PAF49: An RNA Polymerase I subunit essential for rDNA transcription and stabilization of PAF53.

The application of genetic and biochemical techniques in yeast has informed our knowledge of transcription in mammalian cells. Such systems have allowed investigators to determine whether a gene was essential and to determine its function in rDNA transcription. However, there are significant differences in the nature of the transcription factors essential for transcription by Pol I in yeast and mammalian cells, and yeast RNA polymerase I contains 14 subunits while mammalian polymerase contains 13 subunits. We previously reported the adaptation of the auxin-dependent degron that enabled a combination of a "genetics-like" approach and biochemistry to study mammalian rDNA transcription. Using this system, we studied the mammalian orthologue of yeast RPA34.5, PAF49, and found that it is essential for rDNA transcription and cell division. The auxin-induced degradation of PAF49 induced nucleolar stress and the accumulation of P53. Interestingly, the auxin-induced degradation of AID-tagged PAF49 led to the degradation of its binding partner, PAF53, but not vice versa. A similar pattern of co-dependent expression was also found when we studied the non-essential, yeast orthologues. An analysis of the domains of PAF49 that are essential for rDNA transcription demonstrated a requirement for both the dimerization domain and an "arm" of PAF49 that interacts with PolR1B. Further, we demonstrate this interaction can be disrupted to inhibit Pol I transcription in normal and cancer cells which leads to the arrest of normal cells and cancer cell death. In summary, we have shown that both PAF53 and PAF49 are necessary for rDNA transcription and cell growth.

The Mammalian and Yeast A49 and A34 Heterodimers: Homologous but Not the Same.

Ribosomal RNA synthesis is the rate-limiting step in ribosome biogenesis. In eukaryotes, RNA polymerase I (Pol I) is responsible for transcribing the ribosomal DNA genes that reside in the nucleolus. Aberrations in Pol I activity have been linked to the development of multiple cancers and other genetic diseases. Therefore, it is key that we understand the mechanisms of Pol I transcription. Recent studies have demonstrated that there are many differences between Pol I transcription in yeast and mammals. Our goal is to highlight the similarities and differences between the polymerase-associated factors (PAFs) in yeast and mammalian cells. We focus on the PAF heterodimer A49/34 in yeast and PAF53/49 in mammals. Recent studies have demonstrated that while the structures between the yeast and mammalian orthologs are very similar, they may function differently during Pol I transcription, and their patterns of regulation are different.

Dynamics of the RNA polymerase I TFIIF/TFIIE-like subcomplex: a mini-review.

RNA polymerase I (Pol I) is the most specialized eukaryotic Pol. It is only responsible for the synthesis of pre-ribosomal RNA (rRNA), the precursor of 18S, 5.8S and 28S rRNA, the most abundant cellular RNA types. Aberrant Pol I transcription is observed in a wide variety of cancers and its down-regulation is associated with several genetic disorders. The regulation and mechanism of Pol I transcription is increasing in clarity given the numerous high-resolution Pol I structures that have helped bridge seminal genetic and biochemical findings in the field. Here, we review the multifunctional roles of an important TFIIF- and TFIIE-like subcomplex composed of the Pol I subunits A34.5 and A49 in yeast, and PAF49 and PAF53 in mammals. Recent analyses have revealed a dynamic interplay between this subcomplex at nearly every step of the Pol I transcription cycle in addition to new roles in chromatin traversal and the existence of a new helix-turn-helix (HTH) within the A49/PAF53 linker domain that expands its dynamic functions during the Pol I transcription process.

Conditional depletion of the RNA polymerase I subunit PAF53 reveals that it is essential for mitosis and enables identification of functional domains.

Our knowledge of the mechanism of rDNA transcription has benefited from the combined application of genetic and biochemical techniques in yeast. Nomura's laboratory (Nogi, Y., Vu, L., and Nomura, M. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 7026-7030 and Nogi, Y., Yano, R., and Nomura, M. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 3962-3966) developed a system in yeast to identify genes essential for ribosome biogenesis. Such systems have allowed investigators to determine whether a gene was essential and to determine its function in rDNA transcription. However, there are significant differences in both the structures and components of the transcription apparatus and the patterns of regulation between mammals and yeast. Thus, there are significant deficits in our understanding of mammalian rDNA transcription. We have developed a system combining CRISPR/Cas9 and an auxin-inducible degron that enables combining a "genetics-like"approach with biochemistry to study mammalian rDNA transcription. We now show that the mammalian orthologue of yeast RPA49, PAF53, is required for rDNA transcription and mitotic growth. We have studied the domains of the protein required for activity. We have found that the C-terminal, DNA-binding domain (tandem-winged helix), the heterodimerization, and the linker domain were essential. Analysis of the linker identified a putative helix-turn-helix (HTH) DNA-binding domain. This HTH constitutes a second DNA-binding domain within PAF53. The HTH of the yeast and mammalian orthologues is essential for function. In summary, we show that an auxin-dependent degron system can be used to rapidly deplete nucleolar proteins in mammalian cells, that PAF53 is necessary for rDNA transcription and cell growth, and that all three PAF53 domains are necessary for its function.

PAF53 is essential in mammalian cells: CRISPR/Cas9 fails to eliminate PAF53 expression.

When mammalian cells are nutrient and/or growth factor deprived, exposed to inhibitors of protein synthesis, stressed by heat shock or grown to confluence, rDNA transcription is essentially shut off. Various mechanisms are available to accomplish this downshift in ribosome biogenesis. Muramatsu's laboratory (Hanada et al., 1996) first demonstrated that mammalian PAF53 was essential for specific rDNA transcription and that PAF53 levels were regulated in response to growth factors. While S. cerevisae A49, the homologue of vertebrate PAF53, is not essential for viability (Liljelund et al., 1992), deletion of yA49 results in colonies that grow at 6% of the wild type rate at 25°C. Experiments described by Wang et al. (2015) identified PAF53 as a gene "essential for optimal proliferation". However, they did not discriminate genes essential for viability. Hence, in order to resolve this question, we designed a series of experiments to determine if PAF53 was essential for cell survival. We set out to delete the gene product from mammalian cells using CRISPR/CAS9 technology. Human 293 cells were transfected with lentiCRISPR v2 carrying genes for various sgRNA that targeted PAF53. In some experiments, the cells were cotransfected in parallel with plasmids encoding FLAG-tagged mouse PAF53. After treating the transfected cells with puromycin (to select for the lentiCRISPR backbone), cells were cloned and analyzed by western blots for PAF53 expression. Genomic DNA was amplified across the "CRISPRd" exon, cloned and sequenced to identify mutated PAF53 genes. We obtained cell lines in which the endogenous PAF53 gene was "knocked out" only when we rescued with FLAG-PAF53. DNA sequencing demonstrated that in the absence of ectopic PAF53 expression, cells demonstrated unique means of surviving; including recombination or the utilization of alternative reading frames. We never observed a clone in which one PAF53 gene is expressed, unless there was also ectopic expression In the absence of ectopic gene expression, the gene products of both endogenous genes were expressed, irrespective of whether they were partially mutant proteins or not.

Cell cycle and growth stimuli regulate different steps of RNA polymerase I transcription.

Transcription of the ribosomal RNA genes (rDNA) by RNA polymerase I (Pol I) is a major control step for ribosome synthesis and is tightly linked to cellular growth. However, the question of whether this process is modulated primarily at the level of transcription initiation or elongation is controversial. Studies in markedly different cell types have identified either initiation or elongation as the major control point. In this study, we have re-examined this question in NIH3T3 fibroblasts using a combination of metabolic labeling of the 47S rRNA, chromatin immunoprecipitation analysis of Pol I and overexpression of the transcription initiation factor Rrn3. Acute manipulation of growth factor levels altered rRNA synthesis rates over 8-fold without changing Pol I loading onto the rDNA. In fact, robust changes in Pol I loading were only observed under conditions where inhibition of rDNA transcription was associated with chronic serum starvation or cell cycle arrest. Overexpression of the transcription initiation factor Rrn3 increased loading of Pol I on the rDNA but failed to enhance rRNA synthesis in either serum starved, serum treated or G0/G1 arrested cells. Together these data suggest that transcription elongation is rate limiting for rRNA synthesis. We propose that transcription initiation is required for rDNA transcription in response to cell cycle cues, whereas elongation controls the dynamic range of rRNA synthesis output in response to acute growth factor modulation.

A novel role for the Pol I transcription factor UBTF in maintaining genome stability through the regulation of highly transcribed Pol II genes.

Mechanisms to coordinate programs of highly transcribed genes required for cellular homeostasis and growth are unclear. Upstream binding transcription factor (UBTF, also called UBF) is thought to function exclusively in RNA polymerase I (Pol I)-specific transcription of the ribosomal genes. Here, we report that the two isoforms of UBTF (UBTF1/2) are also enriched at highly expressed Pol II-transcribed genes throughout the mouse genome. Further analysis of UBTF1/2 DNA binding in immortalized human epithelial cells and their isogenically matched transformed counterparts reveals an additional repertoire of UBTF1/2-bound genes involved in the regulation of cell cycle checkpoints and DNA damage response. As proof of a functional role for UBTF1/2 in regulating Pol II transcription, we demonstrate that UBTF1/2 is required for recruiting Pol II to the highly transcribed histone gene clusters and for their optimal expression. Intriguingly, lack of UBTF1/2 does not affect chromatin marks or nucleosome density at histone genes. Instead, it results in increased accessibility of the histone promoters and transcribed regions to micrococcal nuclease, implicating UBTF1/2 in mediating DNA accessibility. Unexpectedly, UBTF2, which does not function in Pol I transcription, is sufficient to regulate histone gene expression in the absence of UBTF1. Moreover, depletion of UBTF1/2 and subsequent reduction in histone gene expression is associated with DNA damage and genomic instability independent of Pol I transcription. Thus, we have uncovered a novel role for UBTF1 and UBTF2 in maintaining genome stability through coordinating the expression of highly transcribed Pol I (UBTF1 activity) and Pol II genes (UBTF2 activity).

Regulation of the association of the PAF53/PAF49 heterodimer with RNA polymerase I.

Mammalian PAF49 and PAF53 form a heterodimer and are essential for transcription. However their roles in transcription have not been specifically defined. While the yeast homologues are "not essential" proteins, yeast cells deficient in the homologue of PAF53 grow at 50-66% the wild-type rate at 30°C, but fail to grow at 25°C (Liljelund et al., 1992; Beckouet et al., 2008). There is increasing evidence that these proteins may play important roles in transcription initiation and elongation. We have found that while some cells regulated the protein levels of both PAF53 and PAF49, other cells did not. However, in either case they regulated the nucleolar levels of the PAFs. In addition, we found that the association of PAF49/PAF53 with Pol I is regulated. In examining the mechanism that might regulate this association, we have found that PAF49 is acetylated on multiple sites. The acetylation state of PAF49 does not affect heterodimerization. However, hypoacetylated heterodimer binds to Pol I with greater affinity than acetylated heterodimer. Further, we have found that the heterodimer interacts with Rrn3. We propose a model, in which there is a biochemical interaction between the Pol I-associated heterodimer and Rrn3 and that this interaction facilitates the recruitment of Rrn3 to the polymerase. As the binding of Rrn3 to Pol I is essential to transcription initiation in yeast and mammals, our results provide a greater understanding of the regulation of Rrn3 function and provide biochemical underpinning for the roles of the PAF49/PAF53 heterodimer in transcription initiation and elongation by Pol I.

Selective inhibition of rDNA transcription by a small-molecule peptide that targets the interface between RNA polymerase I and Rrn3.

UNLABELLED: The interface between the polymerase I-associated factor Rrn3 and the 43-kDa subunit of RNA polymerase I is essential to the recruitment of Pol I to the preinitiation complex on the rDNA promoter. In silico analysis identified an evolutionarily conserved 22 amino acid peptide within rpa43 that is both necessary and sufficient to mediate the interaction between rpa43 and Rrn3. This peptide inhibited rDNA transcription in vitro, while a control peptide did not. To determine the effect of the peptide in cultured cells, the peptide was coupled to the HIV TAT peptide to facilitate transduction into cells. The wild-type peptide, but not control peptides, inhibited Pol I transcription and cell division. In addition, the peptide induced cell death, consistent with other observations that "nucleolar stress" results in the death of tumor cells. The 22mer is a small-molecule inhibitor of rDNA transcription that is specific for the interaction between Rrn3 and rpa43, as such it represents an original way to interfere with cell growth. IMPLICATIONS: These results demonstrate a potentially novel pharmaceutical target for the therapeutic treatment of cancer cells.

DNA binding by the ribosomal DNA transcription factor rrn3 is essential for ribosomal DNA transcription.

The human homologue of yeast Rrn3 is an RNA polymerase I-associated transcription factor that is essential for ribosomal DNA (rDNA) transcription. The generally accepted model is that Rrn3 functions as a bridge between RNA polymerase I and the transcription factors bound to the committed template. In this model Rrn3 would mediate an interaction between the mammalian Rrn3-polymerase I complex and SL1, the rDNA transcription factor that binds to the core promoter element of the rDNA. In the course of studying the role of Rrn3 in recruitment, we found that Rrn3 was in fact a DNA-binding protein. Analysis of the sequence of Rrn3 identified a domain with sequence similarity to the DNA binding domain of heat shock transcription factor 2. Randomization, or deletion, of the amino acids in this region in Rrn3, amino acids 382-400, abrogated its ability to bind DNA, indicating that this domain was an important contributor to DNA binding by Rrn3. Control experiments demonstrated that these mutant Rrn3 constructs were capable of interacting with both rpa43 and SL1, two other activities demonstrated to be essential for Rrn3 function. However, neither of these Rrn3 mutants was capable of functioning in transcription in vitro. Moreover, although wild-type human Rrn3 complemented a yeast rrn3-ts mutant, the DNA-binding site mutant did not. These results demonstrate that DNA binding by Rrn3 is essential for transcription by RNA polymerase I.

Characterization of the interactions of mammalian RNA polymerase I associated proteins PAF53 and PAF49.

Masami Muramatsu's laboratory demonstrated the critical role of RNA polymerase I (Pol I)-associated factor PAF53 in mammalian rRNA transcription. They have also identified a second polymerase associated factor, PAF49. Both PAF49 and PAF53 copurify with that fraction of the RNA polymerase I molecules that can function in transcription initiation in vitro. PAF49 and PAF53 are the mammalian homologues of two subunits of yeast RNA polymerase I, A34.5 and A49, that form a TFIIF-related subcomplex in yeast RNA polymerase I. In light of those publications, we investigated the interactions between various deletion and substitution mutants of mammalian PAF49 and PAF53 with the purpose of identifying those domains of the mammalian proteins that interact. Comparison of our results with structural studies on yeast A34.5 and A49 demonstrates that the yeast and mammalian proteins may in fact share structural similarities. In fact, the deletion mutagenesis data confirmed and extended the structural studies. For example, amino acids 41-86 of PAF49 were sufficient to provide the basis for heterodimerization. In silico structural analysis predicted that this region could assume a structure similar to the homologous region of yeast A34.5. Those similarities are insufficient, by themselves, for the proteins to form interspecific heterodimers. However, substitution of amino acids 52-98 of yeast A34.5 with amino acids 41-86 of mammalian PAF49 resulted in a protein that could heterodimerize with mouse PAF53.

Phosphorylation of eukaryotic translation initiation factor 2alpha coordinates rRNA transcription and translation inhibition during endoplasmic reticulum stress.

The endoplasmic reticulum (ER) is the major cellular compartment where folding and maturation of secretory and membrane proteins take place. When protein folding needs exceed the capacity of the ER, the unfolded protein response (UPR) pathway modulates gene expression and downregulates protein translation to restore homeostasis. Here, we report that the UPR downregulates the synthesis of rRNA by inactivation of the RNA polymerase I basal transcription factor RRN3/TIF-IA. Inhibition of rRNA synthesis does not appear to involve the well-characterized mTOR (mammalian target of rapamycin) pathway; instead, PERK-dependent phosphorylation of eIF2alpha plays a critical role in the inactivation of RRN3/TIF-IA. Downregulation of rRNA transcription occurs simultaneously or slightly prior to eIF2alpha phosphorylation-induced translation repression. Since rRNA is the most abundant RNA species, constituting approximately 90% of total cellular RNA, its downregulation exerts a significant impact on cell physiology. Our study demonstrates the first link between regulation of translation and rRNA synthesis with phosphorylation of eIF2alpha, suggesting that this pathway may be broadly utilized by stresses that activate eIF2alpha kinases in order to coordinately regulate translation and ribosome biogenesis during cellular stress.

Mammalian Rrn3 is required for the formation of a transcription competent preinitiation complex containing RNA polymerase I.

Mammalian Rrn3, an essential, polymerase-associated protein, is inactivated when cells are treated with cycloheximide, resulting in the inhibition of transcription by RNA polymerase I. Although Rrn3 is essential for transcription, its function in rDNA transcription has not been determined. For example, it is unclear whether Rrn3 is required for initiation or elongation by RNA polymerase I. Rrn3 has been shown to interact with the 43-kDa subunit of RNA polymerase I and with two of the subunits of SL1. In the current model for transcription, Rrn3 functions to recruit RNA polymerase I to the committed complex formed by SL1 and the rDNA promoter. To examine the question as to whether Rrn3 is required for the recruitment of RNA polymerase I to the template, we developed a novel assay similar to chromatin immunoprecipitation assays. We found that RNA polymerase I can be recruited to a template in the absence of active Rrn3. However, that complex will not initiate transcription, even after Rrn3 is added to the reaction. Interestingly, the complex that forms in the presence of active Rrn3 is biochemically distinguishable from that which forms in the absence of active Rrn3. For example, the functional complex is fivefold more resistant to heparin than that which forms in the absence of Rrn3. Our data demonstrate that Rrn3 must be present when the committed template complex is forming for transcription to occur.

Regulation of cardiac Na+/Ca2+ exchanger by phospholemman.

Phospholemman (PLM) is the first sequenced member of the FXYD family of regulators of ion transport. The mature protein has 72 amino acids and consists of an extracellular N terminus containing the signature FXYD motif, a single transmembrane (TM) domain, and a cytoplasmic C-terminal domain containing four potential sites for phosphorylation. PLM and other members of the FXYD family are known to regulate Na+-K+-ATPase. Using adenovirus-mediated gene transfer into adult rat cardiac myocytes, we showed that changes in contractility and intracellular Ca2+ homeostasis associated with PLM overexpression or downregulation are not consistent with the effects expected from inhibition of Na+-K+-ATPase by PLM. Additional studies with heterologous expression of PLM and cardiac Na+/Ca2+ exchanger 1 (NCX1) in HEK293 cells and cardiac myocytes isolated from PLM-deficient mice demonstrated by co-localization, co-immunoprecipitation, and electrophysiological and radioactive tracer uptake techniques that PLM associates with NCX1 in the sarcolemma and transverse tubules and that PLM inhibits NCX1, independent of its effects on Na+-K+-ATPase. Mutational analysis indicates that the cytoplasmic domain of PLM is required for its regulation of NCX1. In addition, experiments using phosphomimetic and phospho-deficient PLM mutants, as well as activators of protein kinases A and C, indicate that PLM phosphorylated at serine68 is the active form that inhibits NCX1. This is in sharp contrast to the finding that the unphosphorylated PLM form inhibits Na+-K+-ATPase. We conclude that PLM regulates cardiac contractility by modulating the activities of NCX and Na+-K+-ATPase.

Cytoplasmic tail of phospholemman interacts with the intracellular loop of the cardiac Na+/Ca2+ exchanger.

Phospholemman (PLM), a member of the FXYD family of small ion transport regulators, inhibits cardiac Na+/Ca2+ exchanger (NCX1). NCX1 is made up of N-terminal domain consisting of the first five transmembrane segments (residues 1-217), a large intracellular loop (residues 218-764), and a C-terminal domain comprising the last four transmembrane segments (residues 765-938). Using glutathione S-transferase (GST) pull-down assay, we demonstrated that the intracellular loop, but not the N- or C-terminal transmembrane domains of NCX1, was associated with PLM. Further analysis using protein constructs of GST fused to various segments of the intracellular loop of NCX1 suggest that PLM bound to residues 218-371 and 508-764 but not 371-508. Split Na+/Ca2+ exchangers consisting of N- or C-terminal domains with different lengths of the intracellular loop were co-expressed with PLM in HEK293 cells that are devoid of endogenous PLM and NCX1. Although expression of N-terminal but not C-terminal domain alone resulted in correct membrane targeting, co-expression of both N- and C-terminal domains was required for correct membrane targeting and functional exchange activity. NCX1 current measurements indicate that PLM decreased NCX1 current only when the split exchangers contained residues 218-358 of the intracellular loop. Co-immunoprecipitation experiments with PLM and split exchangers suggest that PLM associated with the N-terminal domain of NCX1 when it contained intracellular loop residues 218-358. TM43, a PLM mutant with its cytoplasmic tail truncated, did not co-immunoprecipitate with wild-type NCX1 when co-expressed in HEK293 cells, confirming little to no interaction between the transmembrane domains of PLM and NCX1. We conclude that PLM interacted with the intracellular loop of NCX1, most likely at residues 218-358.

Ribosomal chromatin organization.

Mammalian cells contain approximately 400 copies of the ribosomal RNA genes organized as tandem, head-to-tail repeats spread among 6-8 chromosomes. Only a subset of the genes is transcribed at any given time. Experimental evidence suggests that, in a specific cell type, only a fraction of the genes exists in a conformation that can be transcribed. An increasing body of study indicates that eukaryotic ribosomal RNA genes exist in either a heterochromatic nucleosomal state or in open euchromatic states in which they can be, or are, transcribed. This review will attempt to summarize our current understanding of the structure and organization of ribosomal chromatin.

Altered contractility and [Ca2+]i homeostasis in phospholemman-deficient murine myocytes: role of Na+/Ca2+ exchange.

Phospholemman (PLM) regulates contractility and Ca(2+) homeostasis in cardiac myocytes. We characterized excitation-contraction coupling in myocytes isolated from PLM-deficient mice backbred to a pure congenic C57BL/6 background. Cell length, cell width, and whole cell capacitance were not different between wild-type and PLM-null myocytes. Compared with wild-type myocytes, Western blots indicated total absence of PLM but no changes in Na(+)/Ca(2+) exchanger, sarcoplasmic reticulum (SR) Ca(2+)-ATPase, alpha(1)-subunit of Na(+)-K(+)-ATPase, and calsequestrin levels in PLM-null myocytes. At 5 mM extracellular Ca(2+) concentration ([Ca(2+)](o)), contraction and cytosolic [Ca(2+)] ([Ca(2+)](i)) transient amplitudes and SR Ca(2+) contents in PLM-null myocytes were significantly (P < 0.0004) higher than wild-type myocytes, whereas the converse was true at 0.6 mM [Ca(2+)](o). This pattern of contractile and [Ca(2+)](i) transient abnormalities in PLM-null myocytes mimics that observed in adult rat myocytes overexpressing the cardiac Na(+)/Ca(2+) exchanger. Indeed, we have previously reported that Na(+)/Ca(2+) exchange currents were higher in PLM-null myocytes. Activation of protein kinase A resulted in increased inotropy such that there were no longer any contractility differences between the stimulated wild-type and PLM-null myocytes. Protein kinase C stimulation resulted in decreased contractility in both wild-type and PLM-null myocytes. Resting membrane potential and action potential amplitudes were similar, but action potential duration was much prolonged (P < 0.04) in PLM-null myocytes. Whole cell Ca(2+) current densities were similar between wild-type and PLM-null myocytes, as were the fast- and slow-inactivation time constants. We conclude that a major function of PLM is regulation of cardiac contractility and Ca(2+) fluxes, likely by modulating Na(+)/Ca(2+) exchange activity.

Phospholemman inhibition of the cardiac Na+/Ca2+ exchanger. Role of phosphorylation.

We have demonstrated previously that phospholemman (PLM), a 15-kDa integral sarcolemmal phosphoprotein, inhibits the cardiac Na+/Ca2+ exchanger (NCX1). In addition, protein kinase A phosphorylates serine 68, whereas protein kinase C phosphorylates both serine 63 and serine 68 of PLM. Using human embryonic kidney 293 cells that are devoid of both endogenous PLM and NCX1, we first demonstrated that the exogenous NCX1 current (I(NaCa)) was increased by phorbol 12-myristate 13-acetate (PMA) but not by forskolin. When co-expressed with NCX1, PLM resulted in: (i) decreases in I(NaCa), (ii) attenuation of the increase in I(NaCa) by PMA, and (iii) additional reduction in I(NaCa) in cells treated with forskolin. Mutating serine 63 to alanine (S63A) preserved the sensitivity of PLM to forskolin in terms of suppression of I(NaCa), whereas mutating serine 68 to alanine (S68A) abolished the inhibitory effect of PLM on I(NaCa). Mutating serine 68 to glutamic acid (phosphomimetic) resulted in additional suppression of I(NaCa) as compared with wild-type PLM. These results suggest that PLM phosphorylated at serine 68 inhibited I(NaCa). The physiological significance of inhibition of NCX1 by phosphorylated PLM was evaluated in PLM-knock-out (KO) mice. When compared with wild-type myocytes, I(NaCa) was significant larger in PLM-KO myocytes. In addition, the PMA-induced increase in I(NaCa) was significantly higher in PLM-KO myocytes. By contrast, forskolin had no effect on I(NaCa) in wild-type myocytes. We conclude that PLM, when phosphorylated at serine 68, inhibits Na+/Ca2+ exchange in the heart.

DADLE induces a reversible hibernation-like state in HeLa cells.

[D-Ala(2)-D-Leu(5)-Enkephalin] (DADLE) can induce hibernation when injected into ground squirrels in summer and is able to increase the survival time of explanted organs such as liver and lung. Since cell metabolism is a target of this peptide, we have treated HeLa cells with DADLE and investigated its possible effect on transcription and proliferation as well as the resumption of metabolic activity after treatment. The labelling for Pol I, Pol II and for splicing factors such as snRNPs and SC-35 decreased after treatment as did the nucleolar labelling for UBF. In treated cells, several spherical nuclear bodies were found to be labelled for hnRNPs. In parallel, the number of proliferating cells decreased after treatment with DADLE. After recovery, there was a gradual resumption of cell function: transcription and splicing factors had a distribution similar to that of controls; proliferation resumed; nuclear bodies, representing storage sites for RNPs, disappeared.

Phospholemman overexpression inhibits Na+-K+-ATPase in adult rat cardiac myocytes: relevance to decreased Na+ pump activity in postinfarction myocytes.

Messenger RNA levels of phospholemman (PLM), a member of the FXYD family of small single-span membrane proteins with putative ion-transport regulatory properties, were increased in postmyocardial infarction (MI) rat myocytes. We tested the hypothesis that the previously observed reduction in Na+-K+-ATPase activity in MI rat myocytes was due to PLM overexpression. In rat hearts harvested 3 and 7 days post-MI, PLM protein expression was increased by two- and fourfold, respectively. To simulate increased PLM expression post-MI, PLM was overexpressed in normal adult rat myocytes by adenovirus-mediated gene transfer. PLM overexpression did not affect the relative level of phosphorylation on serine68 of PLM. Na+-K+-ATPase activity was measured as ouabain-sensitive Na+-K+ pump current (Ip). Compared with control myocytes overexpressing green fluorescent protein alone, Ip measured in myocytes overexpressing PLM was significantly (P < 0.0001) lower at similar membrane voltages, pipette Na+ ([Na+]pip) and extracellular K+ ([K+]o) concentrations. From -70 to +60 mV, neither [Na+]pip nor [K+]o required to attain half-maximal Ip was significantly different between control and PLM myocytes. This phenotype of decreased V(max) without appreciable changes in K(m) for Na+ and K+ in PLM-overexpressed myocytes was similar to that observed in MI rat myocytes. Inhibition of Ip by PLM overexpression was not due to decreased Na+-K+-ATPase expression because there were no changes in either protein or messenger RNA levels of either alpha1- or alpha2-isoforms of Na+-K+-ATPase. In native rat cardiac myocytes, PLM coimmunoprecipitated with alpha-subunits of Na+-K+-ATPase. Inhibition of Na+-K+-ATPase by PLM overexpression, in addition to previously reported decrease in Na+-K+-ATPase expression, may explain altered V(max) but not K(m) of Na+-K+-ATPase in postinfarction rat myocytes.