Multisite phosphorylation of Pin1-associated mitotic phosphoproteins revealed by monoclonal antibodies MPM-2 and CC-3

Albert, Alexandra L.; Lavoie, Sébastien B.; Vincent, Michel

doi:10.1186/1471-2121-5-22

Cited by 19 publications

(13 citation statements)

References 76 publications

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“…33 Contrary to CC-3, MPM-2 reacts exclusively with the carboxy-terminal moiety of the p54 nrb protein (Figure 2(d)). These findings confirm that CC-3 and MPM-2 epitopes are different, as previously shown for the RNA polymerase II CTD, 32 and suggest that p54 nrb is a mitotic phosphoprotein. Incidentally, Figure 2(b) shows a slight shift in the migration pattern of p54 nrb when samples were prepared from mitotic cells compared to interphase cells, further suggesting that p54 nrb is phosphorylated in vivo when cells are dividing (see also Figure 6(b)).…”

Section: Resultssupporting

confidence: 90%

“…Recently, several CC-3 mitotic antigens were affinity-purified from HeLa cells and submitted to mass spectrometry analysis for identification. 32 The multifunctional nuclear protein p54 nrb was identified as a protein retained on the CC-3 affinity column. However, as previously reported, 12 p54 nrb binds to the RNA polymerase II C-terminal domain (CTD), which is itself a CC-3 antigen.…”

Section: Resultsmentioning

confidence: 99%

“…31 In a recent work, we have identified the major mitotic antigens of mAb CC-3. 32 They included a few proteins involved in transcription and pre-mRNA processing and many of them also appeared to be novel or already described MPM-2 antigens. One of the CC-3 mitotic antigens was identified as p54 nrb .…”

Section: Introductionmentioning

confidence: 98%

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The Multifunctional Nuclear Protein p54nrb is Multiphosphorylated in Mitosis and Interacts with the Mitotic Regulator Pin1

Proteau

Blier

Albert

et al. 2005

Journal of Molecular Biology

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Section: Resultssupporting

confidence: 90%

Section: Resultsmentioning

confidence: 99%

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The Multifunctional Nuclear Protein p54nrb is Multiphosphorylated in Mitosis and Interacts with the Mitotic Regulator Pin1

Proteau

Blier

Albert

et al. 2005

Journal of Molecular Biology

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“…Pin1 is essential for mitotic progression (11-13) and is required for the DNA replication checkpoint (14). Pin1 substrates are a defined subset of phosphorylated proteins, including many MPM-2 antigens (8,13,(15)(16)(17)(18)(19)(20). Pin1-catalyzed prolyl isomerization regulates the conformation and function of phosphoproteins (15, 17) and also facilitates dephosphorylation (12,15,17).…”

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confidence: 99%

Critical Role of WW Domain Phosphorylation in Regulating Phosphoserine Binding Activity and Pin1 Function

Lu¹,

Zhou²,

Liou³

et al. 2002

Journal of Biological Chemistry

224

261

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Phosphoserine-binding modules help determine the specificity of signal transduction events. One such module, the group IV WW domain, plays an essential role in targeting the phosphorylation-specific prolyl isomerase Pin1 to its substrates. These modules require Ser/Thr phosphorylation of their ligands for binding activity. However, phosphorylation of these modules and its functional significance have not been described, nor is it known whether the function of Pin1 is regulated. Here Compelling evidence indicates that phosphorylation on Ser or Thr residues (pSer/Thr) also promotes the formation of proteinprotein (1-3). Small pSer/Thr-binding protein modules reminiscent of SH2 1 domains involved in recognition of phosphorylated Tyr have been recently described, which include group IV WW domains and FHA domains (2-7). However, the role of phosphorylation of these modules themselves has not been described. The best characterized pSer/Thr-binding WW domain is one in the peptidyl-prolyl isomerase (PPIase) Pin1 (4, 7). Pin1 specifically isomerizes the pSer/Thr-Pro bond (8 -10). Pin1 is essential for mitotic progression (11-13) and is required for the DNA replication checkpoint (14). Pin1 substrates are a defined subset of phosphorylated proteins, including many MPM-2 antigens (8,13,(15)(16)(17)(18)(19)(20). Pin1-catalyzed prolyl isomerization regulates the conformation and function of phosphoproteins (15, 17) and also facilitates dephosphorylation (12,15,17). Thus, Pin1-dependent peptide bond isomerization is a critical post-phosphorylation regulatory mechanism (12). However, it is likely that Pin1 is regulated by a post-translational modification.The first and essential step in Pin1-dependent regulation of targets is the substrate binding activity mediated by its WW domain (10 MATERIALS AND METHODS Interaction of Pin1 Mutants and MPM-2 Antigens orPhosphopeptides-GST fusion proteins were expressed and purified, and their ability to bind mitotic phosphoproteins was assayed as described (8,15). Peptide binding was measured as described (4).Localization Analysis of GFP-Pin1 and Its Mutant Proteins-Pin1 or its mutants were subcloned into pEGFP-C1 or pERFP-N1 (CLON-TECH), respectively. HeLa cells were transfected with pGFP-or pRFPPin1 or its mutants using Superfect (Qiagen). After 6 -24 h, cells were fixed and examined under a fluorescence microscope (Nikon).In Vivo and in Vitro Phosphorylation of Pin1-To detect Pin1 phosphorylation in vivo, HeLa cells were labeled with [ 32 P]orthophosphate, and Pin1 was immunoprecipitated with anti-Pin1 or anti-HA antibodies, followed by SDS-PAGE and autoradiography (4). 32 P-labeled Pin1 proteins were extracted and digested with trypsin followed by twodimensional chromatography (23). In vitro phosphorylation of Pin1 and its mutants were carried out as described (4). To detect the mobility shift of Pin1 during the cell cycle, HeLa cells were arrested at the G 1 /S boundary and released to enter the cell cycle as described (15). Cell lysates were subjected to low bis-SDS-PAGE, followed ...

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“…For instance, PP2A-mediated dephosphorylation of the Pin1-interacting proteins, Raf-1, Cdc25c, Pim-1, Myc, and Tau, critically relies on Pin1 (15)(16)(17)(18). In line with this, juglone prevents the dephosphorylation of MPM2 antigens (19), which constitute a subset of Pin1-interacting mitotic phosphoproteins, as well as of NHERF-1 (20) and Disabled-2 (21). For that matter, it is thought that cis-trans isomerization of p(S/T)-P bonds by Pin1 regulates dephosphorylation of PP2A targets by facilitating the accessibility of this phosphatase to its substrates (see Ref.…”

mentioning

confidence: 63%

Juglone Inactivates Cysteine-rich Proteins Required for Progression through Mitosis

Fila

Metz

Sluijs

2008

Journal of Biological Chemistry

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The parvulin peptidyl-prolyl isomerase Pin1 catalyzes cistrans isomerization of p(S/T)-P bonds and might alter conformation and function of client proteins. Since the trans conformation of p(S/T)-P bonds is preferred by protein phosphatase 2A (PP2A), Pin1 may facilitate PP2A-mediated dephosphorylation. Juglone irreversibly inhibits parvulins and is often used to study the function of Pin1 in vivo. The drug prevents dephosphorylation of mitotic phosphoproteins, perhaps because they bind Pin1 and are dephosphorylated by PP2A. We show here however that juglone inhibited post-mitotic dephosphorylation and the exit of mitosis, independent of Pin1. This effect involved covalent modification of sulfhydryl groups in proteins essential for metaphase/anaphase transition. Particularly cytoplasmic proteins with a high cysteine content were vulnerable to the drug. Alkylation of sulfhydryl groups altered the conformation of such proteins, as evidenced by the disappearance of antibody epitopes on tubulin and the mitotic checkpoint component BubR1. The latter activates the anaphase-promoting complex/ cyclosome, which degrades regulatory proteins, such as cyclin B1 and securins, and is required for mitotic exit. Indeed, juglone-treated cells failed to assemble a mitotic spindle, which correlated with perturbed microtubule dynamics, loss of immunodetectable tubulin, and formation of tubulin aggregates. Juglone also prevented degradation of cyclin B1, independently of the Mps1-controlled mitotic spindle checkpoint. Since juglone affected cell cycle progression at several levels, more specific drugs need to be developed for studies of Pin1 function in vivo. Peptidyl-prolyl isomerases (PPIases)2 accelerate the cis-trans conversion of peptide bonds preceding prolyl residues, which can cause alterations in protein conformation (e.g. see Refs. 1 and 2). PPIases have been grouped into cyclophilin, FK506-binding protein, and parvulin subfamilies (see Ref. 3), for which distinct pharmacological inhibitors are available. The parvulin group is irreversibly inhibited by juglone (4). Pin1 comprises an N-terminal type IV WW domain, which determines phosphorylation-specific protein-protein interactions, and a C-terminal PPIase domain that harbors the catalytic center (see Ref. 5). Pin1 is a unique PPIase, because it preferably binds to side chain-phosphorylated S/T-P moieties in numerous proteins, including crucial cell cycle regulators or proteins that become phosphorylated immediately prior to cell division (6, 7). Isomerization of the p(S/T)-P peptide bond regulates, for instance, localization and phosphorylation status of Pin1 client proteins (see Ref. 5). Pin1 is therefore a regulator that, in concert with proline-directed kinases, phosphatases, and ubiquitin ligases, controls the cell cycle (see Refs. 5 and 8). Pin1 is possibly a cancer target gene, because its overexpression enhances transformed phenotypes induced by oncogenic Ras and Neu (see (5)). Pin1 is overexpressed in many human cancers, and its overexpression correlates with poor...

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Multisite phosphorylation of Pin1-associated mitotic phosphoproteins revealed by monoclonal antibodies MPM-2 and CC-3

Cited by 19 publications

References 76 publications

The Multifunctional Nuclear Protein p54nrb is Multiphosphorylated in Mitosis and Interacts with the Mitotic Regulator Pin1

The Multifunctional Nuclear Protein p54nrb is Multiphosphorylated in Mitosis and Interacts with the Mitotic Regulator Pin1

Critical Role of WW Domain Phosphorylation in Regulating Phosphoserine Binding Activity and Pin1 Function

Juglone Inactivates Cysteine-rich Proteins Required for Progression through Mitosis

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