Phosphorylation of p27Kip1 at Thr-157 interferes with its association with importin alpha during G1 and prevents nuclear re-entry

We have studied mechanisms of Akt-mediated phosphorylation and regulation of cellular localization of p27. Akt phosphorylates Thr-157 in p27 and retains it in the cytosol. In cells arrested in G(1) and then synchronized to enter into S phase, Akt-mediated phosphorylation of Thr-157 p27 occurred in the cytosol during G(1) phase of the cell cycle. Both T157A and S10A p27 mutants localized in the nucleus in all phases of the cell cycle regardless of the expression of active Akt. Thr-157 phosphorylation was undetectable in S10A-p27, suggesting that Ser-10 phosphorylation is required for p27 localization in the cytosol and subsequent phosphorylation at Thr-157. Phosphorylation at Thr-157 interrupted the association of p27 with importin alpha. A T157A-p27 mutant protein exhibited higher association with importin alpha than wild-type-p27. Treatment of transfected and endogenous p27 with alkaline phosphatase rescued its association with importin alpha. Leptomycin B inhibited cytosolic Thr-157 P-p27 staining, implying that CRM1-dependent nuclear export is required for Akt-mediated Thr-157 phosphorylation. Heterokaryon shuttling assays with NIH3T3 (mouse) cells transfected with FLAG-p27 and HeLa (human) cells revealed that both wild type and T157A-p27 shuttled from NIH3T3 to HeLa cell nuclei with similar frequencies. However, S10A-p27 was found only in the NIH3T3 nuclei of NIH3T3-HeLa cell fusions. These results suggest that 1) Ser-10 phosphorylation is required for nuclear export of p27, 2) subsequent Akt-mediated phosphorylation at Thr-157 during G(1) phase corrals p27 in the cytosol, and 3) Thr-157 phosphorylation inhibits the association of p27 with importin alpha thus preventing its re-entry into the nucleus.     

Cyclin D1 production in cycling cells depends on ras in a cell-cycle-specific manner.

BACKGROUND: Cellular Ras and cyclin D1 are required at similar times of the cell cycle in quiescent NIH3T3 cells that have been induced to proliferate, but not in the case of cycling NIH3T3 cells. In asynchronous cultures, Ras activity has been found to be required only during G2 phase to promote passage through the entire upcoming cell cycle, whereas cyclin D1 is required through G1 phase until DNA synthesis begins. To explain these results in molecular terms, we propose a model whereby continuous cell cycle progression in NIH3T3 cells requires cellular Ras activity to promote the synthesis of cyclin D1 during G2 phase. Cyclin D1 expression then continues through G1 phase independently of Ras activity, and drives the G1-S phase transition. RESULTS: We found high levels of cyclin D1 expression during the G2, M and G1 phases of the cell cycle in cycling NIH3T3 cells, using quantitative fluorescent antibody measurements of individual cells. By microinjecting anti-Ras antibody, we found that the induction of cyclin D1 expression beginning in G2 phase was dependent on Ras activity. Consistent with our model, cyclin D1 expression during G1 phase was particularly stable following neutralization of cellular Ras. Finally, ectopic expression of cyclin D1 largely overcame the requirement for cellular Ras activity during the continuous proliferation of cycling NIH3T3 cells. CONCLUSIONS: Ras-dependent induction of cyclin D1 expression beginning in G2 phase is critical for continuous cell cycle progression in NIH3T3 cells.     

Host range and cell cycle activation properties of polyomavirus large T-antigen mutants defective in pRB binding.

We have examined the growth properties of polyomavirus large T-antigen mutants that are unable to bind pRB, the product of the retinoblastoma tumor suppressor gene. These mutants grow poorly on primary mouse cells yet grow well on NIH 3T3 and other established mouse cell lines. Preinfection of primary baby mouse kidney (BMK) epithelial cells with wild-type simian virus 40 renders these cells permissive to growth of pRB-binding polyomavirus mutants. Conversely, NIH 3T3 cells transfected by and expressing wild-type human pRB become nonpermissive. Primary fibroblasts from mouse embryos that carry a homozygous knockout of the RB gene are permissive, while those from normal littermates are nonpermissive. The host range of polyomavirus pRB-binding mutants is thus determined by expression or lack of expression of functional pRB by the host. These results demonstrate the importance of pRB binding by large T antigen for productive viral infection in primary cells. Failure of pRB-binding mutants to grow well in BMK cells correlates with their failure to induce progression from G0 or G1 through the S phase of the cell cycle. Time course studies show delayed synthesis and lower levels of accumulation of large T antigen, viral DNA, and VP1 in mutant compared with wild-type virus-infected BMK cells. These results support a model in which productive infection by polyomavirus in normal mouse cells is tightly coupled to the induction and progression of the cell cycle.     

The oncogenic serine/threonine kinase Pim-1 directly phosphorylates and activates the G2/M specific phosphatase Cdc25C

The proto-oncogene Pim-1 encodes a serine-threonine kinase which is a downstream effector of cytokine signaling and can enhance cell cycle progression by altering the activity of several cell cycle regulators among them the G1 specific inhibitor p21(Waf), the phosphatase Cdc25A and the kinase C-TAK1. Here, we demonstrate by using biochemical assays that Pim-1 can interact with the phosphatase Cdc25C and is able to directly phosphorylate the N-terminal region of the protein. Cdc25C is functionally related to Cdc25A but acts specifically at the G2/M cell cycle transition point and can be inactivated by C-TAK1-mediated phosphorylation. Immuno-fluorescence experiments showed that Pim-1 and Cdc25C co-localize in the cytoplasm of both epithelial and myeloid cells. We find that phosphorylation by Pim-1 enhances the phosphatase activity of Cdc25C and in transfected cells that are arrested in G2/M by bleomycin, Pim-1 can enhance progression into G1. Therefore, we propose that Pim-1 activates Cdc25C by a direct phosphorylation and can thereby assume the function of a positive cell cycle regulator at the G2/M transition.     

Modulation of antioxidant enzymes,reactive oxygen species,and glutathione levels in manganese superoxide dismutase-overexpressing NIH/3T3 fibroblasts during the cell cycle

NIH/3T3 mouse embryo fibroblasts were transfected with the cDNA for manganese superoxide dismutase (MnSOD). Previous studies showed characteristic unique AE profiles in nonsynchronous populations of parental, control plasmid-transfected, and MnSOD-overexpressing NIH/3T3 cell lines. However, the present study showed that during S and M phases of the cell cycle, antioxidant enzyme (AE) levels were altered in MnSOD-overexpressing cell lines towards levels in S and M phases of parental and control plasmid-transfected cells. Because of the demonstration that MnSOD overexpression inhibits cell growth in both nonmalignant and malignant cells, the present study was designed to measure AEs, reactive oxygen species (ROS), and glutathione levels in various phases of the cell cycle in both parental NIH/3T3 cells and NIH/3T3 cells overexpressing MnSOD, to try to determine whether AEs, ROS, and glutathione levels could have a possible regulatory role in cell cycle progression. In all cell lines studied, ROS levels were lower in M than S phase of the cell cycle. Total glutathione and glutathione disulfide levels were greatly increased during the M phase of the cell cycle compared with quiescence and S phase in all cell lines studied. This suggests that oxidative stress exists in M phase of the cell cycle with total glutathione levels increased to decrease oxidative stress. Analysis of MnSOD-overexpressing cell clones showed a correlation of decreased cell growth with an increase in ROS in S phase of the cell cycle and a decrease in glutathione in mitosis. The data strongly suggest that specific levels of cell redox state are necessary for cells to successfully progress through the various phases of the cell cycle. J. Cell. Physiol. 177:148–160, 1998. © 1998 Wiley-Liss, Inc.     

IPP5, a novel protein inhibitor of protein phosphatase 1, promotes G1/S progression in a Thr-40-dependent manner

Protein phosphatase 1 (PP1) is a major serine/threonine phosphatase that controls gene expression and cell cycle progression. Here we describe the characterization of a novel inhibitory molecule for PP1, human inhibitor-5 of protein phosphatase 1 (IPP5). We find that IPP5, containing the PP1 inhibitory subunits, specifically interacts with the PP1 catalytic subunit and inhibits PP1 phosphatase activity. Furthermore, the mutation of Thr-40 within the inhibitory subunit of IPP5 into Ala eliminates the phosphorylation of IPP5 by protein kinase A and its inhibitor activity to PP1, whereas the mutation of Thr-40 within a truncated form of IPP5 into Asp can serve as a dominant active form of IPP5 in inhibiting PP1 activity. In IPP5-negative SW480 and IPP5-highly positive SW620 human colon cancer cells, we find that overexpression of IPP5 promotes the growth and accelerates the G(1)-S transition of SW480 cells in a Thr-40-dependent manner, which could be reversed by downregulation of the PP1 expression. Moreover, silencing of IPP5 inhibits the growth of SW620 cells both in vitro and in nude mice possibly by inducing G(0)/G(1) arrest but not by promoting apoptosis. According to its role in the promotion of cell cycle progression and cell growth, IPP5 up-regulates the expression of cyclin E and the phosphorylated form of retinoblastoma protein. Our findings suggest that IPP5, by acting as an inhibitory molecule for PP1, can promote tumor cell growth and cell cycle progression, and may be a promising target in cancer therapeutics in IPP5-highly expressing tumor cells.     

Ras-induced colony formation and anchorage-independent growth inhibited by elevated expression of Puralpha in NIH3T3 cells

Levels of Puralpha, a conserved, sequence-specific single-stranded DNA and RNA binding protein, fluctuate during the cell cycle, declining at the onset of S-phase and peaking at mitosis. In early G1 Puralpha is associated with the hypophosphorylated form of the retinoblastoma protein, Rb. Microinjection of purified Puralpha into NIH3T3 cells arrests the cell cycle at either G1/S or G2/M checkpoints with distinct morphological consequences. Here we ask whether expression of Puralpha can affect colony formation and anchorage-independent growth in ras-transformed NIH3T3 cells. Two to five-fold elevated levels of Puralpha in stably-transfected cell lines retard entry into and progression through S phase in both ras-transformed and non-transformed cells. Puralpha significantly inhibits colony formation by ras-transformed cells but not by non-transformed cells. In addition, cells transfected to express Puralpha formed only about 1/5 the number of large colonies in soft agar as control-transfected cells, demonstrating a marked inhibition of anchorage-independent growth by Puralpha. Biochemical analysis of nuclear and cytoplasmic Puralpha proteins and confocal microscopic analysis of Puralpha location indicate that access of Puralpha to the nucleus is controlled by both protein modification and sequence domains within the protein. Analyses of deletion mutants identify Puralpha domains mediating nuclear exclusion, including several potential destruction motifs and a PEST sequence at aa's 215-231. In the nucleus Puralpha colocalizes with CDK2 and cyclin A. Puralpha and cyclin D1, however, do not colocalize in the nucleus. At mitosis Puralpha is visualized about the condensed chromosomes and in the cytoplasm, where it colocalizes with cyclin B1. The data indicate that the ability of Puralpha to interact with proteins regulating cell proliferation and transformation is controlled by signals that govern its intracellular localization.     

A Mammalian bromodomain protein,brd4, interacts with replication factor C and inhibits progression to S phase

Brd4 belongs to the BET family of nuclear proteins that carry two bromodomains implicated in the interaction with chromatin. Expression of Brd4 correlates with cell growth and is induced during early G(1) upon mitogenic stimuli. In the present study, we investigated the role of Brd4 in cell growth regulation. We found that ectopic expression of Brd4 in NIH 3T3 and HeLa cells inhibits cell cycle progression from G(1) to S. Coimmunoprecipitation experiments showed that endogenous and transfected Brd4 interacts with replication factor C (RFC), the conserved five-subunit complex essential for DNA replication. In vitro analysis showed that Brd4 binds directly to the largest subunit, RFC-140, thereby interacting with the entire RFC. In line with the inhibitory activity seen in vivo, recombinant Brd4 inhibited RFC-dependent DNA elongation reactions in vitro. Analysis of Brd4 deletion mutants indicated that both the interaction with RFC-140 and the inhibition of entry into S phase are dependent on the second bromodomain of Brd4. Lastly, supporting the functional importance of this interaction, it was found that cotransfection with RFC-140 reduced the growth-inhibitory effect of Brd4. Taken as a whole, the present study suggests that Brd4 regulates cell cycle progression in part by interacting with RFC.     

Expression of c-fos in NIH3T3 cells is very low but inducible throughout the cell cycle.

It has previously been shown that the c-fos proto-oncogene is rapidly and transiently induced following growth factor stimulation of quiescent NIH3T3 mouse fibroblasts. To investigate a possible role of c-fos in growth control mechanisms we have studied its expression and inducibility during the NIH3T3 cell cycle. Two major conclusions can be drawn from this analysis. First, expression of c-fos is not cell cycle-regulated, and is barely detectable in all phases of the cycle. Second, cells at different stages of the cell cycle (except for mitosis) are as sensitive to c-fos induction by growth factors as quiescent cells. These observations suggest that induction of the c-fos gene does not play a role during the continuous cycling of NIH3T3 cells, but they are fully compatible with the hypothesis that a function of c-fos may be associated with the induction of competence in fibroblasts. Through such a function c-fos may contribute to moving cells out of the quiescent state.     

Inhibition of G1 to S cell cycle progression by BCCIP beta

The BCCIP alpha protein was identified as a BRCA2 and CDKN1A (p21, or p21(Waf1/Cip1)) Interacting Protein. It binds to a highly conserved domain proximate to the C-terminus of BRCA2 protein and the C-terminal domain of the CDK-inhibitor p21. Previous reports showed that BCCIP alpha enhances the inhibitory activity of p21 toward CDK2 and that BCCIP alpha inhibits the growth of certain tumor cells. Here we show that a second isoform, BCCIP beta, also binds to p21 and inhibits cell growth. The growth inhibition by BCCIP beta can be partially abrogated in p21 deficient cells. Overexpression of BCCIP beta delays the G1 to S progression and results in an elevated p21 expression. These data suggest BCCIP beta as a new regulator for the G1-S cell cycle progression and cell growth control.     

Inhibition of G1 to S Cell Cycle Progression by BCCIPb

The BCCIPa protein was identified as a BRCA2 and CDKN1A (p21, or p21Waf1/Cip1) Interacting Protein. It binds to a highly conserved domain proximate to the C-terminus of BRCA2 protein and the C-terminal domain of the CDK-inhibitor p21. Previous reports showed that BCCIPa enhances the inhibitory activity of p21 toward CDK2 and that BCCIPa inhibits the growth of certain tumor cells. Here we show that a second isoform, BCCIPb, also binds to p21 and inhibits cell growth. The growth inhibition by BCCIPb can be partially abrogated in p21 deficient cells. Overexpression of BCCIPb delays the G1 to S progression and results in an elevated p21 expression. These data suggest BCCIPb as a new regulator for the G1-S cell cycle progression and cell growth control.     

RACK1 regulates G1/S progression by suppressing Src kinase activity

Cancer genes exert their greatest influence on the cell cycle by targeting regulators of a critical checkpoint in late G(1). Once cells pass this checkpoint, they are fated to replicate DNA and divide. Cancer cells subvert controls at work at this restriction point and remain in cycle. Previously, we showed that RACK1 inhibits the oncogenic Src tyrosine kinase and NIH 3T3 cell growth. RACK1 inhibits cell growth, in part, by prolonging G(0)/G(1). Here we show that RACK1 overexpression induces a partial G(1) arrest by suppressing Src activity at the G(1) checkpoint. RACK1 works through Src to inhibit Vav2, Rho GTPases, Stat3, and Myc. Consequently, cyclin D1 and cyclin-dependent kinases 4 and 2 (CDK4 and CDK2, respectively) are suppressed, CDK inhibitor p27 and retinoblastoma protein are activated, E2F1 is sequestered, and G(1)/S progression is delayed. Conversely, downregulation of RACK1 by short interference RNA activates Src-mediated signaling, induces Myc and cyclin D1, and accelerates G(1)/S progression. RACK1 suppresses Src- but not mitogen-activated protein kinase-dependent platelet-derived growth factor signaling. We also show that Stat3 is required for Rac1 induction of Myc. Our results reveal a novel mechanism of cell cycle control in late G(1) that works via an endogenous inhibitor of the Src kinase.     

Comparative effects of butyrate and N6, 2'-O-dibutyryladenosine-3':5'-cyclic monophosphate on growth, differentiation and gene expression in U937 human monoblastoid cells.

Administration of 1mM sodium butyrate or N6,2'-O-dibutyryladenosine 3':5'-cyclic monophosphate (dbcAMP) inhibits the growth activity of U937 human monoblastoid cells by blocking them at the G1 or at the G1 + G2 phases of the cell cycle, respectively. Both agents induce the differentiation of U937 cells, as proved by the increased expression of the maturation-associated CD11b antigen and by the increased capacity to reduce nitroblue tetrazolium. RNA blot assays indicate that butyrate and dbcAMP decrease the expression of ornithine decarboxylase and c-myc genes, and stimulate the expression of the vimentin gene. However, while dbcAMP induces c-fos mRNA accumulation, butyrate did not affect the expression of this proto-oncogene.     

The initial accumulation of carbamoylphosphate synthetase in embryonic rat hepatocytes, and the cell cycle

The hormone-induced expression of the hepatocyte-specific enzyme carbamoylphosphate synthetase can take place in each phase of the cell cycle and is not restricted to the G1 or the G0 phase. To arrive at this conclusion, the cell cycle parameters of embryonic day 14 rat hepatocytes in vitro were determined by autoradiography after labeling with (3H)-TdR or with (3H)- and (14C)-TdR. An S-phase of approximately 14 h, a G2 + M-phase of 8 h, a G1-phase of 8-13 h and a total cell cycle of 30-35 h were measured. Freshly isolated embryonic hepatocytes have exponential growth parameter values, but shift to a steady state growth under culture conditions in the presence of hormones (glucocorticosteroids, thyroid hormones and cyclic AMP). The length of the S-phase and of the total cell cycle remain constant during the culture time. The time course of accumulation of carbamoylphosphate synthetase protein in embryonic hepatocytes is identical in all phases of the cell cycle. It is suggested that hormones, in particular glucocorticosteroids, simultaneously and independently regulate growth mode and gene expression in developing hepatocytes. The nucleotide-analogue 5-bromodeoxyuridine inhibits the hormone-induced expression of carbamoylphosphate synthetase only in cells that are exposed to the drug during early S-phase, indicating replication of the carbamoylphosphate synthetase gene in that part of the cell cycle.     

Retroviral-mediated gene transfer of the peripheral myelin protein PMP22 in Schwann cells: modulation of cell growth.

The peripheral myelin gene PMP22 is the rat and human homologue of the murine growth arrest-specific gene gas3. Besides a putative role of PMP22 in myelination, a regulatory function in cell growth has been suspected. Here we have investigated both the expression of PMP22 during cell cycle progression of cultured rat Schwann cells and the influence of altered levels of PMP22 on Schwann cell growth. When resting cells were stimulated to begin the cell cycle, the regulation of PMP22 mRNA resembled the growth arrest-specific pattern of gas3 expression observed previously in NIH3T3 fibroblasts. To prove a growth regulatory function of PMP22, we generated Schwann cell cultures by infection with retroviral PMP22 expression vectors that constitutively expressed PMP22 cDNA sequences, in either the sense or antisense orientation. Transduced cells carrying the sense construct overexpressed PMP22 mRNA and protein, whereas in cells infected with an antisense PMP22 expression vector PMP22 mRNA levels were reduced markedly. Altered levels of PMP22 significantly modulated Schwann cell proliferation, as judged by 5-bromo-2'-deoxy-uridine incorporation into replicated DNA. In asynchronously dividing cultures enhanced expression of PMP22 decreased DNA synthesis to 60% of the control level. Conversely, reduced levels of PMP22 mRNA led to enhanced DNA synthesis of approximately 150%. Further cell cycle analyses by flow cytometry revealed that overexpression of PMP22 delayed serum- and forskolin-stimulated entry of resting Schwann cells from G0/G1 into the S + G2/M phases by approximately 8 h, whereas underexpression of PMP22 mRNA slightly increased the proportion of cells that entered the S + G2/M phases.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of Cyclic AMP on Components of the Cell Cycle Machinery Regulating DNA Synthesis in Cultured Astrocytes

Cyclic AMP is a second messenger for various hormones that inhibits cell multiplication and DNA synthesis in cultured astrocytes. We examined the effects of increasing intracellular cyclic AMP on the catalytic (cdks) and regulatory (cyclins and ckis) components of cyclin-dependent protein kinases, which regulate progression of the cell cycle before completion of DNA synthesis, in primary cultured astrocytes and in an astrocytic cell line C.LT.T.1.1. The amount of cdk4 changed little during the cell cycle and was not affected by cyclic AMP. There was little cdk1 and cdk2 in quiescent cells, and their expression increased during the G1-S phases. Cyclic AMP strongly inhibited cdk1 and cdk2 expression. Transforming growth factor beta also inhibited cdk1 expression in primary astrocytes. Cyclic AMP did not affect the two ckis p27KIP1 and p21CIP1. There was little cyclin D1 in quiescent cells, but it increased during the G1 phase and was reduced by cyclic AMP. Cyclin E increased during the G1-S phases and was not affected by cyclic AMP in primary astrocytes. The amount of cyclin A was low in quiescent cells and increased during the G1-S phases. Expression of its mRNA and protein was inhibited by cyclic AMP. The protein kinase activities associated with complexes of cyclins and cdks were increased by growth factors and prevented by cyclic AMP. We conclude that cyclic AMP inhibits progression of the cell cycle in astrocytes at least by preventing the expression of the regulatory subunits, cyclins D1 and A, and catalytic subunits, cdk1 and cdk2, of cyclin-regulated protein kinases. Key Words: Cyclin-dependent protein kinases-Glial cells-Cyclic AMP.