Ramesh C. Adlakha

Phosphorylation of non-histone proteins associated with mitosis in HeLa cells.

Our previous studies indicated that certain non-histone proteins (NHP) extractable with 0.2 M NaCl from mitotic HeLa cells induce germinal vesicle breakdown and chromosome condensation in Xenopus laevis oocytes. Since the maturation-promoting activity of the mitotic proteins is stabilized by phosphatase inhibitors, we decided to examine whether phosphorylation of NHP plays a role in the condensation of chromosomes during mitosis. HeLa cells, synchronized in S phase, were labeled with 32P at the end of S phase, and the cells subsequently collected while they were in G2, mitosis, or G1. Cytoplasmic, nuclear, or chromosomal proteins were extracted and separated by gel electrophoresis. The labeled protein bands were detected by radioautography. The results indicated an 8-10-fold increase in the phosphorylation of NHP from mid-G2 to mitosis, followed by a similar-size decrease as the cells divided and entered G1. The NHP phosphorylation rate increased progressively during G2 traverse and reached a peak in mitosis. Radioautography of the separated NHP revealed eight prominent, extensively phosphorylated protein bands with molecular masses ranging from 27.5 to 100 kD. These NHP were rapidly dephosphorylated during M-G1 transition. Phosphorylation-dephosphorylation of NHP appeared to be a dynamic process, with the equilibrium shifting to phosphorylation during G2-M and dephosphorylation during M-G1 transitions. These results suggest that besides histone H1 phosphorylation, phosphorylation of this subset of NHP may also play a part in mitosis.

Partial purification and characterization of mitotic factors from HeLa cells

Extracts from mitotic HeLa cells, when injected into Xenopus laevis oocytes, exhibit maturation-promoting activity (MPA) as evidenced by the breakdown of the germinal vesicle and the condensation of chromosomes. In this study we have attempted to purify and characterize these mitotic factors. When 0.2 M NaCl-soluble extracts of mitotic HeLa cells were concentrated by ultrafiltration and subjected to affinity chromatography on hydroxylapatite followed by DNA-cellulose, the proteins with MPA eluted as a single peak and their specific activity was increased approx. 200-fold compared with crude extracts. The molecular weight of the mitotic factors was estimated to be 100 kD as determined by chromatography on Sephacryl S-200. SDS-PAGE of the partially-purified mitotic factors indicated the presence of several polypeptides ranging from 40-150 kD with a major band of about 50 kD. The majority of these polypeptides were found to be phosphoproteins as revealed by 32P-labeling and autoradiography. Very little or no phosphorylation was observed at the 50 kD band. Several of these polypeptides were reactive with mitosis-specific monoclonal antibodies, MPM-1 or MPM-2, as shown by immunoblots of these proteins but the major polypeptide band at 50 kD was not. Removal of the immunoreactive polypeptides by precipitation with these antibodies did not destroy the MPA. The MPA of the crude or the partially-purified mitotic factors was destroyed by injection of (but not pretreatment with) alkaline phosphatase within 45 min after injection of mitotic factors. These results are discussed in terms of a possible role of phosphorylation-dephosphorylation of non-histone proteins in the regulation of mitosis and meiosis.

Inhibition of DNA polymerase α by gossypol

Abstract Our earlier studies have shown that gossypol is a specific inhibitor of DNA synthesis in cultured cells at low doses. In an attempt to determine the mechanism for the inhibition of DNA synthesis by gossypol we observed that gossypol does not interact with DNA per se but may affect some of the enzymes involved in DNA replication. These studies indicated that gossypol inhibits both in vivo and in vitro the activity of DNA polymerase α (EC 2.7.7.7), a major enzyme involved in DNA replication, in a time- and dose-dependent manner. Kinetic analysis revealed that gossypol acts as a noncompetitive inhibitor of DNA polymerase α with respect to all four deoxynucleotide triphosphates and to the activated DNA template. Inhibition of DNA polymerase α does not appear to be due to either metal chelation or reduction of sulfhydryl groups on the enzyme. Gossypol also inhibited HeLa DNA polymerase β in a dose-dependent manner, but had no effect on DNA polymerase γ. These results suggest that inhibition of DNA polymerase α may account in part for the inhibition of DNA synthesis and the S-phase block caused by gossypol. The data also raise the possibility that gossypol may interfere with DNA repair processes as well.

Chromosome-bound mitotic factors: release by endonucleases

Additional evidence is presented to support our recently reported conclusion that the mitotic factors of mammalian cells, which induce germinal vesicle breakdown and chromosome condensation when injected into fully grown Xenopus laevis oocytes, are localized on metaphase chromosomes. Chromosomes isolated from mitotic HeLa cells were further purified on sucrose gradients and digested for varying periods with either the micrococcal nuclease or DNase II. At each time point of digestion the amount of mitotic factors released was determined by injecting a supernatant of these fractions, obtained by high-speed centrifugation, into oocytes. The amount of DNA rendered acid soluble under the conditions of digestion used was 3% ot 5% of the total chromosomal DNA. The extent of release of mitotic factors with both nucleases was estimated to be about 30% to 40% as evidenced by the reextraction of the undigested chromosomal pellet with 0.2 M NaC1. Similar results were obtained when nuclei from G2 cells were digested under identical conditions. The release of these chromosome-bound mitotic factors by mild digestion with these nucleases though only partial, clearly demonstrates that a significant proportion of these factors are localized on metaphase chromosomes.

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.

Role of Nonhistone Protein Phosphorylation in the Regulation of Mitosis in Mammalian Cells

The postsynthetic modification of proteins via reversible phosphorylation-dephosphorylation by phosphoprotein kinases and phosphatases has been reported to be an important mechanism in the regulation of numerous intracellular events (15,26,30). A strong correlation between the phosphorylation of histone H1 and (H3) and chromosome condensation and initiation of mitosis has been shown by several investigators (7,8,11,13,14,18,21,23). More recently, it was shown that histone H1 is also phosphorylated during premature chromosome condensation (8,22,27). However, many investigators believe that the superphosphorylation of histone H1 alone is not sufficient for chromosome condensation (10,20,22,27,38). Furthermore, chromosome decondensation could still occur even when dephosphorylation of histone H1 was blocked in mitotic cells (38). There is increasing evidence to suggest that phosphorylation of nonhistone proteins (NHP) may also be required for mitosis-related events. For example, phosphorylation of high mobility group (HMG) proteins has been suggested to be responsible for the shutting off of gene transcription during mitosis (12,28,34), increased phosphorylation of intermediate filament proteins like vimentin at mitosis (17,33), phosphorylation and dephosphorylation of laminar proteins have been implicated in the dissolution and reformation of the nuclear envelope (19,25).