1, 2); these changes may be detectable by quantitative phosphoprotein analysis.The assessment of the phosphorylation status of specific proteins is commonly performed by immunochemical methods using antisera raised against a peptide containing the site of phosphorylation of the target protein. This approach is restricted to the study of known phosphorylation sites and cannot be used to monitor and discover novel phosphorylated proteins and their sites of phosphorylation. To overcome this proble...

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Section: Identification Of Proteins Phosphorylated At Tyr Residues (Amentioning

confidence: 99%

“…MS can be used for large scale protein identification (3, 4) and determination of sites of phosphorylation (5)(6)(7). Most of these studies, however, cannot be used for quantitative analysis.…”

mentioning

confidence: 99%

Quantification of Gel-separated Proteins and Their Phosphorylation Sites by LC-MS Using Unlabeled Internal Standards

Cutillas

Geering

Waterfield

et al. 2005

Molecular & Cellular Proteomics

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“…The purification of six distinct emerin-containing ternary complexes from HeLa cell nuclei (Holaska and Wilson, 2007) suggests that its interactions are differentially regulated. Indeed, emerin can be phosphorylated on Ser or Thr residues, including S49, S66, T67, S120, S163 and S175 (Brill et al, 2004;Hirano et al, 2005;Roberts et al, 2006;Shu et al, 2004). Mitotic phosphorylation of emerin S175 inhibits binding to BAF (Hirano et al, 2005), implicating regions outside the LEM domain in this interaction.…”

Section: Introductionmentioning

confidence: 99%

Tyrosine phosphorylation of nuclear-membrane protein emerin by Src, Abl and other kinases

Tifft

Bradbury

Wilson

2009

Journal of Cell Science

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X-linked recessive Emery-Dreifuss muscular dystrophy (EDMD) is caused by loss of emerin, a nuclear-membrane protein with roles in nuclear architecture, gene regulation and signaling. Phosphoproteomic studies have identified 13 sites of tyrosine phosphorylation in emerin. We validated one study, confirming that emerin is hyper-tyrosine-phosphorylated in Her2-overexpressing cells. We discovered that non-receptor tyrosine kinases Src and Abl each phosphorylate emerin and a related protein, LAP2, directly. Src phosphorylated emerin specifically at Y59, Y74 and Y95; the corresponding triple Yto-F ('FFF') mutation reduced tyrosine phosphorylation bỹ 70% in vitro and in vivo. Substitutions that removed a single hydroxyl moiety either decreased (Y19F, Y34, Y161F) or increased (Y4F) emerin binding to BAF in cells. Y19F, Y34F, Y161F and the FFF mutant also reduced recombinant emerin binding to BAF from HeLa lysates, demonstrating the involvement of both LEM-domain and distal phosphorylatable tyrosines in binding BAF. We conclude that emerin function is regulated by multiple tyrosine kinases, including Her2, Src and Abl, two of which (Her2, Src) regulate striated muscle. These findings suggest roles for emerin as a downstream effector and 'signal integrator' for tyrosine kinase signaling pathway(s) at the nuclear envelope.Key words: Emerin, LEM domain, Nuclear envelope, Emery-Dreifuss muscular dystrophy, Src, Her2, Abl, Barrier-to-autointegration factor (BAF), Cardiomyopathy, Breast cancer, Laminopathy, Neuromuscular junction Journal of Cell Science 3781 Emerin tyrosine phosphorylation receptor tyrosine kinases that can be activated by Her2 or other signaling pathways (Roskoski, 2005;Srinivasan and Plattner, 2006). We confirm Her2-stimulated emerin tyrosine phosphorylation and report that emerin and LAP2 are phosphorylated directly by Src and Abl. We also show that tyrosine phosphorylation of emerin regulates binding to BAF. ResultsWe compiled data from 13 independent proteomic studies (Amanchy et al., 2005;Brill et al., 2004;Cantin et al., 2008; Daub et al., 2008; Guo et al., 2008;Olsen et al., 2006;Pan et al., 2008;Rikova et al., 2007;Rush et al., 2005;Schlosser et al., 2006;Sui et al., 2008;Tao et al., 2005;Tsai et al., 2008), which identified 13 phosphorylated tyrosines in emerin (Fig. 1A). All sites except Y19 and Y41 were identified more than once. All 13 identified phosphorylated Tyr (Tyr-P) residues in human emerin are conserved in mouse, and six (corresponding to Y4, Y41, Y94, Y95, Y105, Y161) are conserved in Xenopus (Fig. 1B). These studies did not reveal responsible pathways or kinases.We validated tyrosine phosphorylation of emerin by immunoprecipitating endogenous emerin from HeLa cells treated with 1 M pervanadate (PV, a tyrosine phosphatase inhibitor) (Huyer et al., 1997) or buffer (PBS) as control, for 30 minutes before lysing cells. Emerin immunoprecipitated from PV-treated cells was specifically recognized by Tyr-P antibodies (not shown; see Fig. 2B). No other tyrosine-phosphorylated bands were dete...

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confidence: 99%

“…Alternatively, the simpler and more sensitive methods of phosphopeptide enrichment by strong cation exchange chromatography (25,26) or immobilized metal ion chromatography (IMAC) (27, 28) have been used successfully for phosphoproteomics. The IMAC method is based on the affinity of di-or trivalent metal ions for phosphate groups, and despite some limitations, including nonspecific binding of peptides (29,30) and the fact that not all phosphopeptides are retained equally efficiently on IMAC resins (26,28,31), this method has shown great promise for large-scale studies (29,32,33).Recently, we have purified mitotic spindles from human HeLa S3 cells and identified a total of 795 proteins, including 151 previously known spindle-associated components (34). In this former study, we have also characterized the purity of the spindle preparations, using differential interference contrast microscopy, Western blot analyses with antibodies against known spindle and control proteins, tandem mass spectrometry (MS͞ MS), and cell biological validation experiments.…”

mentioning

confidence: 99%

Phosphoproteome analysis of the human mitotic spindle

Nousiainen

Silljé

Sauer

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

300

260

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During cell division, the mitotic spindle segregates the sister chromatids into two nascent cells, such that each daughter cell inherits one complete set of chromosomes. Errors in spindle formation can result in both chromosome missegregation and cytokinesis defects and hence lead to genomic instability. To ensure the correct function of the spindle, the activity and localization of spindle associated proteins has to be tightly regulated in time and space. Reversible phosphorylation has been shown to be one of the key regulatory mechanisms for the organization of the mitotic spindle. The relatively low number of identified in vivo phosphorylation sites of spindle components, however, has hampered functional analysis of regulatory spindle networks. A more complete inventory of the phosphorylation sites of spindle-associated proteins would therefore constitute an important advance. Here, we describe the mass spectrometry-based identification of in vivo phosphorylation sites from purified human mitotic spindles. In total, 736 phosphorylation sites were identified, of which 312 could be attributed to known spindle proteins. Among these are phosphorylation sites that were previously shown to be important for the regulation of spindle-associated proteins. Importantly, this data set also comprises 279 novel phosphorylation sites of known spindle proteins for future functional studies. This inventory of spindle phosphorylation sites should thus make an important contribution to a better understanding of the molecular mechanisms that regulate the formation, function, and integrity of the mitotic spindle. mass spectrometry ͉ mitosis ͉ molecular cell biology ͉ phosphorylation ͉ proteomics A t the transition from interphase to mitosis, the microtubule network undergoes a profound change that culminates in the formation of the spindle apparatus. The mitotic spindle then serves to segregate the chromosomes to opposite poles of the cell and to define the plane of cell division. A large number of proteins associate with this microtubule-based structure and regulate its dynamic formation and function (1, 2). Several spindle proteins, including XKCM1 and XMAP215 family members (3, 4), have been identified that either stabilize or destabilize microtubules by mediating the rapid changes between polymerization and depolymerization (5, 6). These proteins play an important role in spindle formation, as illustrated by the striking change in microtubule half-life from 5-10 min in interphase to less than 1 min in mitosis (7), which results in the short and unstable microtubules characteristic of mitosis (5, 6). Another important group of spindle proteins comprises motors of the kinesin and dynein families that are essential for mitotic progression (5,6,8,9). They push the spindle poles away from each other during early mitosis and play crucial roles in capturing chromosomes and positioning them at the metaphase plate. Subsequently, motors also contribute to central spindle formation and cytokinesis. In addition, numerous structural prote...

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Identification of Phosphoproteins and Their Phosphorylation Sites in the WEHI-231 B Lymphoma Cell Line

Cited by 81 publications

References 14 publications

Quantification of Gel-separated Proteins and Their Phosphorylation Sites by LC-MS Using Unlabeled Internal Standards

Quantification of Gel-separated Proteins and Their Phosphorylation Sites by LC-MS Using Unlabeled Internal Standards

Tyrosine phosphorylation of nuclear-membrane protein emerin by Src, Abl and other kinases

Phosphoproteome analysis of the human mitotic spindle

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