Phosphorylation at serine 482 affects stability of NF90 and its functional role in mitosis.

OBJECTIVES: NF90 is a multifunctional double-strand RNA binding protein with documented roles in transcription, mRNA stability, translation, RNA processing and transport, and mitosis. It is a phosphoprotein that interacts with, and is a substrate for, several protein kinases. The study described here was initiated to gain better understanding of specific NF90 phosphorylation sites and their relationship to mechanisms by which NF90 performs its various functions. MATERIALS AND METHODS: Phosphoproteomic studies have identified NF90 serine 482 (S482) as a major phosphorylation site in vivo. Site-specific mutations were introduced at this site and the mutated proteins were expressed in MCF7 cells by transfection. Western blotting was used to examine NF90 expression, stability, and responsiveness to protein kinase activators and inhibitors. Flow cytometry was used to examine effects of NF90 mutation on cell cycle progression. RESULTS: Non-phosphorylatable mutant S482A was unstable compared to phosphomimetic S482E mutant. NF90-S482A expression was greatly enhanced by inhibiting proteasomal degradation or by activating PKC. Identical treatments had little effect on NF90-S482E. In contrast to WT NF90 or NF90-S482E, cells stably expressing NF90-S482A accumulated in M phase when treated with TPA. CONCLUSIONS: Phosphorylation at S482 is important for NF90 stability and in regulating its functional role during mitosis. Based on the sequence surrounding S482, mitotic kinase PLK1 is a strong candidate for the enzyme that phosphorylates NF90 at this site.

The major transcription initiation site of the p27Kip1 gene is conserved in human and mouse and produces a long 5'-UTR.

BACKGROUND: The cyclin-dependent kinase inhibitor p27Kip1 is essential for proper control of cell cycle progression. The levels of p27Kip1 are regulated by several mechanisms including transcriptional and translational controls. In order to delineate the molecular details of these regulatory mechanisms it is important to identify the transcription initiation site within the p27Kip1 gene, thereby defining the promoter region of the gene and the 5'-untranslated region of the p27Kip1 mRNA. Although several previous studies have attempted to map p27Kip1 transcription start sites, the results vary widely for both the mouse and human genes. In addition, even though the mouse and human p27Kip1 gene sequences are very highly conserved, the reported start sites are notably different. RESULTS: In this report, using a method that identifies capped ends of mRNA molecules together with RNase protection assays, we demonstrate that p27Kip1 transcription is initiated predominantly from a single site which is conserved in the human and mouse genes. Initiation at this site produces a 5'-untranslated region of 472 nucleotides in the human p27Kip1 mRNA and 502 nucleotides in the mouse p27Kip1 mRNA. In addition, several minor transcription start sites were identified for both the mouse and human genes. CONCLUSIONS: These results demonstrate that the major transcription initiation sites in the mouse and human p27Kip1 genes are conserved and that the 5'-UTR of the p27Kip1 mRNA is much longer than generally believed. It will be important to consider these findings when designing experiments to identify elements that are involved in regulating the cellular levels of p27Kip1.

Control of cyclin-dependent kinase inhibitor p27 expression by cap-independent translation.

p27 is a key regulator of cell proliferation through inhibition of G(1) cyclin-dependent kinase (CDK) activity. Translation of the p27 mRNA is an important control mechanism for determining cellular levels of the inhibitor. Nearly all eukaryotic mRNAs are translated through a mechanism involving recognition of the 5' cap by eukaryotic initiation factor 4E (eIF4E). In quiescent cells eIF4E activity is repressed, leading to a global decline in translation rates. In contrast, p27 translation is highest during quiescence, suggesting that it escapes the general repression of translational initiation. We show that the 5' untranslated region (5'-UTR) of the p27 mRNA mediates cap-independent translation. This activity is unaffected by conditions in which eIF4E is inhibited. In D6P2T cells, elevated cyclic AMP levels cause a rapid withdrawal from the cell cycle that is correlated with a striking increase in p27. Under these same conditions, cap-independent translation from the p27 5'-UTR is enhanced. These results indicate that regulation of internal initiation of translation is an important determinant of p27 protein levels.

A role for an AP-1-like site in the expression of the myelin basic protein gene during differentiation.

Differentiation of oligodendrocyte progenitors into mature oligodendrocytes involves the timely, cell-type specific expression of a number of different genes. Among these, the expression of the myelin basic protein (MBP) gene closely parallels the course of oligodendrocyte differentiation. To understand how transcription of the myelin basic protein gene is controlled, binding to the distal end of the 5' flanking sequence of the MBP gene was investigated. Specific protein-DNA complexes were localized to an AP-1-like element located between -1230 and -1240. The protein-DNA complexes formed at this site were shown to change as the cells differentiated. In undifferentiated cells two complexes were formed but, as the cells differentiated, binding was nearly completely lost. One of the two complexes was shown to contain a member of the fos family of transcription factors but no jun family members were involved. Mutation of the AP-1-like site resulted in loss of the complex and a change in expression of a reporter construct driven by the mutated promoter sequence. These results demonstrate a role for the AP-1-like site in repression of MBP gene expression in oligodendrocyte progenitor cells.

Mimosine arrests cells in G1 by enhancing the levels of p27(Kip1).

The plant amino acid mimosine, an effective inhibitor of DNA replication, has been demonstrated to arrest cell cycle progression in late G1. To understand further the molecular mechanism by which mimosine affects the cell cycle, we treated quiescent cells with serum in the presence of 800 microM mimosine. The cells did not enter S phase and were completely arrested in G1 phase. Although neither the mitogenic induction of the G1 cyclins nor the protein levels of cdk2 or cdk4 were affected, serum-dependent activation of cdk2 was blocked. This corresponded to elevated levels of the cdk inhibitor p27(Kip1). This was not mediated through inhibition of degradation but rather involved increased synthesis of both p27(Kip1) mRNA and protein. Mimosine did not appear to affect mitogen-dependent signals that normally lead to p27(Kip1) downregulation since the inhibitor was induced to even greater levels in quiescent, unstimulated cells. In the presence of mimosine, actinomycin D treatment for 2 h prevented the increase of p27(Kip1) mRNA, but p27(Kip1) protein levels were still enhanced under these conditions. We propose that mimosine blocks the cell cycle in late G1 phase by upregulation of p27(Kip1) protein levels through transcriptional and posttranscriptional regulatory mechanisms.

The cyclin-dependent kinase inhibitor p27Kip1 is localized to the cytosol in Swiss/3T3 cells.

p27Kip1 plays an important role in cell cycle progression by negatively regulating the activity of cyclin-Cdk complexes. To understand how p27Kip1 functions, the level and subcellular location of p27Kip1 in Swiss/3T3 cells following serum stimulation of quiescent cells was examined. Surprisingly, p27Kip1 was observed exclusively in the cytosol throughout G1 and into early S phase. However, as expected, p27Kip1 in the cytosolic fraction was greatly reduced following serum stimulation and reached very low levels by late G1. The decline in the level of p27Kip1 corresponded in time to an increase in the nuclear level of both Cdk2 and cyclin E. In quiescent 3T3 cells Cdk2 was inactive and co-precipitated with p27Kip1. After serum stimulation, both nuclear and cytosolic Cdk2 was activated and this corresponded to the decline in p27Kip1. Overexpression of p27Kip1 allowed accumulation of the inhibitor in the nucleus but inhibited entry of Cdk2 into the nucleus following serum stimulation. The subcellular localization of p27Kip1 was also examined in a variety of other mammalian cells. In all the cell lines examined the preponderance of p27Kip1 was found in the cytosolic fraction. However, a substantial level of nuclear p27Kip1 was observed for several cell lines. In a primary mixed glial cell culture p27Kip1 was localized to the nucleus. The results suggest that cytosolic p27Kip1 has a functional role in regulating cell cycle progression, possibly through inhibiting transport of cyclin E-Cdk 2 complexes into the nucleus.

ATF-1 is activated in response to UV irradiation in B16 melanoma cells.

We have found that the transferrin receptor gene promoter is strongly activated by exposure of B16 melanoma cells to UV light. This is a delayed event occurring more than 6 h after exposure and requires an AP-1/CRE-like element in the promoter as demonstrated by site-specific mutagenesis. UV irradiation enhances the binding of a nuclear factor to this element and supershift analysis demonstrates that this DNA-protein complex involves ATF-1. No other members of either the AP-1 or CREB/ATF families of transcription factors were found to bind to this DNA element in UV-irradiated B16 cells. Western blots show that the level of ATF-1 does not change following exposure to UV light, indicating that the increased binding of this factor is most likely mediated by posttranslational modifications in response to UV-mediated signaling pathways.

The same xenobiotic response element is required for constitutive and inducible expression of the mammalian aldehyde dehydrogenase-3 gene.

The mammalian aldehyde dehydrogenase-3 gene (ALDH3) exhibits several aspects of tissue-specific expression. Certain normal tissues, such as the cornea, constitutively express ALDH3 at very high levels. Other tissues, such as normal liver, do not express ALDH3. In liver, ALDH3 is inducible by polycyclic aromatic hydrocarbon xenobiotics by an Ah-receptor (AhR)-mediated pathway in which a liganded AhR complexes with nuclear ARNT protein, and the complex binds to a xenobiotic response element (XRE) sequence located near -3.0 kb in the ALDH3 5' flanking region and initiates transcription. We used our recently developed rat corneal epithelium culture model (Boesch et al., J. Biol. Chem. 271, 5150-5157, 1996) to study the molecular basis of constitutive ALDH3 expression. Transient transfection assays of corneal epithelium using a battery of ALDH3 5' flanking region-CAT reporter gene constructs indicate that high constitutive ALDH3 expression involves the same cis-acting elements as xenobiotic-induced ALDH3 expression in liver. These elements include a strong basal promoter region and the XRE located near -3.0 kb. Western analysis confirms the presence of AhR and ARNT proteins in 3-methylcholanthrene-treated rat liver, as well as ARNT protein in rat corneal epithelium. No AhR protein is found in rat cornea. The -3.0-kb ALDH3 XRE region contains multiple overlapping transcription factor binding sequences, including consensus sites for AhR, ARNT, HNF1, HNF4, and C/ebp. Electrophoretic mobility shift assays (EMSAs) indicate that constitutive expression of ALDH3 in cornea involves binding of ARNT, HNF1, and HNF4 to the ALDH3-XRE in an Ah-receptor-independent, ARNT-requiring manner. Transient transfection of ALDH3-CAT reporter gene constructs possessing a mutation in either the ARNT- or HNF4-DNA binding sites of the XRE confirms the functional importance of these sequence motifs in constitutive ALDH3 expression.

Involvement of MAP kinase in the cyclic AMP induction of myelin basic protein gene expression.

Cyclic AMP is involved in the differentiation of oligodendrocyte and Schwann cell progenitors into mature myelin producing cells. The involvement of MAP kinases in this pathway was investigated in the D6P2T cell line. This cell line can be induced to display a differentiated phenotype characterized by myelin basic protein gene expression by increased cyclic AMP. Blocking MAP kinase activity with inhibitors of the activating kinase, MEK, by expression of a dominant negative MAP kinase or by expression of the MAP kinase inactivating phosphatase Mkp-1 all blocked the activation of the myelin basic protein promoter in D6P2T cells. In addition, blocking MAP kinase activation during differentiation of an oligodendrocyte-like cell line, CG4, also leads to inhibition of MBP expression. These findings suggest a role for MAP kinase in the cyclic AMP stimulated expression of the myelin basic protein gene during differentiation.

Cyclin-dependent kinase inhibitor p27kip1 is expressed at high levels in cells that express a myelinating phenotype.

Terminal cellular differentiation is generally accompanied by exit from the cell cycle but the molecular basis of how the two events are coupled is poorly understood. In the central nervous system (CNS) the terminally differentiated, non-proliferating myelin-synthesizing cells, oligodendrocytes, arise from stem cells that are proliferation competent. To study the molecular mechanisms that link oligodendrocyte differentiation and cell cycle control, the D6P2T cell line has been used. This cell line responds similarly to oligodendrocytes in culture in response to increased cyclic AMP (cAMP). Upon increasing cAMP levels, D6P2T cells increase transcription of the endogenous myelin basic protein (MBP) gene. The increase in MBP gene transcription is accompanied by withdrawal of the cells from the cell cycle. The mechanism of cell cycle withdrawal in response to cAMP was found to involve a dramatic increase in the level of the cyclin-dependent kinase (cdk) inhibitor p27kip1 with little or no change in the levels of the cyclins D1 and E. The increase in p27kip1 is at least partially attributable to an increase in the mRNA levels for p27kip1. A striking increase in the cdk inhibitor p27kip1 was also shown to occur in vivo in oligodendrocytes, the cells responsible for myelination in the CNS. In contrast to D6P2T cells, however, this increase in p27kip1 was accompanied by a decrease in the levels of cyclin E.

Phosphatidylinositol 3-kinase inhibitor wortmannin blocks mitogenic activation of the transferrin receptor gene promoter in late G1.

Expression of the transferrin receptor is necessary for cells to progress through S-phase. The transferrin receptor gene promoter is activated as a delayed event following growth factor stimulation of quiescent fibroblasts. Serum stimulation in the presence of vanadate leads to superactivation of the transferrin receptor promoter, suggesting a role for tyrosine phosphorylation. Wortmannin, a potent inhibitor of phosphatidylinositol 3-kinase, a tyrosine kinase-regulated enzyme, blocks mitogen-dependent activation of the transferrin receptor promoter. Furthermore, wortmannin was able to block activation of this promoter when added several hours after serum stimulation of quiescent cells. This suggests that phosphatidylinositol 3-kinase may be required in mid to late G1 and that it is directly involved in a pathway leading to activation of the transferrin receptor promoter. This is further supported by the finding that the transferrin receptor promoter is much less responsive to mitogenic stimulation in cells that have been stably transfected with a dominant negative form of the phosphatidylinositol 3-kinase regulatory subunit. Activation of S6 kinase, an event known to be downstream of phosphatidylinositol 3-kinase activation, appears not to be involved in activation of the transferrin receptor promoter since no effect was observed by treatment of cells with rapamycin.

Characterization of the rat Class 3 aldehyde dehydrogenase gene promoter.

The Class 3 aldehyde dehydrogenase gene (ALDH-3) is differentially expressed. Expression is either constitutive or xenobiotic inducible via an aromatic hydrocarbon (Ah) receptor-mediated pathway, depending upon the tissue. A series of studies were performed to examine the regulation of rat ALDH-3 basal expression. DNase I footprint analysis identified four DNA regions within the proximal 1 kb of the 5' flanking region of rat ALDH-3 which interact with regulatory proteins. Reporter gene and gel mobility shift assays indicate that Sp1-like proteins interact with two proximal DNase I footprinted sites to confer strong promoter activity. Two distal DNase I footprinted sites are found within a region that inhibits rat ALDH-3 promoter activity. This negative region is bound by NF1-like proteins and/or unique proteins. This 1 kb 5' flanking region of rat ALDH-3 may act constitutively in many cell types. In contrast with other Ah receptor regulated genes, no DNA elements or transcription factors acting within this region appear to be involved in regulating xenobiotic-inducible expression of rat ALDH-3.

Mitogenic activation of the transferrin receptor gene promoter is modulated by inhibitors of tyrosine kinases and tyrosine phosphatases.

Transferrin receptor gene expression is coupled to cell proliferation of normal cells and is elevated in nearly all types of tumor cells. A mitogen-responsive region of the transferrin receptor gene promoter has been localized between -78 and -34 relative to the major transcriptional start. The promoter can be activated in quiescent fibroblasts by treatment with either vanadate or phenylarsine oxide, both of which are inhibitors of tyrosine phosphatases and lead to elevated levels of intracellular tyrosine-phosphorylated proteins. Vanadate can act synergistically with other mitogens and, when added together with serum, leads to superactivation of the promoter. Genistein, an inhibitor of certain tyrosine kinases, actually enhances promoter activity in cells treated with either vanadate or phenylarsine oxide. On the other hand, geldanamycin, which also reduces the level of tyrosine-phosphorylated proteins and promoters the morphological reversion of many transformed cell types, is a potent inhibitor of transferrin receptor promoter activation by mitogens. The differential effects of these two tyrosine kinase inhibitors is most likely caused by the specificity of the enzymes that they target. These results indicate that a tyrosine phosphorylation event plays a critical role in the signaling events that lead to activation of the transferrin receptor gene promoter in mitogen-stimulated cells. This is of interest because activation of this promoter is a delayed response that occurs several hours after mitogen addition.

Mitogen induction of nuclear factors that interact with a delayed responsive region of the transferrin receptor gene promoter.

Activation of the human transferrin receptor promoter by mitogenic stimulation of quiescent cells is a delayed event that reaches a maximum several hours after stimulation. Previous results have defined a region of the transferrin receptor gene promoter that is required for increased expression in mitogen-activated cells (W. K. Miskimins and D. B. Brown, Exp. Cell Res., 191: 328-331, 1990; Q. Ouyang et al., Mol. Cell. Biol., 13: 1796-1804, 1993). This region contains two elements (elements A and B) that appear to cooperate in the response to mitogenic stimulation. Serum stimulation of quiescent cells leads to the induction of nuclear factors that bind to both the A and B elements. Induction of these factors is also a delayed response to serum stimulation and reaches a maximum 6-9 h after stimulation. Element A, which is an unusual GC-rich sequence, forms several serum-inducible DNA-protein complexes, all of which depend on contacts within GC boxes. A major inducible complex of element A contains a factor that is supershifted by antibodies against the transcription factor Sp1. The B element appears to have overlapping binding sites for two types of factors. One of these sites binds factors that are competed off by an AP-1 consensus-binding site. The other B element site binds inducible factors that interact with GC boxes, identical to those observed for element A.

Ultraviolet light and serum induce similar delayed responses that lead to activation of a mitogen-responsive promoter.

The transferrin receptor promoter is responsive to growth factors and mitogens. This induction is a delayed response and does not occur until several hours after stimulation of quiescent cells by mitogens. The results described here show that the transferrin receptor promoter is also activated by treatment with ultraviolet (UV) light. Activation of the promoter by UV light is dose dependent and requires the same cis-acting elements that are activated in response to serum and other mitogens. As with serum stimulation, activation of the promoter by UV light is a delayed event and is not initiated until 6 h after treatment. This coincides with the induction of nuclear factors that bind with specificity to the required cis-acting elements. A major GC-box binding factor induced by UV light is also induced by serum and has been shown to be supershifted by antibodies to the Sp1 transcription factor.

A mitogen-responsive promoter region that is synergistically activated through multiple signalling pathways.

A regulatory region of the human transferrin receptor gene promoter was found to be required for increased expression in response to serum or growth factors. This region contains two elements that appear to cooperate for full responsiveness. We found that sodium orthovanadate treatment of cells significantly activated expression of promoter constructs containing these elements. 12-O-Tetradecanoylphorbol-13-acetate alone induced a twofold increase in expression but acted synergistically with vanadate to generate a highly elevated level of expression. Dibutyryl cyclic AMP alone had no effect on expression, but when added together with vanadate and 12-O-tetradecanoylphorbol-13-acetate, led to superinduction of the promoter construct. Induction of expression by these reagents was delayed several hours, and the kinetics were identical to those observed for serum induction.

Interaction of multiple factors with a GC-rich element within the mitogen responsive region of the human transferrin receptor gene.

Nuclear factors from HeLa cells were isolated by elution of DNA-cellulose bound proteins with a double stranded synthetic oligonucleotide corresponding to the region from -34 to -79 of the human transferrin receptor (TR) gene promoter. The eluted proteins were further purified and separated from the oligonucleotide by ion exchange chromatography. Proteins within the resulting fraction bound with specificity to the TR promoter. Retardation gel analysis and competition with specific double-stranded oligonucleotides show that multiple factors present in this fraction compete for binding within the same region of the TR promoter. Footprinting experiments demonstrate that these factors contact a GC-rich element that is within the region that is required for enhanced expression of the gene in proliferating cells. One of the factors protects an extended DNA sequence but still contacts the GC-rich element.

A supercoil-dependent structural alteration within the regulatory region of the human transferrin receptor gene.

The transferrin receptor gene is transcribed at low levels in quiescent cells and at much higher levels in growing or transformed cells. This regulation involves elements located within the first 114 base pairs upstream of the major transcriptional start site. This region is specifically recognized by several transacting factors and contains an element that is composed of alternating purines and pyrimidines. In vitro this element can adopt a non-B DNA conformation in a supercoil-dependent manner. Similar elements, with nearly identical spacing relative to a protein recognition sequence, can be observed in several other proliferation dependent gene promoters.

Partial purification of Xenopus laevis egg extract factor(s) that induce swelling in permeabilized human sperm.

A combination of adsorption (protamine-agarose) and gel filtration (Sephacryl S-300) chromatography was used to enrich for factor(s) in Xenopus laevis frog egg extract that induce nuclear swelling-chromatin decondensation in permeabilized human sperm. It was determined that a 70-fold purification of the factor(s) that induce human sperm nuclear swelling has resulted from the purification scheme. The reduced, active factor(s) have a Kav of 0.12 and an approximate molecular weight of 290,000 daltons. The extract nuclear swelling activity is sensitive to temperatures of 50 degrees C and above, as well as proteolytic treatment. RNase and cycloheximide treatments of the extracts have no effect on the nuclear swelling activity. These data suggest that the egg extract factor(s) that induce nuclear swelling in permeabilized human sperm are protein(s) that are present in the unfertilized frog egg.

Analysis of cis-acting promoter elements using microinjected synthetic oligonucleotides.

The transferrin receptor (TR) is expressed in a proliferation-dependent manner that requires transcriptional activation of its gene. Furthermore TR function is required for the DNA synthetic phase of the cell cycle. Oligonucleotides encoding promoter elements within the human transferrin receptor gene were injected into quiescent fibroblasts. The cells were then serum stimulated and the effect of the oligonucleotides on nuclear labeling with [3H]thymidine was determined. Inhibition was observed for oligonucleotides corresponding to a specific region of the promoter.