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N A Krucher

Publications and source records attributed to N A Krucher.

9 recordsLinked to original sources
PubMed2000

Interaction of the retinoblastoma protein (pRb) with the catalytic subunit of DNA polymerase delta (p125).

The retinoblastoma gene product (pRb) interacts with many cellular proteins to function in the control of cell division, differentiation, and apoptosis. Several pRb binding proteins complex with pRb through an amino acid sequence called the LXCXE motif. The catalytic subunit of DNA polymerase delta (p125) contains a LXCXE motif. To further study the biochemical function of this polymerase, we sought to determine if p125 interacts with pRb. Experiments using GST-pRb fusion proteins showed that p125 from breast epithelial (MCF10A) cell extracts associates with pRb. In addition, GST-p125 fusion proteins bound pRb from the same cell extracts. The pRb that associated with GST-p125 was largely unphosphorylated. Coimmunoprecipitation experiments using cell cycle synchronized cells revealed that p125 and pRb form a complex predominantly during G1 phase, the phase during which pRb is mostly unphosphorylated. In vitro phosphorylation of GST-pRb by the cyclin dependent kinases reduced the ability of p125 to associate with GST-pRh. Addition of the LXCXE containing protein SV40 large T antigen to GST-pRb blocks the ability of p125 to associate with pRb, suggesting that it may be through a LXCXE sequence by which p125 interacts with pRb. Finally, in vitro polymerase assays demonstrate that GST-pRb fusion protein stimulates DNA polymerase delta activity.

Animals↗
PubMed1999

Molecular analysis of selected cell cycle regulatory proteins during aerobic and hypoxic maintenance of human ovarian carcinoma cells.

We have previously reported on the development of an in vitro model system for studying the effect of hypoxia on ovarian carcinoma cell proliferation and invasion (Krtolica and Ludlow, 1996). These data indicate that the cell division cycle is reversibly arrested during the G1 phase. Here, we have continued this study to include the proliferation properties of both aerobic and hypoxic human ovarian carcinoma cells at the molecular level. The growth suppressor product of the retinoblastoma susceptibility gene, pRB, appears to be functional in these cells as determined by SV40 T-antigen binding studies. Additional G1-to-S cell cycle regulatory proteins, cyclins D and E, cyclin-dependent kinases (cdks) 4 and 2, and cdk inhibitors p27 and p18, also appear to be intact based on their apparent molecular weights and cell cycle stage-specific abundance. During hypoxia, there is a decrease in abundance of cyclins D and E, with an increase in p27 abundance. cdk4 activity towards pRB and cdk2 activity towards histone H1 are also decreased. Co-precipitation studies revealed an increased amount of p27 complexing with cyclin E-cdk2 during hypoxia than during aerobic cell growth. In addition, pRB-directed phosphatase activity was found to be greater in hypoxic than aerobic cells. Taken together, a model is suggested to explain hypoxia-induced cell cycle arrest in SKA human ovarian carcinoma cells.

Aerobiosis↗
PubMed1998

Mitogenic activity of steroidogenesis-inducing protein (SIP) during hypoxic stress of human ovarian carcinoma cells.

Steroidogenesis-inducing protein (SIP) is a novel growth factor isolated from human ovarian follicular fluid. While the steroidogenic and mitogenic effects appear to be restricted towards gonadal cell types, we have recently demonstrated that SIP is also a potent mitogen for cell lines derived from ovarian surface epithelial carcinomas. Here, we demonstrate that SIP reverses hypoxia-induced cell proliferation arrest of the human ovarian carcinoma cell line SKA, as determined by flow cytometry and cell proliferation assays. Concomitant with this reversal of proliferation arrest is an increase in expression of cyclins D and E and a reduction in expression of the cyclin-dependent kinase inhibitor p27. Pretreatment of hypoxic SKA cells with SIP is also shown to increase Taxol sensitivity of these cells by two-fold. These studies further characterize the mitogenic activity of SIP at the molecular level and suggest that this protein may be an effective biological response modifier for ovarian carcinoma cells.

Carcinoma↗
PubMed1998

Hypoxia-induced pRB hypophosphorylation results from downregulation of CDK and upregulation of PP1 activities.

Exposure of CV-1P cells to hypoxic conditions results in reversible cell cycle arrest concomitant with accumulation of pRB in the hypophosphorylated, growth suppressive form. Similar to cell cycle arrest induced by serum starvation, we show here that hypoxia-induced arrest is accompanied by a decrease in pRB-directed CDK4 and CDK2 activities, lower cyclin D and E protein levels, and by an increase in p27 protein abundance. Immunoprecipitation studies reveal an increase in p27 association with cyclin E-CDK2 complexes. In contrast to cell cycle arrest induced by serum starvation, hypoxia increases PP1-mediated pRB dephosphorylation. These data reveal that synergy between decreased pRB-directed cyclin/CDK activity and increased pRB-directed phosphatase activity contribute towards inducing and maintaining pRB in its hypophosphorylated, growth suppressive state during hypoxia.

Animals↗
PubMed1997

High molecular weight protein phosphatase type 1 dephosphorylates the retinoblastoma protein.

pRb controls cell proliferation by restricting inappropriate entry of cells into the cell division cycle. As dephosphorylation of pRb during mitotic exit activates its growth suppressive function, identification of the protein phosphatase that dephosphorylates pRb, and characterization of the mechanism of its regulation, are essential to elucidating the mechanisms of cell growth control. By fractionating mitotic CV-1P cell extracts, we identify the protein phosphatase which dephosphorylates pRb as a type 1 serine/threonine phosphoprotein phosphatase (PP1). Molecular sizing analyses indicate that the catalytic enzyme (PP1c) is present in a high molecular weight complex, with a predicted molecular mass of 166 kDa. PP1-interacting proteins in the mitotic cell extracts are identified. Two PP1-interacting proteins (41 and 110 kDa) are shown to form distinct complexes with PP1c from fractions of separated mitotic cell extracts containing phosphorylase phosphatase activity. However, only the 110-kDa PP1-interacting protein is present in fractions containing pRb-directed phosphatase activity, identifying this protein as a putative activator of PP1 function toward pRb during mitosis.

Animals↗
PubMed1997

The cyclin-dependent kinase (cdk) inhibitors, olomoucine and roscovitine, alter the expression of a molluscan circadian pacemaker.

1. In this study, we determined the effects of the cyclin-dependent kinase (cdk) inhibitors, olomoucine and roscovitine, on the circadian rhythm of optic nerve impulse activity recorded from the eye of the marine snail Bulla gouldiana. 2. We found that olomoucine lengthened period and altered circadian phase in a dose-dependent manner without appreciably affecting gene transcription or translation. We also found that the more specific cdk inhibitor, roscovitine, was approximately 10-fold more effective in lengthening circadian period, while the inactive analogue, iso-olomoucine, was ineffective. 3. The current results, along with previous results from our laboratory, are consistent with the hypothesis that the biochemical mechanism responsible for generating the ocular circadian rhythm in B. gouldiana is related to the biochemical mechanism that regulates the eukaryotic cell division cycle, i.e., by modulation of the activity of protein kinases belonging to the cdk family.

Animals↗
PubMed1994

Identification of CDK- and cyclin-like proteins in the eye of Bulla gouldiana.

The ocular circadian rhythm in the eye of Bulla gouldiana is generated by a rhythm in membrane potential of retinal neurons that is driven by alterations in potassium conductance. Since potassium conductance may be modulated by the phosphorylation of potassium channels, the circadian rhythm may reflect rhythmic changes in protein kinase activity. Furthermore, the circadian rhythm recorded from the Bulla eye can be phase shifted by agents that affect protein synthesis and protein phosphorylation on tyrosine residues. Interestingly, the eukaryotic cell division cycle is generated by similar processes. Rhythmic cell division is regulated by periodic synthesis and degradation of a protein, cyclin, and periodic tyrosine phosphorylation of a cyclin-dependent kinase (cdk), p34cdc2. The interaction between these two proteins results in rhythmic kinase activity of p34cdc2. Both cyclin and p34cdc2 are part of two diverse gene families, some of whose members have been localized to post-mitotic cell types with no function yet determined. In the current work, we identify proteins similar to the cdks and cyclin in the eye of Bulla. Neither of these ocular proteins are found in mitotic cells in Bulla, and the cdk-like protein (p40) is specific to the eye. Furthermore, the concentration of the cyclin-like protein (p66) is affected by treatments that phase shift the circadian rhythm. The identification of cdk and cyclin-like proteins in the Bulla eye is consistent with the hypothesis that the biochemical mechanism responsible for generating the ocular circadian rhythm in Bulla is related to the biochemical mechanism that regulates the eukaryotic cell division cycle.

Animals↗