M Phase Phosphoprotein 10 Is a Human U3 Small Nucleolar Ribonucleoprotein Component

We have previously developed a novel technique for isolation of cDNAs encoding M phase phosphoproteins (MPPs). In the work described herein, we further characterize MPP10, one of 10 novel proteins that we identified, with regard to its potential nucleolar function. We show that by cell fractionation, almost all MPP10 was found in isolated nucleoli. By immunofluorescence, MPP10 colocalized with nucleolar fibrillarin and other known nucleolar proteins in interphase cells but was not detected in the coiled bodies stained for either fibrillarin or p80 coilin, a protein found only in the coiled body. When nucleoli were separated into fibrillar and granular domains by treatment with actinomycin D, almost all the MPP10 was found in the fibrillar caps, which contain proteins involved in rRNA processing. In early to middle M phase of the cell cycle, MPP10 colocalized with fibrillarin to chromosome surfaces. At telophase, MPP10 was found in cellular structures that resembled nucleolus-derived bodies and prenucleolar bodies. Some of these bodies lacked fibrillarin, a previously described component of nucleolus-derived bodies and prenucleolar bodies, however, and the bulk of MPP10 arrived at the nucleolus later than fibrillarin. To further examine the properties of MPP10, we immunoprecipitated it from cell sonicates. The resulting precipitates contained U3 small nucleolar RNA (snoRNA) but no significant amounts of other box C/D snoRNAs. This association of MPP10 with U3 snoRNA was stable to 400 mM salt and suggested that MPP10 is a component of the human U3 small nucleolar ribonucleoprotein.     

M phase phosphoprotein 1 is a human plus-end-directed kinesin-related protein required for cytokinesis

The human M phase phosphoprotein 1 (MPP1), previously identified through a screening of a subset of proteins specifically phosphorylated at the G2/M transition (Matsumoto-Taniura, N., Pirollet, F., Monroe, R., Gerace, L., and Westendorf, J. M. (1996) Mol. Biol. Cell 7, 1455-1469), is characterized as a plus-end-directed kinesin-related protein. Recombinant MPP1 exhibits in vitro microtubule-binding and microtubule-bundling properties as well as microtubule-stimulated ATPase activity. In gliding experiments using polarity-marked microtubules, MPP1 is a slow molecular motor that moves toward the microtubule plus-end at a 0.07 microm/s speed. In cycling cells, MPP1 localizes mainly to the nuclei in interphase. During mitosis, MPP1 is diffuse throughout the cytoplasm in metaphase and subsequently localizes to the midzone to further concentrate on the midbody. MPP1 suppression by RNA interference induces failure of cell division late in cytokinesis. We conclude that MPP1 is a new mitotic molecular motor required for completion of cytokinesis.     

Anti-phosphopeptide antibody, P-STM as a novel tool for detecting mitotic phosphoproteins: identification of lamins A and C as two major targets

A polyclonal, phospho-epitope-specific antibody (P-STM) was generated to detect the activated p21-activated kinase 2 (PAK2), based on the regulatory autophosphorylation site Thr(402) of PAK2 [Yu et al., 1998]. In this report, we show that this antibody can also recognize many phosphoproteins in mitotic HeLa and A431 cells. Signal of these phosphoproteins emerged after treating the cells with nocodazole and okadaic acid, and was highly detected in G2-M phase transition of HeLa cells released from double thymidine block. Immunofluorescence analysis revealed that P-STM strongly stained HeLa cells at prometaphase and metaphase, but not at interphase and anaphase. Interestingly, this staining pattern was almost identical to that obtained by staining with MPM2, a monoclonal antibody known to react with phosphoproteins in mitotic HeLa cells. However, the phosphoproteins detected by the two antibodies are quite different. Two-dimensional gel electrophoresis (2DE) and tryptic peptide fingerprint analysis by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry were employed to identify lamins A and C as two of the mitotic cell-specific phosphoproteins recognized by P-STM. Lamins A and C immunoprecipitated from nocodazole-treated cells, but not from untreated cells showed strong reactivity to P-STM, and this reactivity lost completely after protein phosphatase 2A treatment. In summary, our results show that P-STM represents a novel tool for detecting mitotic phosphoproteins, which are different from those recognized by MPM2, and that lamins A and C are the two prominent mitotic phosphoproteins detected by P-STM.     

Immunoelectron microscopic localization of phosphoproteins associated with the mitotic spindle.

We examined the immunogold staining of microtubules and microtubule organizing centers using an improved silver-enhancement reagent for small (1-1.4 nm) gold-conjugated secondary antibodies. First, the staining properties of different commercial preparations of gold-labeled antibodies were compared for sample penetration, label uniformity, and labeling density, and Nanogold 1.4-nm gold-conjugated F(ab') was found to be superior to the other probes examined. However, in samples examined for the localization of alpha- and beta-tubulin, gold staining did not extend through the pericentriolar material nor were the centrioles labeled. This apparent lack of centrosomal staining was not due to problems associated with penetration of the antibody probes, since staining adjacent to and within the centriolar cylinder was observed when phosphoprotein antigens recognized by the MPM-2 antibody were localized. The MPM-2 antibodies also localized to mitotic kinetochores, kinetochore fibers, and midbodies, in addition to mitotic centrosomes. The level of MPM-2 staining of the centrosome varied through the cell cycle. At interphase, this staining was restricted within the centriolar cylinder, whereas in mitotic cells extensive staining throughout the pericentriolar material was also observed. These results established the close relationship of MPM-2-reactive phosphoproteins with the centrosome, and suggest that this technique may be useful for ultrastructural localization of other cytoskeletal proteins.     

Distribution of cytoskeletal proteins sharing a conserved phosphorylated epitope

A group of antigens related by their reactivity with monoclonal antibodies MPM-1 and MPM-2 appear as cells enter mitosis. These antibodies bind to a phosphorylated epitope on certain proteins, and therefore the antigens are presumed to be a group of phosphoproteins. A subset of these proteins has been shown previously to be components of mitotic microtubule organizing centers in PtK1 cells. We present here evidence that the mitosis-specific appearance of these phosphoproteins is a phenomenon common to all eukaryotic cells. The MPM reactive phosphoproteins were localized to mitotic spindle poles regardless of whether the spindle formed in the cytoplasm after nuclear envelope breakdown (open mitosis) or within the nucleus (closed mitosis). This reactivity was not dependent upon the presence of centrioles at the spindle poles. Proteins that contained the phosphorylated epitope were not, however, restricted to mitotic cells. Cells of neuronal derivation and flagellated cells showed specific localization of MPM antibody to the microtubule network and basal bodies respectively. On immunoblots, the MPM antibody reacted with brain MAP-1 among a number of other phosphoproteins. The identification of microtubule-associated protein (MAP)-1 correlates with the localization of the antibody to microtubules of neuroblastoma cells. These results suggest, that different phosphoprotein molecules detected by the MPM antibody may be specific for different mitotic microtubule organizing centers, basal bodies, and other specialized cytoskeletal structures; and the presence of a related phosphorylated domain on these proteins may be important for their proper function and/or interaction with microtubules.     

Simultaneous immunoelectron microscopic visualization of protein B23 and C23 distribution in the HeLa cell nucleolus

The intranucleolar distribution of phosphoproteins B23 and C23 was visualized simultaneously by post-embedding immunoelectron microscopy in HeLa cell nucleoli, using specific antibodies. The data show that proteins B23 and C23 co-localize to the same nucleolar compartments, i.e., the dense fibrillar component and the granular component. Neither of the two antibodies is significantly associated with the fibrillar centers in these cells, although the fibrillar centers appear positive after silver staining. These findings suggest that other unidentified components must be responsible for the silver staining observed in the fibrillar centers of interphase nucleoli. The results are discussed in the light of previously reported data obtained by preembedding immunolabeling techniques and by silver staining, which both suggested a localization of protein C23 inside the fibrillar centers.     

Regulation of a major microtubule-associated protein by MPF and MAP kinase.

The interphase-M phase transition of microtubule dynamics is thought to be induced by phosphorylation reactions mediated by MPF and by MAP kinase functioning downstream of MPF. We have now identified and purified from Xenopus eggs a major microtubule-associated protein, p220, that may be a target protein for these two M phase-activated kinases. p220, when purified from interphase cells, potently bound to microtubules and stimulated tubulin polymerization, whereas p220 purified from M phase cells showed little or no such activities. Cell staining with a monoclonal anti-p220 antibody revealed that p220 is localized on cytoplasmic microtubule networks during interphase, while it is distributed rather diffusely throughout the cell during M phase. We have further found that p220 is phosphorylated specifically in M phase. Moreover, p220 purified from interphase cells served as a good substrate for MAP kinase and MPF in vitro, and two-dimensional phosphopeptide mapping pattern of the p220 phosphorylated in vitro was very similar to that of p220 phosphorylated at M phase in vivo. These results suggest that the drastic change in p220 activity during the transition from interphase to M phase may be induced by its phosphorylation in M phase probably catalyzed by MAP kinase and MPF.     

Increased cell cycle-dependent staining of plant cells by the antibody MPM-2 correlates with preprophase band formation

Cytoplasmic microtubules of animal cells catastrophically depolymerize upon entry into mitosis but in higher plants there is a longer transition during which cortical microtubules form an increasingly narrow preprophase band, and the chromatin gradually condenses. Progression towards mitosis in onion root tip cells was analysed using a CCD camera and image processing to quantify fluorescence staining by the monoclonal antibody MPM-2, which recognizes mitotic phosphoproteins in a range of eukaryotic cells. MPM-2 fluorescence, which was predominantly nuclear, was categorized relative to the stage of the DNA cycle (using DAPI), and to the microtubule cycle (using anti-tubulin) in individual cells. Cells with the characteristic interphase cortical microtubule arrays had a bimodal distribution of DAPI fluorescence, indicating that some were in G1 (2C DNA) whilst the double value suggested the others to be in G2 (4C). There was no difference in MPM-2 fluorescence between 2C and 4C cells possessing the cortical array in which microtubules were evenly distributed. However, in 4C cells possessing a preprophase band MPM-2 values doubled; this relationship applied not only to tight PPBs but to early, broad PPBs in which the individual microtubules could still be distinguished. Since alkaline phosphatase abolished MPM-2 reactivity it is concluded that mitotic phosphoproteins do not necessarily begin to accumulate in G2 per se , but during that part of G2 when the preprophase band first becomes recognizable as a distinct entity.     

Mitogen-induced tyrosine-phosphorylated 41- and 43-kDa proteins are family members of extracellular signal-regulated kinases/microtubule-associated protein 2 kinases.

Two antipeptide antibodies, one against the peptide corresponding to residues 307-327 (alpha Y91) and one against the peptide corresponding to the C-terminal portion (alpha C92) of the deduced amino acid sequence of the extracellular signal-regulated kinase 1 (ERK1), precipitated two 41-kDa and/or two 43-kDa phospho-proteins from mitogen-stimulated Swiss 3T3 cells. Electrophoretic mobilities on two-dimensional gels of the immunoprecipitated 41- and 43-kDa phosphoproteins were similar to those of the 41- and 43-kDa cytosol proteins, whose increased tyrosine phosphorylation we and others had originally identified in various mitogen-stimulated cells (Cooper, J. A., Sefton, B. M., and Hunter, T. (1984) Mol. Cell. Biol. 4, 30-37; Kohno, M. (1985) J. Biol. Chem. 260, 1771-1779); phosphopeptide map analysis revealed that they were respectively identical molecules. All those phosphoproteins contained phosphotyrosine, and the more acidic forms contained additional phosphothreonine. Immunoprecipitated 41- and 43-kDa phosphoproteins had serine/threonine kinase activity toward myelin basic protein (MBP) and microtuble-associated protein 2 (MAP2). With the combination of two-dimensional gel electrophoresis and the kinase assay in MBP-containing polyacrylamide gels of the alpha Y91 immunoprecipitates, with or without phosphatase 2A treatment, we showed that only their acidic forms were active. These results clearly indicate that 41- and 43-kDa proteins, the increased tyrosine phosphorylation of which is rapidly and commonly induced by mitogen stimulation of fibroblasts, are family members of ERKs/MAP2 kinases and that phosphorylation both on tyrosine and threonine residues is necessary for their activation.     

Changes in distribution of nuclear matrix antigens during the mitotic cell cycle

We examined the distribution of nonlamin nuclear matrix antigens during the mitotic cell cycle in mouse 3T3 fibroblasts. Four monoclonal antibodies produced against isolated nuclear matrices were used to characterize antigens by the immunoblotting of isolated nuclear matrix preparations, and were used to localize the antigens by indirect immunofluorescence. For comparison, lamins and histones were localized using human autoimmune antibodies. At interphase, the monoclonal antibodies recognized non-nucleolar and nonheterochromatin nuclear components. Antibody P1 stained the nuclear periphery homogeneously, with some small invaginations toward the interior of the nucleus. Antibody I1 detected an antigen distributed as fine granules throughout the nuclear interior. Monoclonals PI1 and PI2 stained both the nuclear periphery and interior, with some characteristic differences. During mitosis, P1 and I1 were chromosome-associated, whereas PI1 and PI2 dispersed in the cytoplasm. Antibody P1 heavily stained the periphery of the chromosome mass, and we suggest that the antigen may play a role in maintaining interphase and mitotic chromosome order. With antibody I1, bright granules were distributed along the chromosomes and there was also some diffuse internal staining. The antigen to I1 may be involved in chromatin/chromosome higher-order organization throughout the cell cycle. Antibodies PI1 and PI2 were redistributed independently during prophase, and dispersed into the cytoplasm during prometaphase. Antibody PI2 also detected antigen associated with the spindle poles.     

Structural evidence for a polymorphic or allelic form of the heavy chain variable region.

The heavy (H) chains of anti-phosphocholine (PC) antibodies from C57BL/6J and CBA/J were sequenced through the N-terminal 36 residues and compared with previously published sequences of A/J anti-PC antibody and BALB/c PC-binding myeloma proteins T15, M603, and M511. Each of these antibody preparations contained molecules having light (L) chains and idiotypic determinants of T15, M511, and M603 indicating the presence of at least three different anti-PC antibodies in each pool. The structures of the C57BL/6J and CBA/J H chains each revealed a single sequence from positions 1 to 36 (which includes the first complementarity determining region (CDR), and they were identical. The first CDR was identical to that previously found for BALB/c and A/J indicating that this portion of these antibody molecules is highly conserved throughout inbred mice and is probably critical to PC-binding. A surprising finding was that both C57NL/6 and CBA sequence differed from the BALB/c and A/J sequences at two positions, residue 14 and 16. Since each of these strains differs at the allotype locus, the data indicates that the evolution of allotypy in mice occurred after variable region diversity for the particular genes.     

Monoclonal antibodies that recognize lacto-N-fucopentaose III (CD15) react with the adhesion-promoting glycoprotein family (LFA-1/HMac-1/gp 150,95) and CR1 on human neutrophils

A variety of monoclonal antibodies has been used to study the roles of surface proteins in neutrophil function. Many monoclonal antibodies that bind to human neutrophils react with the oligosaccharide lacto-N-fucopentaose III. Sequential immunoprecipitation of radiolabeled proteins from extracts of neutrophils labeled at the cell surface with 125I, and partial proteolysis peptide mapping studies were used to compare the proteins recognized by several widely used monoclonal antibodies that react with human neutrophils. The monoclonal antibodies that react with lacto-N-fucopentaose III (CD15) immunoprecipitated five distinct neutrophil surface proteins. The data indicate that CD15 monoclonal antibodies react with a subset of the LFA-1/HMac-1/gp 150,95 glycoprotein family as well as with CR1 on human neutrophils. The CD15 antibodies studied differed in their avidities for these proteins. The molecules immunoprecipitated by the CD15 antibodies tested were more resistant to proteolysis than the homologous proteins immunoprecipitated by the other monoclonal antibodies studied that react directly with the alpha M (CD11) or beta (CD18) chains of the LFA-1/HMac-1/gp 150,95 glycoprotein family. Some of the differences in antibody reactivity and protease sensitivity of the membrane proteins recognized by these antibodies may be due to differences in glycosylation. The data suggest that the antibodies studied can detect differences in post-translational modification among copies of certain surface proteins.     

Monoclonal antibodies specific for tight-binding human chromatin antigens reveal structural rearrangements within the nucleus during the cell cycle

The class of nonhistone chromosomal proteins that remains bound to DNA in chromatin in the presence of 2.5 M NaCl-5 M urea has proven refractile to biochemical analysis. In order to study its role in chromatin organization, we have produced monoclonal antibodies that are specific for the HeLa DNA-protein complex that remains after extraction of chromatin with high salt and urea. The antibody-producing clones were identified with an ELISA assay. Of the six clones selected, five were stabilized by limiting dilution. All clones are IgG producers. None cross-react significantly with native DNA, core histones, or the high-mobility group nonhistone proteins. All antibodies are specific for nuclear or juxtanuclear antigens. Indirect immunofluorescence shows that three antibodies, which are nonidentical, stain three different nuclear networks. Available evidence indicates that two of these networks are the nuclear matrix. A fourth antibody reveals structures reminiscent of chromocenters. A fifth antibody, AhNA-1, binds to interphase HeLa chromatin and specifically decorates metaphase chromosomes. AhNA-1 similarly recognizes rat chromosomes. Each of these monoclonal antibodies also reveals a changing pattern of nuclear staining as cells progress through the cell cycle. Presumably, this reflects the rearrangement of the cognate antigens.     

Multiple antibodies to titin immunoreact with AHNAK and localize to the mitotic spindle machinery.

Recently, the large filamentous striated-muscle protein titin has been observed in non-muscle cells, and, in one instance, has been proposed to have a nuclear function as a chromosomal component contributing to structure and elasticity. In this study, we sought to further characterize the presumptive nuclear isoform of titin. Immunofluorescence microscopy with multiple titin-specific monoclonal antibodies shows localization to the nucleus in interphase cells and to the spindle machinery in mitotic cells in all cell types examined; localization to condensed chromosomes is not observed. An abundant 700-kDa phosphoprotein is the predominant species immunoprecipitated with these antibodies. Sequencing of peptide fragments of the immunopurified protein reveals identity to AHNAK, a nuclear phosphoprotein, an identification that was confirmed by Western blot analysis with antibodies to AHNAK and peptide fragmentation patterns. Sequence comparison suggests similarities between the repetitive heptad phi+/-phiP+/-phi+/- motif in AHNAK and the PEVK region of titin, potentially explaining the cross-reactivity observed between AHNAK antibodies and titin antibodies. Interestingly, although some AHNAK antibodies stain interphase nuclei, no evidence of mitotic spindle localization is seen, suggesting that the identity of the protein at the latter location is more closely related to titin than AHNAK. This concept is further supported by observations that cell lines not expressing AHNAK have similar antititin antibody localization to the mitotic spindle. We conclude that (1) multiple titin antibodies, particularly those recognizing the PEVK region, cross-react with AHNAK, and (2) the mitotic spindle staining observed with antititin antibodies is most likely due to the association of titin or a titin-like molecule with this structure.     

A transforming ras gene can provide an essential function ordinarily supplied by an endogenous ras gene.

Microinjection of monoclonal antibody Y13-259, which reacts with all known mammalian and yeast ras-encoded proteins, has previously been shown to prevent NIH 3T3 cells from entering the S phase (L. S. Mulcahy, M. R. Smith, and D. W. Stacey, Nature [London] 313:241-243, 1985). We have now found several transformation-competent mutant v-rasH genes whose protein products in transformed NIH 3T3 cells are not immunoprecipitated by this monoclonal antibody. These mutant proteins are, however, precipitated by a different anti-ras antibody. Each of these mutants lacks Met-72 of v-rasH. In contrast to the result for cells transformed by wild-type v-rasH, Y13-259 microinjection of NIH 3T3 cells transformed by these mutant ras genes did not prevent the cells from entering the S phase. These results imply that a transformation-competent ras gene can supply a normal essential function for NIH 3T3 cells. When the proteins encoded by the mutant ras genes were overproduced in Escherichia coli, several mutant proteins that lacked Met-72 failed to bind Y13-259 in a Western blot. However, a ras protein from a mutant lacking amino antibody, but a ras protein from a mutant lacking amino acids 72 to 84 did not. These results suggest that Y13-259 may bind to a higher ordered structure that has been restored in the mutant lacking amino acids 72 to 82.     

Nuclear and mitotically enhanced epitope.

Salt-extracted proteins of taxol-stabilized microtubules from Chinese hamster ovary cells arrested at mitosis were used to immunize mice for hybridoma production. From a group of related monoclonal antibodies (MAbs), one, C9, recognized an epitope on antigens localized by immunofluorescence microscopy to interphase centrosomes and nuclei. The availability of the nuclear antigen was cell cycle-dependent; however, permeabilization of cells before fixation revealed that the antigen was present throughout the cell cycle. The nuclear antigen was exposed during prophase and was released from the nucleus upon nuclear envelope breakdown filling the cytoplasm of the mitotic cell. Antigenic material re-accumulated at daughter nuclei and was concealed during G1 phase. Detergent extraction of the cytoplasmic antigen from mitotic cells enabled localization of antigens to centrosomes, kinetochores, and the furrowing region/midbody. Immunoblot analysis of cells of a variety of species of origin identified an approximate 250 kD polypeptide as corresponding to the nuclear antigen, whereas polypeptides of 107/117 kD as well as approximately 250 kD accounted for the mitotic cytoplasmic antigens. No polypeptides could be associated with antigens at centrosomes, kinetochores, or midbodies. This MAb joins the antibody preparations previously reported that describe nuclear antigens, or epitopes on antigens, enhanced at mitosis.     

Production of a monospecific antiserum against the early region 1A proteins of adenovirus 12 and adenovirus 5 by an adenovirus 12 early region 1A-beta-galactosidase fusion protein antigen expressed in bacteria.

Antisera were prepared against the amino acid sequences encoded within the N-terminal half of the adenovirus 12 (Ad12) early region 1A (E1A) gene. This was accomplished by construction of a plasmid vector which encoded the N-terminal 131 amino acids of Ad12 E1A joined in frame to the coding sequence of beta-galactosidase. After induced synthesis in Escherichia coli, the Ad12 E1A-beta-galactosidase fusion protein (12-1A-FP) was extracted with urea and used to raise antibodies in rabbits. The 12-1A-FP antisera immunoprecipitated major phosphoproteins of 39,000 and 37,000 apparent molecular weights from Ad12-transformed and infected cells. The 12-1A-FP antisera also immunoprecipitated E1A phosphoproteins from Ad5-transformed and infected cells. Immunospecificity of the 12-1A-FP antisera was demonstrated by the ability of 12-1A-FP antigen to block immunoprecipitation of E1A proteins. Furthermore, E1A proteins immunoprecipitated from in vivo-labeled cells comigrated with those translated in vitro by RNA that had been hybridization selected to E1A DNA.     

Antibodies define multiple proteins with epitopes exposed on the surface of live Babesia bigemina merozoites

Babesia bigemina is one of several tick-borne hemoparasitic diseases of cattle that are inadequately controlled and cause substantial livestock production losses in tropical and subtropical climates. Recovery from acute babesiosis is associated with development of protective immunity against subsequent challenge with both homologous and heterologous parasites. Viable and infectious merozoites, the intraerythrocytic stage of B. bigemina responsible for clinical disease, were separated from contaminating host cells by density gradient centrifugation. Monoclonal antibodies developed against gradient-separated merozoites were screened for surface reactivity against live merozoites in an immunofluorescent binding assay. Surface-reactive antibodies immunoprecipitated five major biosynthetically radiolabeled merozoite proteins with relative m.w. of 72,000, 58,000, 55,000, 45,000, and 36,000 in SDS-PAGE. Two additional proteins immunoprecipitated with the 45,000 m.w. protein were unreactive with monoclonal antibody in western blots and are apparently part of a membrane complex co-precipitated by this antibody. In contrast, additional proteins of m.w. of 36,000, 35,000, and 33,000, immunoprecipitated with the 58,000 protein, all contain the surface-exposed epitope bound by monoclonal antibody. Immune serum from an animal that had recovered from infection with a Mexico isolate of B. bigemina immunoprecipitated five radiolabeled proteins from the Mexico isolate that co-migrated in SDS-PAGE with major proteins precipitated by surface-reactive monoclonal antibodies. In addition, antibodies against a Kenya isolate of B. bigemina immunoprecipitated the same co-migrating proteins from radio-labeled Mexico isolate, demonstrating epitope conservation between surface proteins of geographically different isolates. The identification of proteins with epitopes exposed on the surface of live merozoites and accessible to antibody provides candidates to be tested as protective immunogens in cattle.     

Threonine phosphorylation is associated with mitosis in HeLa cells

Phosphorylation and dephosphorylation of proteins play an important role in the regulation of mitosis and meiosis. In our previous studies we have described mitosis-specific monoclonal antibody MPM-2 that recognizes a family of phosphopeptides in mitotic cells but not in interphase cells. These peptides are synthesized in S phase but modified by phosphorylation during G2/mitosis transition. The epitope for the MPM-2 is a phosphorylated site. In this study, we attempted to determine which amino acids are phosphorylated during the G2-mitosis (M) transition. We raised a polyclonal antibody against one of the antigens recognized by MPM-2, i.e. a protein of 55 kDa, that is present in interphase cells but modified by phosphorylation during mitosis. This antibody recognizes the p55 protein in both interphase and mitosis while it is recognized by the monoclonal antibody MPM-2 only in mitotic cells. Phosphoamino acid analysis of protein p55 from 32P-labeled S-phase and M-phase HeLa cell extracts after immunoprecipitation with anti-p55 antibodies revealed that threonine was extensively phosphorylated in p55 during G2-M but not in S phase, whereas serine was phosphorylated during both S and M phases. Tyrosine was not phosphorylated. Identical results were obtained when antigens recognized by MPM-2 were subjected to similar analysis. As cells completed mitosis and entered G1 phase phosphothreonine was completely dephosphorylated whereas phosphoserine was not. These results suggest that phosphorylation of threonine might be specific to some of the mitosis-related events.