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The Y-box binding protein 1 (YB-1) belongs to the cold-shock domain protein superfamily, one of the most evolutionarily conserved nucleic acid-binding proteins currently known. YB-1 performs a wide variety of cellular functions, including transcriptional and translational regulation, DNA repair, drug resistance, and stress responses to extracellular signals. Inasmuch as the level of YB-1 drastically increases in tumor cells, this protein is considered to be one of the most indicative markers of malignant tumors. Here, we present evidence that ΔNp63α, the predominant p63 protein isoform in squamous epithelia and YB-1, can physically interact. Into the nucleus, ΔNp63α and YB-1 cooperate in PI3KCA gene promoter activation. Moreover, ΔNp63α promotes YB-1 nuclear accumulation thereby reducing the amount of YB-1 bound to its target transcripts such as that encoding the SNAIL1 protein. Accordingly, ΔNp63α enforced expression was associated with a reduction of the level of SNAIL1, a potent inducer of epithelial to mesenchymal transition. Furthermore, ΔNp63α depletion causes morphological change and enhanced formation of actin stress fibers in squamous cancer cells. Mechanistic studies indicate that ΔNp63α affects cell movement and can reverse the increase of cell motility induced by YB-1 overexpression. These data thus suggest that ΔNp63α provides inhibitory signals for cell motility. Deficiency of ΔNp63α gene expression promotes cell mobilization, at least partially, through a YB-1-dependent mechanism.

Background: γ-Tubulin moderates the expression of E2F-regulated promoters by direct binding to DNA. Results: RB1 and γ-tubulin proteins moderate each other's expression by binding to their respective gene promoters. Conclusion: Reduction of γ-tubulin protein levels in tumors with nonfunctional RB1 leads to induction of apoptosis. Significance: The RB1/γ-tubulin signal network can be considered as a new target for cancer treatment., In various tumors inactivation of growth control is achieved by interfering with the RB1 signaling pathway. Here, we describe that RB1 and γ-tubulin proteins moderate each other's expression by binding to their respective gene promoters. Simultaneous reduction of RB1 and γ-tubulin protein levels results in an E2F1-dependent up-regulation of apoptotic genes such as caspase 3. We report that in various tumors types, there is an inverse correlation between the expression levels of γ-tubulin and RB1 and that in tumor cell lines with a nonfunctioning RB1, reduction of γ-tubulin protein levels leads to induction of apoptosis. Thus, the RB1/γ-tubulin signal network can be considered as a new target for cancer treatment.

The human SLC39A13 gene encodes ZIP13, a member of the LZT (LIV-1 subfamily of ZIP zinc transporters) family. The ZIP13 protein is important for connective tissue development, and its loss of function is causative for the spondylocheiro dysplastic form of Ehlers-Danlos syndrome. However, this protein has not been characterized in detail. Here we report the first detailed biochemical characterization of the human ZIP13 protein using its ectopic expressed and the purified recombinant protein. Protease accessibility, microscopic, and computational analyses demonstrated that ZIP13 contains eight putative transmembrane domains and a unique hydrophilic region and that it resides with both its N and C termini facing the luminal side on the Golgi. Analyses including cross-linking, immunoprecipitation, Blue Native-PAGE, and size-exclusion chromatography experiments indicated that the ZIP13 protein may form a homo-dimer. We also demonstrated that ZIP13 mediates zinc influx, as assessed by monitoring the expression of the metallothionein gene and by detecting the intracellular zinc level with a zinc indicator, FluoZin-3. Our data indicate that ZIP13 is a homo-dimerized zinc transporter that possesses some domains that are not found in other LZT family members. This is the first biochemical characterization of the physiologically important protein ZIP13 and the demonstration of homo-dimerization for a mammalian ZIP zinc transporter family member. This biochemical characterization of the human ZIP13 protein provides important information for further investigations of its structural characteristics and function.

Recently, mitochondria have been suggested to act in tumor suppression. However, the underlying mechanisms by which mitochondria suppress tumorigenesis are far from being clear. In this study, we have investigated the link between mitochondrial dysfunction and the tumor suppressor protein p53 using a set of respiration-deficient (Res(-)) mammalian cell mutants with impaired assembly of the oxidative phosphorylation machinery. Our data suggest that normal mitochondrial function is required for γ-irradiation (γIR)-induced cell death, which is mainly a p53-dependent process. The Res(-) cells are protected against γIR-induced cell death due to impaired p53 expression/function. We find that the loss of complex I biogenesis in the absence of the MWFE subunit reduces the steady-state level of the p53 protein, although there is no effect on the p53 protein level in the absence of the ESSS subunit that is also essential for complex I assembly. The p53 protein level was also reduced to undetectable levels in Res(-) cells with severely impaired mitochondrial protein synthesis. This suggests that p53 protein expression is differentially regulated depending upon the type of electron transport chain/respiratory chain deficiency. Moreover, irrespective of the differences in the p53 protein expression profile, γIR-induced p53 activity is compromised in all Res(-) cells. Using two different conditional systems for complex I assembly, we also show that the effect of mitochondrial dysfunction on p53 expression/function is a reversible phenomenon. We believe that these findings will have major implications in the understanding of cancer development and therapy.

Glutathione peroxidase 4 (Gpx4) is an essential antioxidant enzyme having multiple functions. A long form Gpx4 protein and a short form Gpx4 protein, which are distinguishable by the presence or lack of a mitochondrial signal peptide at the N terminus, are generated from the Gpx4 gene. In this study, we generated transgenic mice using mutated GPX4 genes encoding either the long form Gpx4 (lGPX4 gene) or the short form Gpx4 (sGPX4 gene). Our results showed that transgenic mice with the sGPX4 gene had increased Gpx4 protein in all tissues and were protected against diquat-induced apoptosis in liver. Moreover, the sGPX4 gene was able to rescue the lethal phenotype of the mouse Gpx4-null mutation. In contrast, transgenic mice with the lGPX4 gene had increased Gpx4 protein only in the testes, and the lGPX4 gene failed to rescue the lethal phenotype of the mouse Gpx4-null mutation. In Gpx4-null mice rescued by the sGPX4 gene, the Gpx4 protein was present in mitochondria isolated from somatic tissues, and the submitochondrial distribution pattern of the Gpx4 protein in these mice was identical to that in wild-type mice. Interestingly, the male Gpx4-null mice rescued by the sGPX4 gene were infertile and exhibited sperm malformation. Together, our results demonstrated for the first time that the short form Gpx4 protein is present in somatic tissue mitochondria and is essential for survival and protection against apoptosis in mice, whereas the long form Gpx4 protein is important for male fertility.

To determine the mechanisms of spermatogenesis, it is essential to identify and characterize germ cell-specific genes. Here we describe a protein encoded by a novel germ cell-specific gene, Mm.290718/ZFP541, identified from the mouse spermatocyte UniGene library. The protein contains specific motifs and domains potentially involved in DNA binding and chromatin reorganization. An antibody against Mm.290718/ZFP541 revealed the existence of the protein in testicular spermatogenic cells (159 kDa) but not testicular and mature sperm. Immunostaining analysis of cells at various stages of spermatogenesis consistently showed that the protein is present in spermatocytes and round spermatids only. Transfection assays and immunofluorescence studies indicate that the protein is localized specifically in the nucleus. Proteomic analyses performed to explore the functional characteristics of Mm.290718/ZFP541 showed that the protein forms a unique complex. Other major components of the complex included histone deacetylase 1 (HDAC1) and heat-shock protein A2. Disappearance of Mm.290718/ZFP541 was highly correlated with hyperacetylation in spermatids during spermatogenesis, and specific domains of the protein were involved in the regulation of interactions and nuclear localization of HDAC1. Furthermore, we found that premature hyperacetylation, induced by an HDAC inhibitor, is associated with an alteration in the integrity of Mm.290718/ZFP541 in spermatogenic cells. Our results collectively suggest that the Mm.290718/ZFP541 complex is implicated in chromatin remodeling during spermatogenesis, and we provide further information on the previously unknown molecular mechanism. Consequently, we re-designate Mm.290718/ZFP541 as “SHIP1” representing spermatogenic cell HDAC-interacting protein 1.

B23 (NPM/nucleophosmin) is a multifunctional nucleolar protein and a member of the nucleoplasmin superfamily of acidic histone chaperones. B23 is essential for normal embryonic development and plays an important role in genomic stability, ribosome biogenesis, and anti-apoptotic signaling. Altered protein expression or genomic mutation of B23 is encountered in many different forms of cancer. Although described as multifunctional, a genuine molecular function of B23 is not fully understood. Here we show that B23 is associated with a protein complex consisting of ribosomal proteins and ribosome-associated RNA helicases. A novel, RNA-independent interaction between ribosomal protein S9 (RPS9) and B23 was further investigated. We found that S9 binding requires an intact B23 oligomerization domain. Depletion of S9 by small interfering RNA resulted in decreased protein synthesis and G1 cell cycle arrest, in association with induction of p53 target genes. We determined that S9 is a short-lived protein in the absence of ribosome biogenesis, and proteasomal inhibition significantly increased S9 protein level. Overexpression of B23 facilitated nucleolar storage of S9, whereas knockdown of B23 led to diminished levels of nucleolar S9. Our results suggest that B23 selectively stores, and protects ribosomal protein S9 in nucleoli and therefore could facilitate ribosome biogenesis.

To effectively direct targeted repression, the class I histone deacetylases (HDACs) associate with many important regulatory proteins. In this paper we describe the molecular characterization of a member of the Jumonji domain 2 (JMJD2) family of proteins, and demonstrate its binding to both class I HDACs and the retinoblastoma protein (pRb). JMJD2 proteins are characterized by the presence of two leukemia-associated protein/plant homeodomain (LAP/PHD) zinc fingers, one JmjN, one JmjC (containing an internal retinoblastoma-binding protein 2 (RBBP2)-like sequence), and two Tudor domains. The first member of this group, JMJD2A, is widely expressed in human tissues and cell lines, and high endogenous expression of JMJD2A mRNA was found in several cell types, including human T-cell lymphotropic virus 1 (HTLV-1)-infected cell lines. JMJD2A and JMJD2B exhibit cell type-specific responses to the HDAC inhibitor trichostatin A. We show that the JMJD2A protein associates in vivo with pRb and class I HDACs, and mediates repression of E2F-regulated promoters. In HTLV-1 virus-infected cells, we find that JMJD2A binds to the viral Tax protein. Antibodies to JMJD2A recognize the native protein but also a half-sized protein fragment, the latter up-regulated in THP-1 cells during the G(2)/M phase of the cell cycle. The ability of JMJD2A to associate with pRb and HDACs and potentiate pRb-mediated repression of E2F-regulated promoters implies an important role for this protein in cell proliferation and oncogenesis.

Members of the Sir2 family of NAD-dependent protein deacetylases regulate diverse cellular processes including aging, gene silencing, and cellular differentiation. Here, we report that the distant mammalian Sir2 homolog SIRT6 is a broadly expressed, predominantly nuclear protein. Northern analysis of embryonic samples and multiple adult tissues revealed mouse SIRT6 (mSIRT6) mRNA peaks at day E11, persisting into adulthood in all eight tissues examined. At the protein level, mSIRT6 was readily detectable in the same eight tissue types, with the highest levels in muscle, brain, and heart. Subcellular localization studies using both C- and N-terminal green fluorescent protein fusion proteins showed mSIRT6-green fluorescent protein to be a predominantly nuclear protein. Indirect immunofluorescence using antibodies to two different mSIRT6 epitopes confirmed that endogenous mSIRT6 is also largely nuclear. Consistent with previous findings, we did not observe any NAD+-dependent protein deacetylase activity in preparations of mSIRT6. However, purified recombinant mSIRT6 did catalyze the robust transfer of radiolabel from [32P]NAD to mSIRT6. Two highly conserved residues within the catalytic core of the protein were required for this reaction. This reaction is most likely mono-ADP-ribosylation because only the modified form of the protein was recognized by an antibody specific to mono-ADP-ribose. Surprisingly, we observed that the catalytic mechanism of this reaction is intra-molecular, with individual molecules of mSIRT6 directing their own modification. These results provide the first characterization of a Sir2 protein from phylogenetic class IV.

The protein RAS association domain family protein 1A (RASSF1A), which is encoded by a gene that is frequently silenced in many types of sporadic tumor, functions in mitosis as a regulator of the anaphase-promoting complex (APC). With the use of a yeast two-hybrid screen, we identified a human protein, previously designated C19ORF5, that interacts with RASSF1A. This protein, here redesignated RASSF1A-binding protein 1 (RABP1), contains two microtubule-associated protein domains, and its association with RASSF1A was confirmed in mammalian cells by immunoprecipitation and immunofluorescence analyses. RABP1 was found to be localized to the centrosome throughout the cell cycle in a manner dependent on its microtubule-associated protein domains. Ectopic expression of RABP1 induced both stabilization of mitotic cyclins and mitotic arrest at prometaphase in a RASSF1A-dependent manner. It also increased the extent of association between RASSF1A and Cdc20. Conversely depletion of RABP1 by RNA interference prevented both the localization of RASSF1A to the spindle poles as well as its binding to Cdc20, resulting in premature destruction of mitotic cyclins and acceleration of mitotic progression. These findings indicate that RABP1 is required for the recruitment of RASSF1A to the spindle poles and for its inhibition of APC-Cdc20 activity during mitosis.

Gene 4 protein (gp4) of bacteriophage T7 provides two essential functions at the T7 replication fork, primase and helicase activities. Previous studies have shown that the single-stranded DNA-binding protein of T7, encoded by gene 2.5, interacts with gp4 and modulates its multiple functions. To further characterize the interactions between gp4 and gene 2.5 protein (gp2.5), we have examined the effect of wild-type and altered gene 2.5 proteins as well as Escherichia coli single-stranded DNA-binding (SSB) protein on the ability of gp4 to synthesize primers, hydrolyze dTTP, and unwind duplex DNA. Wild-type gp2.5 and E. coli SSB protein stimulate primer synthesis and DNA-unwinding activities of gp4 at low concentrations but do not significantly affect single-stranded DNA-dependent hydrolysis of dTTP. Neither protein inhibits the binding of gp4 to single-stranded DNA. The variant gene 2.5 proteins, gp2.5-F232L and gp2.5-Delta26C, inhibit primase, dTTPase, and helicase activities proportional to their increased affinities for DNA. Interestingly, wild-type gp2.5 stimulates the unwinding activity of gp4 except at very high concentrations, whereas E. coli SSB protein is highly inhibitory at relative low concentrations.

Smad4 is a key intracellular mediator for the transforming growth factor-beta (TGF-beta) superfamily of growth factors and is also an important tumor suppressor. The receptor-regulated Smad (R-Smad) proteins are regulated by ubiquitin-mediated degradation, yet the precise control of Smad4 protein stability is unclear. We have identified SCF(beta-TrCP1), a ubiquitin (E3) ligase, as a critical determinant for the protein degradation of Smad4 protein. F-box protein beta-TrCP1 in this E3 ligase interacts with Smad4 both in yeast and in mammalian cells, but has no interaction with Smad2 and has weak interaction with Smad3. The beta-TrCP1/Smad3 interaction was abolished by Smad4 gene silencing, indicating the interaction is indirect and is through Smad4. Ectopic expression of SCF complex containing beta-TrCP1 is sufficient to induce the ubiquitination and degradation of Smad4. Furthermore, small interfering RNA-triggered endogenous beta-TrCP1 suppression increases the expression of Smad4 protein. Consistent with these results, cells that overexpress the SCF complex display an inhibited TGF-beta-dependent transcriptional activity and an impaired cell cycle arrest function. Thus, SCF(beta-TrCP1) abrogates TGF-beta function in vivo by decreasing Smad4 stability.

To study the mechanisms of protein secretion as well as the cellular responses to impaired protein folding and transport in filamentous fungi, we have analyzed Trichoderma reesei cultures treated with chemical agents that interfere with these processes, dithiothreitol, brefeldin A, and the Ca2+-ionophore A23187. The effects of the drugs on the kinetics of protein synthesis and transport were characterized using metabolic labeling of synthesized proteins. Cellobiohydrolase I (CBHI, Cel7A), the major secreted cellulase, was analyzed as a model protein. Northern analysis showed that under conditions where protein transport was inhibited (treatments with dithiothreitol or brefeldin A) the unfolded protein response pathway was activated. The active form of the hac1 mRNA that mediates unfolded protein response signaling was induced, followed by induction of the foldase and chaperone genes pdi1 and bip1. Concomitant with the activation of the unfolded protein response pathway, the transcript levels of genes encoding secreted proteins, like cellulases and xylanases, were drastically decreased, suggesting a novel type of feedback mechanism activated in response to impairment in protein folding or transport (repression under secretion stress (RESS)). By studying expression of the reporter gene lacZ under cbh1 promoters of different length, it was shown that the feedback response was mediated through the cellulase promoter.

PTEN phosphatase is one of the most commonly targeted tumor suppressors in human cancers and a key regulator of cell growth and apoptosis. We have found that PTEN is cleaved by caspase-3 at several target sites, located in unstructured regions within the C terminus of the molecule. Cleavage of PTEN was increased upon TNFalpha-cell treatment and was negatively regulated by phosphorylation of the C-terminal tail of PTEN by the protein kinase CK2. The proteolytic PTEN fragments displayed reduced protein stability, and their capability to interact with the PTEN interacting scaffolding protein S-SCAM/MAGI-2 was lost. Interestingly, S-SCAM/MAGI-2 was also cleaved by caspase-3. Our findings suggest the existence of a regulatory mechanism of protein stability and PTEN-protein interactions during apoptosis, executed by caspase-3 in a PTEN phosphorylation-regulated manner.

The p53 family includes three members that share significant sequence homology, yet exhibit fundamentally different functions in tumorigenesis. Whereas p53 displays all characteristics of a classical tumor suppressor, its homologues p63 and p73 do not. We have previously shown, that NH(2)-terminally truncated isoforms of p73 (Delta TA-p73), which act as dominant-negative inhibitors of p53 are frequently overexpressed in cancer cells. Here we provide evidence that Delta TA-p73 isoforms also affect the retinoblastoma protein (RB) tumor suppressor pathway independent of p53. Delta TA-p73 isoforms inactivate RB by increased phosphorylation, resulting in enhanced E2F activity and proliferation of fibroblasts. By inactivating the two major tumor suppressor pathways in human cells they act functionally analogous to several viral oncoproteins. These findings provide an explanation for the fundamentally different functions of p53 and p73 in tumorigenesis.

Gene 17 of the Bacillus subtilis phage Φ29 is expressed early after infection, and it has been shown to be required at the very beginning of phage replication under conditions of low but not high multiplicity of infection. It has been proposed that, at the beginning of the infection, protein p17 could be recruiting limiting amounts of initiation factors at the viral origins. Once the infection process is established and the replication proteins reach optimal concentration, protein p17 becomes dispensable. In this paper we focused, on the one hand, on the study of protein p17 dimerization and the role of a putative coiled-coil region. On the other hand, we focused on its interaction with the viral origin-binding protein p6. Based on our results we propose that protein p17 function is to optimize binding of protein p6 at the viral DNA ends, thus favoring the initiation of replication and negatively modulating its own transcription.

The serum and glucocorticoid-induced protein kinase gene (sgk-1) encodes a multifunctional kinase that can be phosphorylated and activated through a phosphatidylinositol 3-kinase-dependent signaling pathway. In many cell types, endogenous SGK-1 steady-state protein levels are very low but can be acutely up-regulated after glucocorticoid receptor-mediated transcriptional activation; in breast epithelial and cancer cell lines, this up-regulation is associated with promotion of cell survival. We and others have noted that ectopically introduced full-length SGK-1 is poorly expressed, although SGK-1 lacking the first 60 amino acids (delta60SGK-1) is expressed at much higher-fold protein levels than wild-type SGK-1 in both human embryonic kidney 293T and MCF10A mammary epithelial cells. In this report, we demonstrate for the first time that the low steady-state expression level of SGK-1 is due to polyubiquitination and subsequent degradation by the 26S proteasome. Deletion of the amino-terminal 60 amino acids of SGK-1 results in a mutant SGK-1 protein that is neither efficiently polyubiquitinated nor degraded by the 26S proteasome, accounting for the higher steady-state levels of the truncated protein. We also demonstrate that a subset of SGK-1 localizes to the plasma membrane and that the polyubiquitin-modified SGK-1 localizes to a membrane-associated fraction of the cell. Taken together, these data suggest that a significant fraction of SGK-1 is membrane-associated and ubiquitinated. These findings are consistent with the recently described role of SGK-1 in phosphorylating the membrane-associated protein Nedd4-2 and the integral membrane Na+/H+ exchanger isoform 3 (NHE3) and suggest a novel mechanism of regulation of SGK-1.

POB1 was previously identified as a RalBP1-binding protein. POB1 and RalBP1 function downstream of small G protein Ral and regulate receptor-mediated endocytosis. To look for additional functions of POB1, we screened for POB1-binding proteins using a yeast two-hybrid method and found that POB1 interacts with mouse ASAP1, which is a human PAG2 homolog. PAG2 is a paxillin-associated protein with ADP-ribosylation factor GTPase-activating protein activity. POB1 formed a complex with PAG2 in intact cells. The carboxyl-terminal region containing the proline-rich motifs of POB1 directly bound to the carboxyl-terminal region including the SH3 domain of PAG2. Substitutions of Pro(423) and Pro(426) with Ala (POB1(PA)) impaired the binding of POB1 to PAG2. Expression of PAG2 inhibited fibronectin-dependent migration and paxillin recruitment to focal contacts of CHO-IR cells. Co-expression with POB1 but not with POB1(PA) suppressed the inhibitory action of PAG2 on cell migration and paxillin localization. These results suggest that POB1 interacts with PAG2 through its proline-rich motif, thereby regulating cell migration.

A physical and functional interaction between the Ca2+-binding protein Mts1 (S100A4) and the tumor suppressor p53 protein is shown here for the first time. We demonstrate that Mts1 binds to the extreme end of the C-terminal regulatory domain of p53 by several in vitro and in vivoapproaches: co-immunoprecipitation, affinity chromatography, and far Western blot analysis. The Mts1 protein in vitro inhibits phosphorylation of the full-length p53 and its C-terminal peptide by protein kinase C but not by casein kinase II. The Mts1 binding to p53 interferes with the DNA binding activity of p53 in vitro and reporter gene transactivation in vivo, and this has a regulatory function. A differential modulation of the p53 target gene (p21/WAF, bax, thrombospondin-1, and mdm-2) transcription was observed upon Mts1 induction in tet-inducible cell lines expressing wild type p53. Mts1 cooperates with wild type p53 in apoptosis induction. Our data imply that the ability of Mts1 to enhance p53-dependent apoptosis might accelerate the loss of wild type p53 function in tumors. In this way, Mts1 can contribute to the development of a more aggressive phenotype during tumor progression.

p120 GTPase-activating protein (GAP) down-regulates Ras by stimulating GTP hydrolysis of active Ras. In addition to its association with Ras, GAP has been shown to bind to several tyrosine-phosphorylated proteins in cells stimulated by growth factors or expressing transforming tyrosine kinase variants. Here we report the cloning and characterization of a novel GAP-binding protein, mTid-1, a DnaJ chaperone protein that represents the murine homolog of the Drosophila tumor suppressor l(2)tid gene. Three alternatively spliced variants of mTid-1 were isolated, two of which correspond to the recently identified hTid-1(L) and hTid-1(S) forms of the human TID1 gene that exhibit opposing effects on apoptosis. We demonstrate that both cytoplasmic precursor and mitochondrial mature forms of mTid-1 associate with GAP in vivo. Interestingly, although mTid-1 is found tyrosine-phosphorylated in v-src-transformed fibroblast cells, GAP selectively binds to the unphosphorylated form of mTid-1. In immunofluorescence experiments, GAP and Tid-1 were shown to colocalize at perinuclear mitochondrial membranes in response to epidermal growth factor stimulation. These findings raise the possibility that Tid chaperone proteins may play a role in governing the conformation, activity, and/or subcellular distribution of GAP, thereby influencing its biochemical and biological activity within cells.

Overexpression of the transcription factor YY1 activates DNA synthesis in differentiated primary human coronary artery smooth muscle cells. Overexpression of the retinoblastoma protein together with YY1 blocked this effect. In growth-arrested cells, YY1 resides in a complex with the retinoblastoma protein, but the complex is not detected in serum-stimulated S phase cultures, indicating that the interaction of the retinoblastoma protein and YY1 is cell cycle-regulated. Recombinant retinoblastoma protein directly interacts with YY1, destabilizing the interaction of YY1 with DNA and inhibiting its transcription initiator function in vitro. We conclude that in differentiated cells elevation of the nuclear level of YY1 protein favors progression into the S phase, and we propose that this activity is regulated by its interaction with the retinoblastoma protein.

The catalytic subunits of protein kinase CK2, CK2alpha and CK2alpha', are closely related to each other but exhibit functional specialization. To test the hypothesis that specific functions of CK2alpha and CK2alpha' are mediated by specific interaction partners, we used the yeast two-hybrid system to identify CK2alpha- or CK2alpha'-binding proteins. We report the identification and characterization of a novel CK2-interacting protein, designated CKIP-1, that interacts with CK2alpha, but not CK2alpha', in the yeast two-hybrid system. CKIP-1 also interacts with CK2alpha in vitro and is co-immunoprecipitated from cell extracts with epitope-tagged CK2alpha and an enhanced green fluorescent protein fusion protein encoding CKIP-1 (i.e. EGFP-CKIP-1) when they are co-expressed. CK2 activity is detected in anti-CKIP-1 immunoprecipitates performed with extracts from non-transfected cells indicating that CKIP-1 and CK2 interact under physiological conditions. The CKIP-1 cDNA is broadly expressed and encodes a protein with a predicted molecular weight of 46,000. EGFP-CKIP-1 is localized within the nucleus and at the plasma membrane. The plasma membrane localization is dependent on the presence of an amino-terminal pleckstrin homology domain. We postulate that CKIP-1 is a non-enzymatic regulator of one isoform of CK2 (i.e. CK2alpha) with a potential role in targeting CK2alpha to a particular cellular location.

Brain-enriched isoforms of skeletal proteins in the spectrin and ankyrin gene families have been described. Here we characterize protein 4.1B, a novel homolog of erythrocyte protein 4.1R that is encoded by a distinct gene. In situ hybridization revealed high level, focal expression of 4.1B mRNA in select neuronal populations within the mouse brain, including Purkinje cells of the cerebellum, pyramidal cells in hippocampal regions CA1-3, thalamic nuclei, and olfactory bulb. Expression was also detected in adrenal gland, kidney, testis, and heart. 4.1B protein exhibits high homology to the membrane binding, spectrin-actin binding, and C-terminal domains of 4.1R, including motifs for interaction with NuMA and FKBP13. cDNA characterization and Western blot analysis revealed multiple spliceoforms of protein 4.1B, with functionally relevant heterogeneity in the spectrin-actin and NuMA binding domains. Regulated alternative splicing events led to expression of unique 4. 1B isoforms in brain and muscle; only the latter possessed a functional spectrin-actin binding domain. By immunofluorescence, 4. 1B was localized specifically at the plasma membrane in regions of cell-cell contact. Together these results indicate that 4.1B transcription is selectively regulated among neuronal populations and that alternative splicing regulates expression of 4.1B isoforms possessing critical functional domains typical of other protein 4.1 family members.

Acyl-CoA-binding protein, a 20-kDa homodimer that exerts many physiological functions, promotes activation of the classic calpain forms, most markedly that of the m-isozyme. This protein factor was purified from rat skeletal muscle and was also expressed in Escherichia coli. Both native and recombinant acyl-CoA-binding proteins show the same molecular properties and an identical capacity to decrease the [Ca(2+)] required for m-calpain activity. The binding of long-chain acyl-CoAs to acyl-CoA-binding protein does not modify the activating effect on calpains. Acyl-CoA-binding protein seems to be involved in the m-calpain regulation process, whereas the previously identified UK114 activator is a specific modulator of micro-calpain. Acyl-CoA-binding protein is proposed as a new component of the Ca(2+)-dependent proteolytic system. A comparative analysis among levels of classic calpains and their activator proteins is also reported.

Overexpression of mutant p53 has been reported to promote tumorigenicity in several cancers. However, despite its potential importance, the signals regulating mutant p53 protein expression are not known. Here we show that a form of p53 that is incapable of binding DNA is overexpressed in the acute promyelocytic leukemia NB4 cell line. Our results demonstrate that treatment of NB4 cells with bryostatin-1, which induces differentiation in this cell line, leads to hyperphosphorylation of this DNA binding-impaired form of p53 via mitogen-activated protein kinase. After this phosphorylation, the p53 protein is degraded by the ubiquitin/proteasome pathway. Furthermore, we show that inhibition of p53 hyperphosphorylation blocks p53 protein degradation and cell differentiation. In addition, inhibition of the ubiquitin/proteasome pathway also blocks p53 protein degradation and cell differentiation. These findings suggest a role for mitogen-activated protein kinase in the degradation of the DNA binding-impaired form of p53 protein and in the bryostatin-induced differentiation observed in this cell line. The implications of these results with respect to the functional significance of p53 phosphorylation and degradation in cell differentiation are discussed.

The Wilms' tumor suppressor gene, wt1, encodes a zinc-finger transcription factor, WT1, that plays an important role in controlling urogenital development. Previously, WT1 has been shown to inhibit cell growth and to repress transcription initiated from the promoters of a number of growth-promoting genes. However, few physiological target genes that are transcriptionally activated by WT1 have been established. Using suppression subtractive hybridization polymerase chain reaction, we isolated a WT1 target gene that is up-regulated about 15-fold in cells expressing WT1. The gene was identified as retinoblastoma suppressor (Rb)-associated protein 46 (RbAp46), a nuclear protein that interacts physically with Rb and is a component of the human mSin3 co-repressor complex. Cells transfected with RbAp46 cDNA formed fewer colonies than the control cells, and RbAp46 suppressed the growth rate (by about 2-fold) of transfected cells. In the developing kidney and gonad, RbAp46 exhibits an expression pattern similar to that of WT1. We conclude that RbAp46 has strong growth inhibition activity and may function as an important mediator of WT1's function.

In budding yeast Saccharomyces cerevisiae CDC45 is an essential gene required for initiation of DNA replication. A structurally related protein Tsd2 is necessary for DNA replication in Ustilago maydis. We have identified and cloned the gene for a human protein homologous to the fungal proteins. The human gene CDC45L is 30 kilobases long and contains 15 introns. The 16 exons encode a protein of 566 amino acids. The human protein is 52 and 49.5% similar to CDC45p and Tsd2p, respectively. The level of CDC45L mRNA peaks at G1-S transition, but total protein amount remains constant throughout the cell cycle. Consistent with a role of CDC45L protein in the initiation of DNA replication it co-immunoprecipitates from cell extracts with a putative replication initiator protein, human ORC2L. In addition, subcellular fractionation indicates that the association of the protein with the nuclear fraction becomes labile as S phase progresses. The CDC45L gene is located to chromosome 22q11.2 region by cytogenetics and by fluorescence in situ hybridization. This region, known as DiGeorge syndrome critical region, is a minimal area of 2 megabases, which is consistently deleted in DiGeorge syndrome and related disorders. The syndrome is marked by parathyroid hypoplasia, thymic aplasia, or hypoplasia and congenital cardiac abnormalities. CDC45L is the first gene mapped to the DiGeorge syndrome critical region interval whose loss may negatively affect cell proliferation.

The serum response element is one of the major promoter elements of the immediate early response to extracellular signals. The serum response element includes two main binding sites for proteins: the Ets box, which binds p62(TCF), and the CArG box, which binds p67(SRF). These two proteins are direct targets for signal transduction pathways; p62(TCF) is a nuclear end point of the Ras/mitogen-activated protein kinase pathway, and p67(SRF) is targeted by the Rho/Rac small G-proteins. The mechanism by which the signal is further transduced from the transcription factors to the basal transcriptional machinery is poorly understood. Recent data have suggested that the cAMP-responsive element-binding protein (CREB)-binding protein, a transcriptional adaptor involved in the transactivation through a wide variety of enhancer elements, participates in p62(TCF) activity. We here show that the CREB-binding protein also cooperates in the process of transactivation by p67(SRF). Cotransfections of expression vectors for the CREB-binding protein increased the expression, in response to serum, of reporters under the control of the c-fos serum response element. Interestingly, the C-terminal moiety of the CREB-binding protein was not necessary to observe this effect. The cooperation did not require the Ets box in the serum response element, and the CArG box was sufficient, indicating that the CREB-binding protein is able to cooperate with p67(SRF) in the absence of an Ets protein. Co-immunoprecipitation experiments using cell extracts showed that p67(SRF) could be retained with antibodies directed against the CREB-binding protein, suggesting that the two proteins form a multimolecular complex in live cells. The physical interaction between p67(SRF) and the CREB-binding protein was further confirmed by two-hybrid assays in mammalian cells. Our results indicate that the CREB-binding protein cooperates with p67(SRF) and, thus, suggest that the serum response element is regulated by a multimolecular complex, which includes the CREB-binding protein, p67(SRF), and p62(TCF), with multiple interactions between the components of the complex.

Distinct subcellular localization of activated protein kinase C (PKC) isozymes is mediated by their binding to isozyme-specific RACKs (receptors for activated C-kinase). Our laboratory has previously isolated one such protein, RACK1, and demonstrated that this protein displays specificity for PKCbeta. We have recently shown that at least part of the PKCepsilon RACK-binding site on PKCepsilon lies within the unique V1 region of this isozyme (Johnson, J. A., Gray, M. O., Chen, C.-H., and Mochly-Rosen, D. (1996) J. Biol. Chem. 271, 24962-24966). Here, we have used the PKCepsilon V1 region to clone a PKCepsilon-selective RACK, which was identified as the COPI coatomer protein, beta'-COP. Similar to RACK1, beta'-COP contains seven repeats of the WD40 motif and fulfills the criteria previously established for RACKs. Activated PKCepsilon colocalizes with beta'-COP in cardiac myocytes and binds to Golgi membranes in a beta'-COP-dependent manner. A role for PKC in control of secretion has been previously suggested, but this is the first report of direct protein/protein interaction of PKCepsilon with a protein involved in vesicular trafficking.

Both protein kinase C and the retinoblastoma tumor suppressor protein have been linked to the regulation of cell growth and cell death, suggesting the differential roles these factors play in mediating cell fate. In some cells, protein kinase C-induced activation of the retinoblastoma protein results in G1 arrest. However, inducible overexpression and activation of the protein kinase Calpha isozyme or the addition of 12-O-tetradecanoylphorbol-13-acetate in the prostate epithelial cell line, LNCaP, resulted in apoptosis preceded by induction of p21 and dephosphorylation of the retinoblastoma protein. Consistent with a role for the retinoblastoma growth suppressor protein in protein kinase C-induced apoptosis, DU145 cells, which do not express functional retinoblastoma protein or LNCaP cells, which have been transfected with the retinoblastoma inhibitor, E1a, were resistant to apoptosis. LNCaP apoptosis was initiated by a unique conflict between the growth-suppressive activity of the retinoblastoma protein and growth-promoting mitogenic signals. Thus, when this conflict was prevented by serum depletion, apoptosis was suppressed. The caspase family of cysteine proteases is believed to encompass the execution machinery of mammalian apoptosis, and addition of the cell-permeable caspase inhibitor, Z-Val-Ala-Asp-fluoromethylketone, afforded nearly total protection from protein kinase C-signaled apoptosis. This protection correlated with the total loss of caspase activity as measured by the proteolytic cleavage of nuclear poly(ADP-ribose) polymerase. On the basis of these results, we propose that protein kinase C regulates a novel cell death pathway that is initiated by a cellular conflict between retinoblastoma growth-suppressive signals and serum mitogenic signals in proliferating prostate epithelial cells and that is executed by the caspase family of cysteine proteases.

We have previously identified distinct nuclear and mitochondrial isoforms of dUTPase in human cells, reporting the cDNA sequence of the nuclear isoform (DUT-N). We now report a cDNA corresponding to the mitochondrial isoform (DUT-M). The DUT-M cDNA contains an 252-amino acid open reading frame, encoding a protein with a predicted M r of 26,704. The amino-terminal region of the protein contains an arginine-rich, 69-residue mitochondrial targeting presequence that is absent in the mature protein. In vitro transcription and translation of the DUT-M cDNA results in the production of a precursor protein with an apparent molecular mass of 31 kDa as judged by SDS-polyacrylamide gel electrophoresis. The DUT-M precursor is enzymatically active and immunoreacts with a dUTPase-specific monoclonal antibody. Mitochondrial import and processing studies demonstrate that the DUT-M precursor is processed into a 23-kDa protein and imported into mitochondria in vitro. Isoelectric focusing experiments demonstrate that the DUT-N has a pI of 6.0, while the processed form of DUT-M has a more basic pI of 8.1, measurements that are in agreement with predicted values. Studies aimed at understanding the expression of these isoforms were performed utilizing quiescent and replicating 34Lu human lung fibroblasts as a model cell culture system. Northern blot analysis, employing an isoform-specific probe, demonstrates that DUT-N and DUT-M are encoded by two distinct mRNA species of 1.1 and 1.4 kilobases, respectively. Western and Northern blot analysis reveal that DUT-M protein and mRNA are expressed in a constitutive fashion, independent of cell cycle phase or proliferation status. In contrast, DUT-N protein and mRNA levels are tightly regulated to coincide with nuclear DNA replication status. Because DUT-N and DUT-M have identical amino acid and cDNA sequences in their overlapping regions, we set out to determine if they were encoded by the same gene. The 5′ region of the gene encoding dUTPase was isolated and characterized by a combination of Southern hybridization and DNA sequencing. These analyses demonstrate that the dUTPase isoforms are encoded by the same gene with isoform-specific transcripts arising through the use of alternative 5′ exons. This finding represents the first example in humans of alternative 5′ exon usage to generate differentially expressed nuclear and mitochondrial specific protein isoforms.

The RAD54 and RAD51 genes are involved in genetic recombination and double-strand break repair in the yeast Saccharomyces cerevisiae. The Rad51 protein is thought to be a yeast analogue of the Eschericia coli recA gene product and catalyzes strand exchange between homologous single- and double-stranded DNAs in vitro. RAD54 exhibits homologies to several known ATPases and is a member of the SWI2/MOT1 family. We show here that the Rad54 protein interacts with the Rad51 protein in vivo and in vitro and that the NH2-terminal 115 residues of the Rad54 protein are necessary for this interaction. Combined with previously reported results, these data imply that the Rad54 protein is part of a multiprotein yeast recombination complex.

p202, an interferon-inducible murine protein, is a member of the "200 family" of proteins and is primarily nuclear. p202 is a modulator of transcription; it binds several transcription factors, including NF-kappaB p50 and p65, AP-1 c-Fos and c-Jun, and E2F1, and inhibits their transcriptional activity. p202 also binds pRb, the retinoblastoma protein, and if overexpressed it retards cell proliferation. Here we report that using the yeast two-hybrid assay we found that p202 bound the murine homolog of the human p53-binding protein 1 (53BP1), a protein shown to interact with the DNA binding domain of the p53 tumor suppressor protein. p202 bound a 98amino acid segment from 53BP1. This binding was inhibited by the replacement in p202 of a histidine (from the M(F/L)HATVA(T/S) sequence that is conserved among all of the 200 family proteins) by phenylalanine. We also report that overexpression of p202 inhibited the p53-dependent expression of reporter genes containing p53-activable segments from the mdm2 and p21 genes, whereas a decrease in the p202 level (in consequence of the expression of 202 antisense RNA) resulted in an increase in the p53-dependent expression of these reporters. Expression of the 53BP1 segment binding to p202 overcame the inhibition by overexpressed p202 of the transcription of reporters mediated by the p53 or the AP-1 transcription factors and of the proliferation of yeast.

The breast cancer susceptibility gene BRCA1 encodes an 1863-amino acid protein that acts as a tumor suppressor. The biochemical function of BRCA1 is unknown, and there are conflicting results describing its subcellular location. We have identified a 220-kDa protein, which is reactive with three antibodies raised against the amino- and carboxyl-terminal regions of BRCA1. Immunoflourescence staining with an antibody to the carboxyl terminus of BRCA1 localized the protein to the nucleus of breast, ovarian, and cervical carcinoma-derived cell lines. A similar result was observed by biochemical subcellular fractionation that indicated that the 220-kDa protein was localized primarily to the nucleus of cell lines established from breast carcinomas. In addition to the 220-kDa protein, one antibody, C-20, also recognized a 180-kDa protein in MDA-MB-468 total cell lysates that was not detected by the other two antibodies. Several observations suggest the 180-kDa protein is the epidermal growth factor (EGF) receptor: (i) C-20 reacted avidly with a 180-kDa protein immunoprecipitated by an antibody to the EGF receptor; (ii) an EGF receptor antibody detected a 180-kDa protein immunoprecipitated by C-20; (iii) the affinity purified EGF receptor was both immunoprecipitated and detected on immunoblots by the C-20 antibody but not another BRCA1 antibody; (iv) similar phosphopeptide maps were generated from the EGF receptor and the 180-kDa protein immunoprecipitated by C-20, and this peptide map was distinct from the 220-kDa phosphoprotein; and (v) the C-20 immunizing peptide bears sequence identity to the EGF receptor. These results indicate that BRCA1 is a 220-kDa nuclear protein and that the 180-kDa protein reported previously may be unrelated to BRCA1.

Several lines of evidence indicate that serine/threonine protein phosphatases may act as negative regulators of cellular growth. For example, treatment of cells with the tumor-promoter okadaic acid, an inhibitor of certain types of these phosphatases, resulted in the increased expression of several proto-oncogenes, indicating a negative role of the respective phosphatases in gene regulation. However, it was puzzling to find that okadaic acid-treated cells, even in the presence of highly expressed proto-oncogenes, did not proliferate, but were arrested at certain points of the cell cycle. To further analyze this discrepancy, we investigated the involvement of protein phosphatases in the control of other cell cycle regulatory genes, such as cdc2 which encodes an essential cell cycle regulatory kinase. We found that cdc2 gene expression was blocked by okadaic acid, but stimulated by protein phosphatase 2A. Protein phosphatase 2A is shown to be a positive regulator of cdc2 gene activity and to be required for cdc2 expression. Thus, our findings identify protein phosphatase 2A as a positive regulator of a major cell cycle regulatory gene and therefore suggest a stimulatory role of this enzyme in this aspect of cellular growth control.

Interferon-inducible membrane proteins of approximately 17 kDa have been suggested to play a role in the antiproliferative activity of interferons based on (1) their pattern of induction in interferon-sensitive and -resistant cell lines and (2) the ability of a membrane fraction enriched in 17-kDa proteins to inhibit cell growth. To gain insight into the nature of the proteins that mediate the antiproliferative activity of interferons, a monoclonal antibody, 13A5, was generated that reacted specifically with a 17-kDa interferon-inducible cell surface protein. The expression pattern of this 17-kDa protein by human cell lines correlated with sensitivity to the antiproliferative activity of interferons. To obtain information regarding the structure of this protein, the 13A5 antibody was used to screen COS cells transfected with a human cDNA expression library. Sequence analysis of a full-length cDNA clone revealed identity with the 9-27 cDNA, previously isolated on the basis of its interferon inducibility by differential screening. In addition, the 17-kDa protein encoded by the 9-27 gene was shown to be identical to the Leu-13 antigen. Leu-13 was previously identified as a 16-kDa interferon-inducible protein in leukocytes and endothelial cells and is a component of a multimeric complex involved in the transduction of antiproliferative and homotypic adhesion signals. These results suggest a novel level of cellular regulation by interferons involving a membrane protein, encoded by the interferon-inducible 9-27 gene, which associates with other proteins at the cell surface, forming a complex relaying growth inhibitory and aggregation signals.

Using a monoclonal antibody, IVd4, that recognizes a novel group of hyaluronan-binding proteins, we have immunoscreened a cDNA library constructed from embryonic chick heart muscle mRNA. One of the cDNAs isolated from the library encodes a 29.3-kDa protein homologous to Cdc37, an essential cell cycle regulatory factor previously characterized genetically in yeast and Drosophila; this is the first vertebrate CDC37 gene to be cloned to date. We also present evidence for the existence of a second chick isoform that is identical to the 29.3-kDa protein over the first 175 amino acids but is entirely different at the carboxyl terminus and lacks the IVd4 epitope. The avian Cdc37 binds hyaluronan, chondroitin sulfate and heparin in vitro, and both isoforms contain glycosaminoglycan-binding motifs previously described in several hyaluronan-binding proteins. These findings suggest a role for glycosaminoglycans in cell division control.

Many of the antimicrobial, immunomodulatory, and cell growth regulatory activities of the interferons are mediated by interferon-inducible proteins. One family of such murine proteins is encoded by six or more adjacent and structurally related genes (gene 200 cluster). Two homologous human genes have also been reported. p202, encoded by the Ifi202 gene in the gene 200 cluster, is a 52-kDa nuclear phosphoprotein. Constitutive overexpression of p202 in transfected cells is growth-inhibitory. We report here that p202 binds the cell growth regulatory retinoblastoma protein (pRb) in vitro and in vivo. The binding is due to direct interaction between the two proteins. p202 has two nonoverlapping segments for binding pRb, and pRb has two nonoverlapping segments (one of them including the pocket region) for binding p202. The hypophosphorylated form of pRb binds to p202, p202 is the first interferon-inducible protein found to bind pRb.

The rad21 gene of Schizosaccharomyces pombe is involved in the repair of double-strand breaks in DNA and is essential for mitotic growth (Birkenbihl, R. P., and Subramani, S.(1992) Nucleic Acids Res. 20, 6605-6611). We show that the [Medline] Rad21 protein migrates with an aberrantly slow mobility, has a thrombin cleavage site, and is multiply phosphorylated mainly at serine residues. The expression of the rad21 mRNA and the Rad21 protein is cell cycle-regulated, with the peak of mRNA and protein expression occurring near the G1 to S transition. Following translation of the protein, hypophosphorylated forms of the protein appear. However, the most phosphorylated form of Rad21 appears only later in the cell cycle (in S to G2). Analysis of the radiosensitive mutant rad21-45 revealed that the mutant protein is permanently hypophosphorylated. The Rad21 protein is nuclear during the cell cycle. The nuclear localization signal was identified in the C-terminal third of the protein. Upon repression of the Rad21 protein expressed from the repressible nmt1 promoter, the unphosphorylated and hypophosphorylated forms of Rad21 disappeared first. When the concentration of the most highly phosphorylated form of Rad21 sank under a critical level, the cells underwent aberrant mitoses. They exhibited loss of proper nuclear organization and abnormal septation.

Genes 41 and 59 of bacteriophage T4 are involved in DNA recombination as well as in DNA replication. The 41 protein has a DNA helicase activity. The 59 protein has been recently purified and found to have a specific affinity for both 32 protein (single-stranded DNA-binding protein) and 41 protein (Yonesaki 1994, J. Biol. Chem. 269, 1284-1289). We examined the effects of 59 protein on ssDNA-dependent ATPase activity and DNA helicase activity of 41 protein in the presence or absence of 32 protein. The ATPase activity of 41 protein was strongly inhibited by 32 protein over a wide range of amounts from subsaturation to oversaturation of ssDNA. The 32 protein was also inhibitory toward DNA helicase activity. Addition of 59 protein effectively eliminated these inhibitory effects of 32 protein. Moreover, 59 protein facilitated 41 protein to overcome the barrier to initiate the unwinding reaction with a duplex flanking a single-stranded DNA gap. Intriguingly, 32 protein at an amount optimal for saturation of ssDNA stimulated the overcoming of the barrier when 59 protein was present. For the best circumvention of this initiation barrier, only eight monomers of 59 protein/one DNA substrate molecule containing 2900 nucleotides of ssDNA were required. These results strongly suggest that 59 protein modulates 41 protein activities by forming a complex with 41 protein and that 41 protein can produce recombinogenic ssDNA with the aid of 32 and 59 proteins.

Protein 4.1 is the prototype of a family of proteins that include ezrin, talin, brain tumor suppressor merlin, and tyrosine phosphatases. All members of the protein 4.1 superfamily share a highly conserved N-terminal 30-kDa domain whose biological function is poorly understood. It is believed that the attachment of the cytoskeleton to the membrane may be mediated via this 30-kDa domain, a function that requires formation of multiprotein complexes at the plasma membrane. In this investigation, synthetically tagged peptides and bacterially expressed proteins were used to map the protein 4.1 binding site on human erythroid glycophorin C, a transmembrane glycoprotein, and on human erythroid p55, a palmitoylated peripheral membrane phosphoprotein. The results show that the 30-kDa domain of protein 4.1 binds to a 12-amino acid segment within the cytoplasmic domain of glycophorin C and to a positively charged, 39-amino acid motif in p55. Sequences similar to this charged motif are conserved in other members of the p55 superfamily, including the Drosophila discs-large tumor suppressor protein. Our data provide new insights into how protein 4.1, glycophorin C, p55, and their non-erythroid homologues, interact with the cytoskeleton to exert their physiological effects.

GADD45 (growth arrest and DNA damage) is a DNA-damage-inducible gene regulated in part by the tumor suppressor p53. A role in negative growth control has recently been suggested based on significant (more than 75%) reduction of colony formation following over expression of Gadd45. To better understand the role of Gadd45, we have developed specific rabbit and murine antibodies raised against the human recombinant protein. Using these antibodies, we have found that in ML-1 cells Gadd45 is predominantly a nuclear protein. MyD118, a protein induced by terminal differentiation sharing 57% homology with Gadd45, does not cross-react with any of the antibodies produced. As expected, the induction of Gadd45 protein by ionizing radiation (IR) was also found to be dependent on a wild type p53 phenotype. Interestingly, WI-L2-NS, a human lymphoid cell line, showed very high basal levels of Gadd45 mRNA and protein in addition to a high constitutive level of a mutated p53 protein. In this cell line, the high levels of GADD45 did not inhibit cellular growth in spite of the fact that no mutations were found in GADD45 sequence. These results indicate that some cell line(s) can tolerate high levels of Gadd45 and abrogate its growth suppression function.

Several genes critical to the uptake, sequestration, and utilization of iron are regulated at the post-transcriptional level. The mRNAs encoded by these genes contain highly conserved stem-loop structures called iron-responsive elements (IREs). IREs function as the nucleic acid-binding sites for a cytosolic RNA-binding protein called the IRE-binding protein or IRE-BP. Binding of the IRE-BP to IREs is reversibly regulated by the iron status of the cell. The IRE-BP is highly conserved among human, rat, mouse, and rabbit, and it is identical to the cytosolic form of aconitase. In this study, we demonstrate that a distinct human gene encoding a protein which is 57% identical to the initially described IRE-BP, now referred to as iron regulatory protein 1 or IRP1, is also capable of binding to IREs with the same in vitro affinity and specificity the originally identified protein. This second gene product, which we call IRP2, is expressed in many tissues, but its mRNA abundance and tissue distribution are different from IRP1. In most cell lines tested, levels of IRP2 are inversely regulated by iron levels due to iron-dependent regulation of the half-life of the protein. In addition to changes in total amounts of IRP2, we demonstrate that the IRE binding activity of IRP2 can also vary up to 4-fold in the absence of any change in IRP2 protein levels. The possible reasons for the existence of a second IRP are discussed.

The acute response of steroidogenic cells to trophic hormone stimulation is the mobilization of cholesterol from cellular stores to the mitochondrial outer membrane and the transfer of this cholesterol to the mitochondrial inner membrane where the first enzymatic step in steroidogenesis occurs. The transfer of cholesterol across the mitochondrial membranes is dependent upon de novo protein synthesis, and this is the regulated step in the process. Although the newly synthesized regulatory protein(s) have yet to be identified, we previously have proposed a candidate protein which we identified in MA-10 cells that is synthesized in response to luteinizing hormone stimulation and that is localized to the mitochondria. In the present study, we report the isolation of a cDNA that encodes this luteinizing hormone-induced protein. Analysis of the cDNA and protein sequences reveals this is a novel protein. Importantly, we demonstrate for the first time that expression of the protein in MA-10 cells in the absence of hormone stimulation is sufficient to induce steroid production. We conclude that this protein is required in the acute regulation of steroidogenesis and propose to call this protein the Steroidogenic Acute Regulatory protein (StAR).

A rise in extracellular calcium (Ca2+e) suppresses not only secretion of parathyroid hormone (PTH) but also expression of the PTH gene to ensure constant plasma Ca2+ level. A nuclear protein(s) in a wide variety of cells bound to the specific DNA elements (negative Ca2+ responsive elements, nCaREs) in the human PTH gene in sequence-specific and Ca2+e concentration-dependent manners. Our Southwestern cloning revealed that a redox factor protein (ref1), which was known to activate several transcription factors via alterations of their redox state, belonged to an nCaRE binding protein. The level of ref1 mRNA as well as of its protein was elevated by an increase in Ca2+e concentration. In gel shift assay, anti-ref1 antibody eliminated formation of the nCaRE-protein complex. We also found that there was another protein(s) interacting with nCaREs and ref1. Further, experiments with an antisense-ref cDNA expression vector introduced into cultured cells suggested that DNA (nCaRE)-ref1 interaction led to Ca2+e-mediated transcriptional suppression. Thus, it is concluded that ref1 possesses transcription repressor activity in addition to its function as a transcriptional auxiliary protein.

Human protein C and activated protein C are shown to bind to endothelium specifically, selectively and saturably (Kd = 30 nM, 7000 sites per cell) in a Ca(2+)-dependent fashion. Expression cloning revealed a 1.3-kilobase pair cDNA that coded for a novel type 1 transmembrane glycoprotein capable of binding protein C. This protein appears to be a member of the CD1/major histocompatibility complex superfamily. Like thrombomodulin, the receptor involved in protein C activation, the endothelial cell protein C receptor function and message are both down-regulated by exposure of endothelium to tumor necrosis factor. Identification of endothelial cell protein C receptor as a member of the CD1/major histocompatibility complex superfamily provides insights into the role of protein C in regulating the inflammatory response.

Nuclear respiratory deficient mutants of Saccharomyces cerevisiae have been screened for lesions in genes affecting ubiquinol-cytochrome c reductase activity. In the present study we describe a new gene, CBP4, whose encoded product is absolutely essential for the activity and expression of the respiratory enzyme. We have cloned and sequenced CBP4, which encodes a 20-kDa protein having no obvious homology to any known protein. cbp4 mutants are unable to respire due to specific loss of ubiquinol-cytochrome c reductase activity. cbp4 mutants demonstrate a pleiotropic reduction in the steady state levels of four subunits of ubiquinol-cytochrome c reductase, namely core 4, core 5, the Rieske protein and cytochrome b. Cytochrome b is not spectrally visible in the mutants, although transcription and translation of the apoprotein is normal. Antiserum prepared against a trpE/CBP4 gene fusion react with an 18.4-kDa mitochondrial protein in wild type yeast, but the protein is not found in a mutant containing a deletion of 86% of the CBP4 coding region. CBP4 protein is tightly associated with the mitochondrial membrane as evidenced by the association of the protein with the membrane fraction following carbonate extraction. The phenotype of cbp4 mutants is similar to that of cbp3 mutants. CBP3 encodes a second protein essential for ubiquinol-cytochrome c reductase activity. We found that loss of CBP3 protein does not affect the membrane stability or steady state concentration of CBP4 protein. A double mutant containing a deletion in both CBP3 and CBP4 was constructed. Our study shows that the phenotype of the double mutant is identical to the phenotypes of the individual single mutants.

The mts-1 gene is associated with the expression of the metastatic phenotype of tumor cells. The protein product of the mts-1 gene belongs to the S100 family of Ca(2+)-binding proteins with unknown biochemical function. In the present work, monoclonal anti-Mts-1 antibodies were used to isolate and characterize Mts-1 protein possible targets. Mts-1 protein can be immunoprecipitated by both anti-Mts-1 and anti-myosin antibodies as a complex with myosin from lysates of different mouse and human cell lines. Precipitation of myosin by anti-Mts-1 antibodies is specific and depends on the presence of Mts-1 protein. Ca(2+)-dependent association between Mts-1 protein and the heavy chain of non-muscle myosin was demonstrated by blot overlay technique. Furthermore, association between myosin and Mts-1 was confirmed by sucrose gradient analysis. Finally, immunofluorescent staining of the mouse mammary adenocarcinoma cell line showed that Mts-1 protein is co-localized with the myosin complex. The data suggest that the target for Mts-1 protein is a heavy chain of non-muscle myosin.

The tumor suppressor protein p53 is extremely unstable in most cell lines. In contrast, many mutant and oncogenic species of the protein are stable. The degradation of p53 in vivo requires metabolic energy; however, the proteolytic system(s) involved have not been identified. The ubiquitin system has been implicated in the degradation of p53 in vitro. The degradation is stimulated significantly by the human papillomavirus (HPV) oncoprotein E6 that associates with p53 and facilitates conjugate formation and subsequent degradation. Complex formation between E6 and p53 is promoted by a cellular protein designated E6-associated protein (E6-AP). Initial dissection of the conjugation process have demonstrated a role for the ubiquitin-activating enzyme, E1, but the ubiquitin-carrier protein (E2, UBC) and the ubiquitin protein ligase, E3, have not been identified. In this study, we report that a novel species of ubiquitin-carrier protein designated E2-F1 (Blumenfeld, N., Gonen, H., Mayer, A., Smith, C., Siegel, N.R., Schwartz, A.L., and Ciechanover, A. (1994) J. Biol. Chem. 269, 9574-9581) is involved in the conjugation and degradation of p53. This E2 enzyme recognizes non-"N-end rule" protein substrates and appears to mediate their conjugation via a novel species of E3. The process of recognition appears to be selective; E2-F1 is not required for the conjugation and degradation of human N-myc. The involvement of E2-F1 in the in vitro process appears to be physiologically meaningful and to reproduce the in vivo process; mutant species of p53 that do not interact with E6 and are stable in vivo are not recognized by the cell free system.

Amplification and overexpression of the c-erbB-2 gene appears to play a role in the pathogenesis of human breast and other cancers. Frequent amplification (20-30%) of the c-erbB-2 gene was observed in human adenocarcinomas of kidney, pancreas, lung, ovarian, and breast cancer. The gene product is a 185-kDa glycoprotein that has intrinsic tyrosine kinase activity and is believed to be a receptor. Several candidate ligands have been described. In the present study we have identified and purified a novel DNA-binding protein from malignant human breast tissues. The protein binds to a core element (-22 to +9, +1 being the transcription start site) of the c-erbB-2 promoter region in a sequence-specific manner. The affinity-purified protein has the ability to induce DNA synthesis in quiescent NIH/3T3 cells, suggesting that the factor has mitogenic activity. The purified protein induces c-erbB-2 expression on the surface of microinjected NIH/3T3 cells. This DNA-binding protein is a sequence-specific cellular factor that is associated with high level expression of the c-erbB-2 gene and appears to play a role in cell transformation. Understanding the control and expression of this DNA-binding protein may shed light on the mechanism(s) of c-erbB-2 gene regulation and its potential role in the pathogenesis of human adenocarcinomas.