Phosphorylation Of E1 Research Articles

Pyruvate dehydrogenase complex deficiency is linked to regulatory loop disorder in the αV138M variant of human pyruvate dehydrogenase

The pyruvate dehydrogenase multienzyme complex (PDHc) connects glycolysis to the tricarboxylic acid cycle by producing acetyl-CoA via the decarboxylation of pyruvate. Because of its pivotal role in glucose metabolism, this complex is closely regulated in mammals by reversible phosphorylation, the modulation of which is of interest in treating cancer, diabetes, and obesity. Mutations such as that leading to the αV138M variant in pyruvate dehydrogenase, the pyruvate-decarboxylating PDHc E1 component, can result in PDHc deficiency, an inborn error of metabolism that results in an array of symptoms such as lactic acidosis, progressive cognitive and neuromuscular deficits, and even death in infancy or childhood. Here we present an analysis of two X-ray crystal structures at 2.7-Å resolution, the first of the disease-associated human αV138M E1 variant and the second of human wildtype (WT) E1 with a bound adduct of its coenzyme thiamin diphosphate and the substrate analogue acetylphosphinate. The structures provide support for the role of regulatory loop disorder in E1 inactivation, and the αV138M variant structure also reveals that altered coenzyme binding can result in such disorder even in the absence of phosphorylation. Specifically, both E1 phosphorylation at αSer-264 and the αV138M substitution result in disordered loops that are not optimally oriented or available to efficiently bind the lipoyl domain of PDHc E2. Combined with an analysis of αV138M activity, these results underscore the general connection between regulatory loop disorder and loss of E1 catalytic efficiency.

Intracellular Requirements for Passive Proton Transport through the Na+,K+-ATPase

The Na+,K+-ATPase (NKA or Na/K pump) hydrolyzes one ATP to exchange three intracellular Na+ (Na+i) for two extracellular K+ (K+o) across the plasma membrane by cycling through a set of reversible transitions between phosphorylated and dephosphorylated conformations, alternately opening ion-binding sites externally (E2) or internally (E1). With subsaturating [Na+]o and [K+]o, the phosphorylated E2P conformation passively imports protons generating an inward current (IH), which may be exacerbated in NKA-subunit mutations associated with human disease. To elucidate the mechanisms of IH, we studied the effects of intracellular ligands (transported ions, nucleotides, and beryllium fluoride) on IH and, for comparison, on transient currents measured at normal Na+o (QNa). Utilizing inside-out patches from Xenopus oocytes heterologously expressing NKA, we observed that 1) in the presence of Na+i, IH and QNa were both activated by ATP, but not ADP; 2) the [Na+]i dependence of IH in saturating ATP showed K0.5,Na = 1.8 ± 0.2 mM and the [ATP] dependence at saturating [Na+]i yielded K0.5,ATP = 48 ± 11 μM (in comparison, Na+i-dependent QNa yields K0.5,Na = 0.8 ± 0.2 mM and K0.5,ATP = 0.43 ± 0.03 μM; 3) ATP activated IH in the presence of K+i (∼15% of the IH observed in Na+i) only when Mg2+i was also present; and 4) beryllium fluoride induced maximal IH even in the absence of nucleotide. These data indicate that IH occurs when NKA is in an externally open E2P state with nucleotide bound, a conformation that can be reached through forward Na/K pump phosphorylation of E1, with Na+i and ATP, or by backward binding of K+i to E1, which drives the pump to the occluded E2(2K), where free Pi (at the micromolar levels found in millimolar ATP solutions) promotes external release of occluded K+ by backdoor NKA phosphorylation. Maximal IH through beryllium-fluorinated NKA indicates that this complex mimics ATP-bound E2P states.

Phosphorylation of bovine papillomavirus E1 by the protein kinase CK2 near the nuclear localization signal does not influence subcellular distribution of the protein in dividing cells.

The bovine papillomavirus E1 helicase is essential for viral replication. In dividing cells, DNA replication maintains, but does not increase, the viral genome copy number. Replication is limited by low E1 expression and an E1 nucleocytoplasmic shuttling mechanism. Shuttling is controlled in part by phosphorylation of E1 by cellular kinases. Here we investigate conserved sites for phosphorylation by kinase CK2 within the E1 nuclear localization signal. When these CK2 sites are mutated to either alanine or aspartic acid, no change in replication phenotype is observed, and there is no effect on the subcellular distribution of E1, which remains primarily nuclear. This demonstrates that phosphorylation of E1 by CK2 at these sites is not a factor in regulating viral DNA replication in dividing cells.

Heterogeneous nuclear ribonucleoprotein E1 regulates protein disulphide isomerase translation in oxidized low-density lipoprotein-activated endothelial cells.

Endothelium-derived protein disulphide isomerase (PDI) is required for thrombus formation in vivo. But, how to control PDI overproduction in oxidized low-density lipoprotein (oxLDL)-activated vascular endothelial cells (VECs) is not well understood. In this study, we try to answer this question using our newly identified activator of mTOC1 3-benzyl-5-((2-nitrophenoxy) methyl)-dihydrofuran-2 (3H)-one (3BDO) that has been shown to protect VECs. First, we performed a proteomics analysis on the oxLDL-activated vascular VECs in the presence or absence of 3BDO. Next, we constructed the heterogeneous nuclear ribonucleoprotein E1 (hnRNP E1) mutants at Ser43 and used the RNA-ChIP technique to investigate the relationship between hnRNP E1 and PDI production. Furthermore, we examined the effect of 3BDO on oxLDL-altered phosphorylation of Akt1 and Akt2. Finally, we studied the effect of 3BDO on oxLDL-altered PDI protein level in apolipoprotein E(-/-) mice with advanced atherosclerosis. In VECs, oxLDL-increased PDI protein level, induced hnRNP E1 phosphorylation at Ser43, suppressed the binding of hnRNP E1 to PDI 5'UTR and induced the phosphorylation of Akt2 but not Akt1. All ofthese processes were blocked by 3BDO. Importantly, Ser43 mutant of hnRNP E1 inhibited the increase of PDI protein level and the decrease of the binding of hnRNP E1 and PDI 5'UTR induced by oxLDL. Furthermore, 3BDO suppressed oxLDL-induced PDI protein increase in the serum and plaque endothelium of apolipoprotein E(-/-) mice. hnRNP E1 is a new regulator of PDI translation in oxLDL-activated VECs, and 3BDO is a powerful agent for controlling PDI overproduction.

Methods to assess the nucleocytoplasmic shuttling of the HPV E1 helicase and its effects on cellular proliferation and induction of a DNA damage response.

Replication of the human papillomavirus (HPV) double-stranded DNA genome in the nucleus of infected cells relies on the viral proteins E1 and E2 in conjunction with the host DNA replication machinery. This process is tightly linked to the replication of cellular DNA, in part through the cyclin-dependent phosphorylation of E1, which inhibits its export out of the nucleus to promote its accumulation in this compartment during S-phase. It has been recently shown that accumulation of E1 in the nucleus, while a prerequisite for viral DNA replication, leads to the inhibition of cellular proliferation and the activation of a DNA damage response (DDR). Here we describe methods to monitor the subcellular localization of E1 and to assess the deleterious effects of its nuclear accumulation on cellular proliferation, cell cycle progression and the induction of a DDR, using a combination of colony formation assays, immunofluorescence microcopy, and flow cytometry approaches.

Establishment of a TGFβ-Induced Post-Transcriptional EMT Gene Signature

A major challenge in the clinical management of human cancers is to accurately stratify patients according to risk and likelihood of a favorable response. Stratification is confounded by significant phenotypic heterogeneity in some tumor types, often without obvious criteria for subdivision. Despite intensive transcriptional array analyses, the identity and validation of cancer specific ‘signature genes’ remains elusive, partially because the transcriptome does not mirror the proteome. The simplification associated with transcriptomic profiling does not take into consideration changes in the relative expression among transcripts that arise due to post-transcriptional regulatory events. We have previously shown that TGFβ post-transcriptionally regulates epithelial-mesenchymal transition (EMT) by causing increased expression of two transcripts, Dab2 and ILEI, by modulating hnRNP E1 phosphorylation. Using a genome-wide combinatorial approach involving expression profiling and RIP-Chip analysis, we have identified a cohort of translationally regulated mRNAs that are induced during TGFβ-mediated EMT. Coordinated translational regulation by hnRNP E1 constitutes a post-transcriptional regulon inhibiting the expression of related EMT-facilitating genes, thus enabling the cell to rapidly and coordinately regulate multiple EMT-facilitating genes.

Identification of an mRNP Complex Regulating Tumorigenesis at the Translational Elongation Step

Transcript-selective translational regulation of epithelial-mesenchymal transition (EMT) by transforming growth factor-β (TGF-β) is directed by the hnRNP E1-containing TGF-β-activated-translational (BAT) mRNP complex. Herein, eukaryotic elongation factor-1 A1 (eEF1A1) is identified as an integral component of the BAT complex. Translational silencing of Dab2 and ILEI, two EMT transcripts, is mediated by the binding of hnRNP E1 and eEF1A1 to their 3'UTR BAT element, whereby hnRNP E1 stalls translational elongation by inhibiting the release of eEF1A1 from the ribosomal A site. TGF-β-mediated hnRNP E1 phosphorylation, through Akt2, disrupts the BAT complex, thereby restoring translation of target EMT transcripts. Attenuation of hnRNP E1 expression in two noninvasive breast epithelial cells (NMuMG and MCF-7) not only induced EMT but also enabled cells to form metastatic lesions invivo. Thus, translational regulation by TGF-β at the elongation stage represents a critical checkpoint coordinating the expression of EMT transcripts required during development and in tumorigenesis and metastatic progression.

Phosphorylation of the Human Papillomavirus Type 16 E1^E4 Protein at T57 by ERK Triggers a Structural Change That Enhances Keratin Binding and Protein Stability

The E1--E4 protein of human papillomavirus type 16 (HPV16) causes cytokeratin reorganization in the middle and upper epithelial layers and is thought to contribute to multiple facets of the virus life cycle. Although little is known as to how HPV16 E1--E4 (16E1--E4) functions are controlled following the first expression of this protein, the finding that low-risk E1--E4 proteins can be phosphorylated in vivo suggests an important role for kinases. Here, we show that 16E1--E4 is phosphorylated by cyclin-dependent kinase 1 (CDK1) and CDK2, extracellular signal-regulated kinase (ERK), protein kinase A (PKA), and PKC alpha, with CDK1/2 serine 32 and ERK threonine 57 phosphorylations representing the two primary events seen in cells in cycle. Interestingly, T57 phosphorylation was found to trigger a structural change in the 16E1--E4 protein that compacts the central fold region, leading to an increase in 16E1--E4 stability and overall abundance in the cell. When compared to wild-type 16E1--E4, a T57D phosphomimic was found to have greatly enhanced keratin-binding ability and an ability to modulate the binding of the unphosphorylated form, with keratin binding protecting the T57-phosphorylated form of 16E1--E4 from proteasomal degradation. In HPV16 genome-containing organotypic rafts, the T57-phosphorylated form was specifically detected in the intermediate cell layers, where productive infection occurs, suggesting that T57 phosphorylation may have a functional role at this stage of the viral life cycle. Interestingly, coexpression with 16E5 and ERK activation enhanced T57 phosphorylation, suggesting that E1--E4 and E5 may work together in vivo. Our data suggest a model in which the expression of 16E5 from the major E1--E4-E5 mRNA promotes T57 phosphorylation of E1--E4 and keratin binding, with dephosphorylation occurring following the switch to late poly(A) usage. Other forms of E1--E4, with alternative functional roles, may then increase in prevalence in the upper layers of the epithelium.

The conformation of H,K‐ATPase determines the nucleoside triphosphate (NTP) selectivity of active proton transport.

The gastric H,K-ATPase is related to other cation transport ATPases, i.e., Na,K-ATPase and Ca-ATPase, which are called E1-E2 ATPases in recognition of conformational transitions during their respective transport and catalytic cycles. Generally, these ATPases cannot utilize NTPs other than ATP for net pumping activity. For example, under standard assay conditions rates of NTP hydrolysis and H+ pumping by the H,K-ATPase for CTP are about 10% of those for ATP and undetectable with GTP, ITP and UTP. We observed that H,K-ATPase will catalyze NTP/ADP phosphate exchange at similar rates for all of these NTPs. An initial hypothesis that all NTPs form a phosphoenzyme intermediate but only those formed by ATP can catalyze proton transport has been modified by additional studies. Our new results demonstrate that under two conditions that promote the conformational change from E2.K to E1 all of these NTPs will support H+ transport at 30–50% of the rate with ATP. 1) K+-stimulated NTPase activity and H+ pumping activity occur when NTP and ADP are present. 2) Luminal K+ stimulates NTP-dependent H+ pumping when cytoplasmic K+ is ~zero. These data support structural models for E1-E2 ATPases whereby adenosine binding promotes the E2.K to E1 conformational change and K+ de-occlusion, and then there is less nucleotide specificity for the phosphorylation of E1.

Nuclear Import of Bovine Papillomavirus Type 1 E1 Protein Is Mediated by Multiple Alpha Importins and Is Negatively Regulated by Phosphorylation near a Nuclear Localization Signal

Papillomavirus DNA replication occurs in the nucleus of infected cells and requires the viral E1 protein, which enters the nuclei of host epithelial cells and carries out enzymatic functions required for the initiation of viral DNA replication. In this study, we investigated the pathway and regulation of the nuclear import of the E1 protein from bovine papillomavirus type 1 (BPV1). Using an in vitro binding assay, we determined that the E1 protein interacted with importins alpha3, alpha4, and alpha5 via its nuclear localization signal (NLS) sequence. In agreement with this result, purified E1 protein was effectively imported into the nucleus of digitonin-permeabilized HeLa cells after incubation with importin alpha3, alpha4, or alpha5 and other necessary import factors. We also observed that in vitro binding of E1 protein to all three alpha importins was significantly decreased by the introduction of pseudophosphorylation mutations in the NLS region. Consistent with the binding defect, pseudophosphorylated E1 protein failed to enter the nucleus of digitonin-permeabilized HeLa cells in vitro. Likewise, the pseudophosphorylation mutant showed aberrant intracellular localization in vivo and accumulated primarily on the nuclear envelope in transfected HeLa cells, while the corresponding alanine replacement mutant displayed the same cellular location pattern as wild-type E1 protein. Collectively, our data demonstrate that BPV1 E1 protein can be transported into the nucleus by more than one importin alpha and suggest that E1 phosphorylation by host cell kinases plays a regulatory role in modulating E1 nucleocytoplasmic localization. This phosphoregulation of nuclear E1 protein uptake may contribute to the coordination of viral replication with keratinocyte proliferation and differentiation.

Impaired nucleotide excision repair upon macrophage differentiation is corrected by E1 ubiquitin-activating enzyme.

Global nucleotide excision repair is greatly attenuated in terminally differentiated mammalian cells. We observed this phenomenon in human neurons and in macrophages, noting that the transcription-coupled repair pathway remains functional and that there is no significant reduction in levels of excision repair enzymes. We have discovered that ubiquitin-activating enzyme E1 complements the repair deficiency in macrophage extracts, and although there is no reduction in the concentration of E1 upon differentiation, our results indicate a reduction in phosphorylation of E1. In preliminary studies, we have identified the basal transcription factor TFIIH as the potential target for ubiquitination. We suggest that this unusual type of regulation at the level of the E1 enzyme is likely to affect numerous cellular processes and may represent a strategy to coordinate multiple phenotypic changes upon differentiation by using E1 as a "master switch."

Nucleocytoplasmic Shuttling of Bovine Papillomavirus E1 Helicase Downregulates Viral DNA Replication in S Phase

The papillomavirus E1 protein is essential for the initiation of viral replication. We previously showed that the bovine papillomavirus E1 protein is unstable and becomes resistant to ubiquitin-mediated degradation when tightly bound to cyclin E-cyclin-dependent kinase 2 (Cdk2) before the start of DNA synthesis. However, neither the protection nor the targeted degradation of E1 appears to depend on its phosphorylation by Cdk. Here, we report that Cdk phosphorylation of E1 is also not a prerequisite for the initiation of viral DNA replication either in vitro or in vivo. Nevertheless, we found that phosphorylation of one Cdk site, Ser283, abrogates E1 replicative activity only in a cellular context. We show that this site-specific phosphorylation of E1 drives its export from the nucleus and promotes its continuous nucleocytoplasmic shuttling. In addition, we find that E1 shuttling occurs in S phase, when cyclin A-Cdk2 is activated. E1 interacts with the active cyclin A-Cdk2 complex and is phosphorylated on Ser283 by this kinase. These data suggest that the phosphorylation of E1 on Ser283 is a negative regulatory event that is involved in preventing the amplification of viral DNA during S phase. This finding reveals a novel facet of E1 regulation that could account for the variations of the viral replication capacity during different cell cycle phases, as well as in different stages of the viral cycle.

Cyclin/CDK Regulates the Nucleocytoplasmic Localization of the Human Papillomavirus E1 DNA Helicase

Cyclin-dependent kinases (CDKs) play key roles in eukaryotic DNA replication and cell cycle progression. Phosphorylation of components of the preinitiation complex activates replication and prevents reinitiation. One mechanism is mediated by nuclear export of critical proteins. Human papillomavirus (HPV) DNA replication requires cellular machinery in addition to the viral replicative DNA helicase E1 and origin recognition protein E2. E1 phosphorylation by cyclin/CDK is critical for efficient viral DNA replication. We now show that E1 is phosphorylated by CDKs in vivo and that phosphorylation regulates its nucleocytoplasmic localization. We identified a conserved regulatory region for localization which contains a dominant leucine-rich nuclear export sequence (NES), the previously defined cyclin binding motif, three serine residues that are CDK substrates, and a putative bipartite nuclear localization sequence. We show that E1 is exported from the nucleus by a CRM1-dependent mechanism unless the NES is inactivated by CDK phosphorylation. Replication activities of E1 phosphorylation site mutations are reduced and correlate inversely with their increased cytoplasmic localization. Nuclear localization and replication activities of most of these mutations are enhanced or restored by mutations in the NES. Collectively, our data demonstrate that CDK phosphorylation controls E1 nuclear localization to support viral DNA amplification. Thus, HPV adopts and adapts the cellular regulatory mechanism to complete its reproductive program.

R-Lipoic Acid Inhibits Mammalian Pyruvate Dehydrogenase Kinase

The four pyruvate dehydrogenase kinase (PDK) and two pyruvate dehydrogenase phosphatase (PDP) isoenzymes that are present in mammalian tissues regulate activity of the pyruvate dehydrogenase complex (PDC) by phosphorylation/dephosphorylation of its pyruvate dehydrogenase (E1) component. The effect of lipoic acids on the activity of PDKs and PDPs was investigated in purified proteins system. R-lipoic acid, S-lipoic acid and R-dihydrolipoic acid did not significantly affect activities of PDPs and at the same time inhibited PDKs to different extents (PDK1 > PDK4 ∼ PDK2 > PDK3 for R-LA). Since lipoic acids inhibited PDKs activity both when reconstituted in PDC and in the presence of E1 alone, dissociation of PDK from the lipoyl domains of dihydrolipoamide acetyltransferase in the presence of lipoic acids is not a likely explanation for inhibition. The activity of PDK1 towards phosphorylation sites 1, 2 and 3 of E1 was decreased to the same extent in the presence of R-lipoic acid, thus excluding protection of the E1 active site by lipoic acid from phosphorylation. R-lipoic acid inhibited autophosphorylation of PDK2 indicating that it exerted its effect on PDKs directly. Inhibition of PDK1 by R-lipoic acid was not altered by ADP but was decreased in the presence of pyruvate which itself inhibits PDKs. An inhibitory effect of lipoic acid on PDKs would result in less phosphorylation of E1 and hence increased PDC activity. This finding provides a possible mechanism for a glucose (and lactate) lowering effect of R-lipoic acid in diabetic subjects.

Facilitated Interaction between the Pyruvate Dehydrogenase Kinase Isoform 2 and the Dihydrolipoyl Acetyltransferase

The dihydrolipoyl acetyltransferase (E2) has an enormous impact on pyruvate dehydrogenase kinase (PDK) phosphorylation of the pyruvate dehydrogenase (E1) component by acting as a mobile binding framework and in facilitating and mediating regulation of PDK activity. Analytical ultracentrifugation (AUC) studies established that the soluble PDK2 isoform is a stable dimer. The interaction of PDK2 with the lipoyl domains of E2 (L1, L2) and the E3-binding protein (L3) were characterized by AUC. PDK2 interacted very weakly with L2 (Kd approximately 175 microM for 2 L2/PDK2) but much tighter with dimeric glutathione S-transferase (GST)-L2 (Kd approximately 3 microM), supporting the importance of bifunctional binding. Reduction of lipoyl groups resulted in approximately 8-fold tighter binding of PDK2 to GST-L2red, which was approximately 300-fold tighter than binding of 2 L2red and also much tighter than binding by GST-L1red and GST-L3red. The E2 60-mer bound approximately 18 PDK2 dimers with a Kd similar to GST-L2. E2.E1 bound more PDK2 (approximately 27.6) than E2 with approximately 2-fold tighter affinity. Lipoate reduction fostered somewhat tighter binding at more sites by E2 and severalfold tighter binding at the majority of sites on E2.E1. ATP and ADP decreased the affinity of PDK2 for E2 by 3-5-fold and adenosine 5'-(beta,gamma-imino)triphosphate or phosphorylation of E1 similarly reduced PDK2 binding to E2.E1. Reversible bifunctional binding to L2 with the mandatory singly held transition fits the proposed "hand-over-hand" movement of a kinase dimer to access E1 without dissociating from the complex. The gain in binding interactions upon lipoate reduction likely aids reduction-engendered stimulation of PDK2 activity; loosening of binding as a result of adenine nucleotides and phosphorylation may instigate movement of lipoyl domain-held kinase to a new E1 substrate.

Functional analysis of a carboxyl-terminal phosphorylation mutant of the bovine papillomavirus E1 protein

The papillomavirus E1 protein is essential for viral DNA replication, and phosphorylation of E1 appears to regulate protein function and DNA replication. Serine 584 of bovine papillomavirus E1 is in a conserved motif resembling a CK2 consensus site, and is phosphorylated by CK2 in vitro. Mutation of serine 584 to alanine eliminates replication of the viral genome in transient replication assays. Wild-type and mutant E1 proteins were expressed from recombinant baculoviruses and used to assess biochemical functions of the amino acid 584 substitution. Helicase enzyme activity, E1 binding to the viral E2 protein and to cellular DNA polymerase alpha-primase were all unaffected in the mutant protein. Binding of E1 to viral replication origin DNA sequences was reduced in the mutant, but not eliminated. The carboxyl-terminal region of the protein appears to play a role in regulating E1 function, and adds to a complex picture emerging for papillomavirus DNA replication control.

A carboxyl-terminal serine of the bovine papillomavirus E1 protein is phosphorylated in vivo and in vitro

The E1 protein of bovine papillomavirus (BPV) plays several key roles in viral DNA replication. E1 binds the viral origin, unwinds template DNA at the replication fork and recruits cellular replication machinery to the viral DNA. E1 is phosphorylated at multiple sites, and phosphorylation of E1 regulates E1 function and viral DNA replication. A consensus motif for the cellular kinase CK2 was identified at serine 584 near the carboxyl terminus of BPV E1, and found to be highly conserved among papillomavirus E1 proteins. Serine 584 was identified as a substrate of CK1 and CK2 in vitro by mutational and biochemical analysis, and was phosphorylated by a cellular kinase in cultured cells.

Reaction Mechanism for Mammalian Pyruvate Dehydrogenase Using Natural Lipoyl Domain Substrates

The pyruvate dehydrogenase (E1) component of the pyruvate dehydrogenase complex (PDC) catalyzes a two-step reaction. Recombinant production of substrate amounts of the lipoyl domains of the dihydrolipoyl transacetylase (E2) component of the mammalian PDC allowed kinetic characterization of the rapid physiological reaction catalyzed by E1. Using either the N-terminal (L1) or the internal (L2) lipoyl domain of E2 as a substrate, analyses of steady state kinetic data support a ping pong mechanism. Using standard E1 preparations, Michaelis constants (Km) were 52 ± 14 μM for L1 and 24.8 ± 3.8 μM for pyruvate and kcat was 26.3 s−1. With less common, higher activity preparations of E1, the Km values were ≥160 μM for L1 and ≥35 μM for pyruvate and kcat was ≥70 s−1. Similar results were found with the L2 domain. The best synthetic lipoylated-peptide (L2 residues 163–177) was a much poorer substrate (Km ≥ 15 mM, kcat ≈ 5 s−1; kcat/Km decreased >1500-fold) than L1 or L2, but a far better substrate in the E1 reaction than free lipoamide (kcat/Km increased >500-fold). Each lipoate source was an effective substrate in the dihydrolipoyl dehydrogenase (E3) reaction, but E3 had a lower Km for the L2 domain than for lipoamide or the lipoylated peptides. In contrast to measurements with slow E1 model reactions that use artificial acceptors, we confirmed that the natural E1 reaction, using lipoyl domain acceptors, was completely inhibited (>99%) by phosphorylation of E1 and the phosphorylation strongly inhibited the reverse of the second step catalyzed by E1. The mechanisms by which phosphorylation interferes with E1 activity is interpreted based on accrued results and the location of phosphorylation sites mapped onto the 3-D structure of related α-keto acid dehydrogenases.

Interaction between cyclin-dependent kinases and human papillomavirus replication-initiation protein E1 is required for efficient viral replication.

We have identified the human papillomavirus (HPV) DNA replication initiation protein E1 as a tight-binding substrate of cyclin E/cyclin-dependent kinase (Cdk) complexes by using expression cloning. E1, a DNA helicase, collaborates with the HPV E2 protein in ori-dependent replication. E1 formed complexes with cyclin E in insect and mammalian cells, independent of Cdks and E2. Additional cyclins, including A-, B-, and F-type (but not D-type), interacted with the E1/E2 complex, and A- and E-type cyclin kinases were capable of phosphorylating E1 and E2 in vitro. Association with cyclins and efficient phosphorylation of E1 required the presence of a cyclin interaction motif (the RXL motif). E1 lacking the RXL motif displayed defects in E2-dependent HPV ori replication in vivo. Consistent with a role for Cdk-mediated phosphorylation in E1 function, an E1 protein lacking all four candidate Cdk phosphorylation sites still associated with E2 and cyclin E but was impaired in HPV replication in vitro and in vivo. Our data reveal a link between cyclin/Cdk function and activation of HPV DNA replication through targeting of Cdk complexes to the E1 replication-initiation protein and suggest a functional role for E1 phosphorylation by Cdks. The use of cyclin-binding RXL motifs is now emerging as a major mechanism by which cyclins are targeted to key substrates.

Modulation of Bovine Papillomavirus DNA Replication by Phosphorylation of the Viral E1 Protein

E1 is the DNA replication origin recognition protein for bovine papillomavirus (BPV), and it carries out enzymatic functions required for initiation of viral DNA replication. Cellular mechanisms likely play a role in regulating BPV DNA replication. We are investigating the role of phosphorylation of E1 on viral replicationin vivoand on E1 activityin vitro.Serine 109 is a phosphoacceptorin vivoand is targeted by protein kinase A and protein kinase Cin vitro.A viral genome carrying a serine 109 to alanine mutation replicates more efficiently than wild-typein vivoin a transient replication assay. Furthermore, purified mutant protein, while having wild-type levels of ATPase activity, is able to bind more origin-containing DNA than wild-type E1. Phosphorylation therefore appears to play a selective role in modulating a specific E1 function during viral DNA replication.