Monoclonal antibody CC-3 recognizes phosphoproteins in interphase and mitotic cells

Thibodeau, Alain; Vincent, Michel

doi:10.1016/0014-4827(91)90510-2

Cited by 27 publications

(28 citation statements)

References 44 publications

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“…2B). Comparative western blots using two other mitotic phosphorylation-specific antibodies, MPM2 (Davis et al, 1983) and CC-3 (Thibodeau and Vincent, 1991), showed that P190 reacts with a distinct subset of mitotic phosphoproteins (Fig. 2B).…”

Section: Resultsmentioning

confidence: 99%

Identification of a novel mitotic phosphorylation motif associated with protein localization to the mitotic apparatus

Yang

Camp

Gritsenko

et al. 2007

Journal of Cell Science

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The chromosomal passenger complex (CPC) is a crucial regulator of chromosome, cytoskeleton and membrane dynamics during mitosis. Here, using liquid chromatography coupled to mass spectrometry (LC-MS), we identified phosphopeptides and phosphoprotein complexes recognized by a phosphorylation-specific antibody that labels the CPC. A mitotic phosphorylation motif {PX[G/T/S][L/M]S(P) P or WGLS(P) P} was identified by MS in 11 proteins, including FZR1 (Cdh1) and RIC8A–two proteins with potential links to the CPC. Phosphoprotein complexes contained the known CPC components INCENP, Aurora-B (Aurkb) and TD-60 (Rcc2, RCC1-like), as well as SMAD2, 14-3-3 proteins, PP2A and Cdk1 (Cdc2a), a probable kinase for this motif. Protein sequence analysis identified phosphorylation motifs in additional proteins, including SMAD2, PLK3 and INCENP. Mitotic SMAD2 and PLK3 phosphorylation was confirmed using phosphorylation-specific antibodies, and, in the case of Plk3, phosphorylation correlated with its localization to the mitotic apparatus and the midbody. A mutagenesis approach was used to show that INCENP phosphorylation is required for its localization to the midbody. These results provide evidence for a shared phosphorylation event that regulates localization of crucial proteins during mitosis.

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Section: Resultsmentioning

confidence: 99%

Identification of a novel mitotic phosphorylation motif associated with protein localization to the mitotic apparatus

Yang

Camp

Gritsenko

et al. 2007

Journal of Cell Science

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“…Antibodies Used-The mAb CC3 (IgG 2a ) was previously obtained by immunizing Balb/C mice with chick embryo proteins (29) and shown to recognize a phosphoepitope located on the carboxyl-terminal domain (CTD) of the hyperphosphorylated RNA polymerase II largest subunit (pol IIo) (23,30). The mAb Pol3/3 recognizes a conserved region located outside of the CTD of the largest subunit of the RNA polymerase II (RNA pol II LS) and was kindly provided by E. K. F. Bautz (31).…”

Section: Methodsmentioning

confidence: 99%

Direct Interaction of the KRAB/Cys2-His2Zinc Finger Protein ZNF74 with a Hyperphosphorylated Form of the RNA Polymerase II Largest Subunit

Grondin

Côté

Bazinet

et al. 1997

Journal of Biological Chemistry

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We previously identified ZNF74 as a developmentally expressed gene commonly deleted in DiGeorge syndrome. ZNF74 encodes an RNA-binding protein tightly associated with the nuclear matrix and belongs to a large subfamily of Cys 2 -His 2 zinc finger proteins containing a KRAB (Kruppel-associated box) repressor motif. We now report on the multifunctionality of the zinc finger domain of ZNF74. This nucleic acid binding domain is shown here to function as a nuclear matrix targeting sequence and to be involved in protein-protein interaction. By far-Western analysis and coimmunoprecipitation studies, we demonstrate that ZNF74 interacts, via its zinc finger domain, with the hyperphosphorylated largest subunit of RNA polymerase II (pol IIo) but not with the hypophosphorylated form. The importance of the phosphorylation in this interaction is supported by the observation that phosphatase treatment inhibits ZNF74 binding. Double immunofluorescence experiments indicate that ZNF74 colocalizes with the pol IIo and the SC35 splicing factor in irregularly shaped subnuclear domains. Thus, ZNF74 sublocalization in nuclear domains enriched in pre-mRNA maturating factors, its RNA binding activity, and its direct phosphodependent interaction with the pol IIo, a form of the RNA polymerase functionally associated with premRNA processing, suggest a role for this member of the KRAB multifinger protein family in RNA processing.Zinc finger proteins of the TFIIIA/Kruppel type belong to the largest known family of transcription factors (1, 2). These proteins are characterized by Cys 2 -His 2 zinc finger motifs often repeated in tandem that fold around zinc ions and function as nucleic acid binding domains (3-6). About one-third of mammalian Cys 2 -His 2 zinc finger proteins contain a conserved domain of approximately 75 amino acids called KRAB (Kruppelassociated box) (7). The KRAB domain, located at the N terminus of Cys 2 -His 2 multifinger proteins, can confer strong distance-independent transcriptional repression of both activated and basal RNA polymerase II promoter activity (8 -14). Since they encode a repression motif and a potential DNA binding domain, members from the large KRAB/Cys 2 -His 2 protein family are presently thought to function as transcriptional regulators of gene expression.We previously cloned ZNF74, a gene that encodes a KRAB/ Cys 2 -His 2 protein (15). ZNF74 lies a few kilobases proximal to a polymorphic CA repeat (D22S264) (16) that was shown to be a distal marker for 22q11.2 deletions associated with increased susceptibility to schizophrenia (17). ZNF74 is also one of the few genes found hemizygously deleted in the majority of patients with the DiGeorge syndrome, a microdeletion disorder associated with a wide variety of congenital malformations including cardiac defects, thymic hypoplasia, and hypocalcemia (18 -20). To date, however, both the role of embryologically expressed ZNF74 in the DiGeorge syndrome (19) and its biochemical and cellular functions remain unclear.Although the presence of a KRAB motif and of 1...

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“…H5 and H14 are IgMs directed against phosphoepitopes within the CTD (39 -41) and were obtained from Covance. CC3 is an IgG isolated in a screen for chicken proteins with developmentally regulated expression (42). B3 is an IgM directed against nuclear matrix components (43).…”

Section: Materials-[␥-mentioning

confidence: 99%

TFIIF-associating Carboxyl-terminal Domain Phosphatase Dephosphorylates Phosphoserines 2 and 5 of RNA Polymerase II

Lin

Dubois

Dahmus

2002

Journal of Biological Chemistry

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The carboxyl-terminal domain (CTD) of the largest RNA polymerase (RNAP) II subunit undergoes reversible phosphorylation throughout the transcription cycle. The unphosphorylated form of RNAP II is referred to as IIA, whereas the hyperphosphorylated form is known as IIO. Phosphorylation occurs predominantly at serine 2 and serine 5 within the CTD heptapeptide repeat and has functional implications for RNAP II with respect to initiation, elongation, and transcription-coupled RNA processing. In an effort to determine the role of the major CTD phosphatase (FCP1) in regulating events in transcription that appear to be influenced by serine 2 and serine 5 phosphorylation, the specificity of FCP1 was examined. FCP1 is capable of dephosphorylating heterogeneous RNAP IIO populations of HeLa nuclear extracts. The extent of dephosphorylation at specific positions was assessed by immunoreactivity with monoclonal antibodies specific for phosphoserine 2 or phosphoserine 5. As an alternative method to assess FCP1 specificity, RNAP IIO isozymes were prepared in vitro by the phosphorylation of purified calf thymus RNAP IIA with specific CTD kinases and used as substrates for FCP1. FCP1 dephosphorylates serine 2 and serine 5 with comparable efficiency. Accordingly, the specificity of FCP1 is sufficiently broad to dephosphorylate RNAP IIO at any point in the transcription cycle irrespective of the site of serine phosphorylation within the consensus repeat. Reversible phosphorylation of the carboxyl-terminal domain (CTD)1 of the largest RNA polymerase (RNAP) II subunit plays an important role in the regulation of gene expression. The CTD of mammalian RNAP II is comprised of 52 repeats of the consensus sequence 1 YSPTSPS 7 (for a review, see Ref. 1). RNAP IIA, which contains an unmodified CTD, is actively recruited to the promoter as part of the preinitiation complex (2-5), whereas RNAP IIO, which contains a hyperphosphorylated CTD, is responsible for transcript elongation (6, 7). Therefore, protein kinases and phosphatases that alter the state of CTD phosphorylation can serve as transcriptional activators or repressors depending on the point in the transcription cycle at which they function.CTD phosphorylation occurs predominantly at serines 2 and 5 within the heptapeptide repeat. Genetic evidence indicates that the roles of serines in positions 2 and 5 are different. First, the partial substitution of serines in either position 2 or 5 have different effects on viability (8). Second, SRB (suppressors of RNA Polymerase IIB) mutations suppress the lethal effect of position 2 substitutions but not position 5 substitutions (9). Biochemical evidence has also confirmed differences between the two predominant serine positions. Serine 5 but not serine 2 phosphorylation recruits and activates the 5Ј-capping machinery (10, 11). Furthermore, nutritional stress and heat shock can independently alter the pattern of CTD phosphorylation, indicating that phosphoserine 2 and phosphoserine 5 are functionally different (12)(13)(14).A recent study using...

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Monoclonal antibody CC-3 recognizes phosphoproteins in interphase and mitotic cells

Cited by 27 publications

References 44 publications

Identification of a novel mitotic phosphorylation motif associated with protein localization to the mitotic apparatus

Identification of a novel mitotic phosphorylation motif associated with protein localization to the mitotic apparatus

Direct Interaction of the KRAB/Cys2-His2Zinc Finger Protein ZNF74 with a Hyperphosphorylated Form of the RNA Polymerase II Largest Subunit

TFIIF-associating Carboxyl-terminal Domain Phosphatase Dephosphorylates Phosphoserines 2 and 5 of RNA Polymerase II

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