N-terminal Phosphorylation Sites Research Articles

Phosphoregulation in the N-terminus of NRT2.1 affects nitrate uptake by controlling the interaction of NRT2.1 with NAR2.1 and kinase HPCAL1 in Arabidopsis.

NRT2.1, the major high affinity nitrate transporter in roots, can be phosphorylated at five different sites within the N- and the C-terminus. Here, we characterized the functional relationship of two N-terminal phosphorylation sites, S21 and S28, in Arabidopsis. Based on a site-specific correlation network, we identified a receptor kinase (HPCAL1, AT5G49770), phosphorylating NRT2.1 at S21 and resulting in active nitrate uptake. HPCAL1 itself was regulated by phosphorylation at S839 and S870 within its kinase domain. In the active state, when S839 was dephosphorylated and S870 was phosphorylated, HPCAL1 was found to interact with the N-terminus of NRT2.1, mainly when S28 was dephosphorylated. Phosphorylation of NRT2.1 at S21 resulted in a reduced interaction of NRT2.1 with its activator NAR2.1, but nitrate transport activity remained. By contrast, phosphorylated NRT2.1 at S28 enhanced the interaction with NAR2.1, but reduced the interaction with HPCAL1. Here we identified HPCAL1 as the kinase affecting this phospho-switch through phosphorylation of NRT2.1 at S21.

Identification of sucrose synthase from Micractinium conductrix to favor biocatalytic glycosylation.

Sucrose synthase (SuSy, EC 2.4.1.13) is a unique glycosyltransferase (GT) for developing cost-effective glycosylation processes. Up to now, some SuSys derived from plants and bacteria have been used to recycle uridine 5'-diphosphate glucose in the reactions catalyzed by Leloir GTs. In this study, after sequence mining and experimental verification, a SuSy from Micractinium conductrix (McSuSy), a single-cell green alga, was overexpressed in Escherichia coli, and its enzymatic properties were characterized. In the direction of sucrose cleavage, the specific activity of the recombinant McSuSy is 9.39 U/mg at 37°C and pH 7.0, and the optimum temperature and pH were 60°C and pH 7.0, respectively. Its nucleotide preference for uridine 5'-diphosphate (UDP) was similar to plant SuSys, and the enzyme activity remained relatively high when the DMSO concentration below 25%. The mutation of the predicted N-terminal phosphorylation site (S31D) significantly stimulated the activity of McSuSy. When the mutant S31D of McSuSy was applied by coupling the engineered Stevia glycosyltransferase UGT76G1 in a one-pot two-enzyme reaction at 10% DMSO, 50 g/L rebaudioside E was transformed into 51.06 g/L rebaudioside M in 57 h by means of batch feeding, with a yield of 76.48%. This work may reveal the lower eukaryotes as a promising resource for SuSys of industrial interest.

In silico analysis of the possible crosstalk between O-linked β-GlcNAcylation and phosphorylation sites of Disabled 1 adaptor protein in vertebrates.

Disabled 1 (Dab1) is an adaptor protein with essential functions regulated by reelin signaling and affects many biological processes in the nervous system, including cell motility, adhesion, cortical development, maturation, and synaptic plasticity. Posttranslational modifications directly guide the fates of cytoplasmic proteins to complete their functions correctly. Reciprocal crosstalk between O-GlcNAcylation and phosphorylation is a dynamic modification in cytoplasmic proteins. It modulates the functions of the proteins by regulating their interactions with other molecules in response to the continuously changeable cell microenvironment. Although Dab1 contains conserved recognition sites for phosphorylation in their N-terminal protein interaction domain, the O-β-GlcNAcylation and phosphorylation sites of human Dab1 sequence, their reciprocal crosstalk, and potential kinases catalyzing the phosphorylation remain unknown. In this study, we determined potential thirty-seven O-β-GlcNAcylation and sixty-seven phosphorylation sites. Conserved twenty-one residues of these glycosylated sites were also phosphorylated with various kinases, including ATM, CKI, DNAPK, GSK3, PKC, PKG, RSK, cdc2, cdk5, and p38MAPK. In addition, we analyzed these conserved sites at our constructed two- and three-dimensional structures of human Dab1 protein. Dab1 protein models were frequently composed of coil structures as well as α-helix and β-strands. Many of these conserved crosstalk sites between O-β-GlcNAcylation and phosphorylation were localized at the coil region of the protein model. These findings may guide biochemical, genetic, and glyco-biology based on further experiments about the Dab1 signaling process. Understanding these modifications might change the point of view of the Dab1 signaling process and treatment for pathological conditions in neurodegenerative diseases such as Alzheimer's disease.

Research advances on the structure, function and regulation of NIP aquaporins in plants

<p indent="0mm">Aquaporin protein (AQP) family, as a “carrier” of water, micronutrients, and solute macromolecules in plants, plays an important role in material transport. Unlike other members of the AQP family, nodulin 26-like intrinsic proteins (NIPs) mainly penetrate small molecules, such as water, glycerin, silicic acid, boric acid, and urea. In addition, it is involved in plant cell osmoregulation, seed germination, lateral root production, leaf and flower growth and development, and response to biotic and abiotic (drought, chilling, salinity, metalloid toxicity, hypoxia, etc.) stresses. So far, structures of the NIP family have been partially reported. The NIPs can be divided into three classes based on the pore size of the ar/R selective filter, which determines the absorption and transportation of different substrates. Moreover, the NIPs contain two NPA motifs, which are located in loop B and loop E and have a typical ring-like hourglass structure. Therefore, different structures of the NIP subfamily play different roles in plant membrane solute transport and water balance. The <italic>NIP</italic> gene family has been identified in <italic>Arabidopsis thaliana</italic>,<italic> Oryza sativa</italic>, and other plants, whose function needs to be achieved through a variety of signaling pathways and the synergistic effect of physiological processes. In recent years, more and more reports have shown how the <italic>NIP</italic> gene participates in regulating gene expression levels under various stress conditions and interacts with a variety of stress proteins to jointly regulate the water and osmotic balance of plants and improve plant resistance and adaptability. The co-expression of <italic>NIPs</italic> with <italic>AtBOR1</italic>, <italic>AtNodGS</italic>, and <italic>AtACR3</italic> genes can enhance the transport of solute and aquaporin. Besides, the interaction between NIP and 14-3-3f proteins can stabilize cell structure and substance synthesis in vivo, thus enhancing plant cold resistance. Interestingly, <italic>NIP</italic> is regulated by WRKY transcription factors and plays an active role in the regulation and distribution of metalloid elements in plants. In addition, the <italic>NIP </italic>genes are involved in the response of various hormone signaling pathways in plants, and their gating mechanisms and osmotic stress responses are regulated by the C- and N-terminal phosphorylation sites of NIP proteins, but the specific response mechanisms remain to be further explored. <italic>NIPs</italic> are a large multi-gene family with functional redundancy among members, and the similarities and differences in functions and mechanisms of different subfamilies of <italic>NIP</italic> genes need to be investigated. One of our main subsequent research directions is to explore the stress resistance network of the <italic>NIP</italic> genes, especially their response mechanisms to biotic stress. What’s more, NIPs can interact with PIP proteins belonging to the AQP family and affect the diffusion of water molecules by changing their positions. Therefore, whether NIPs interact with other members of the AQP family, such as SIPs and TIPs, deserves to be validated further. In summary, the origin and evolution, structure, classification, biological function, and regulation mechanism of the NIP family in plants are reviewed, which provides new insights and references for further research.

Cardiac Troponin I N-Terminal Pseudo-Phosphorylation Regulates Power Output and Stretch Activation

Beat-to-beat heart function is modulated by β-adrenergic stimulation, which elicits PKA phosphorylation of several cardiac myocyte proteins including cardiac troponin I (cTnI). PKA phosphorylation of the cTnI N-terminal region is known to regulate myofilament Ca2+ sensitivity of force. Our lab has also observed PKA phosphorylation increases cardiac myofilament power and augments transient force overshoot. However, it is unknown if cTnI N-terminal phosphorylation per se affects myofilament power and stretch activation. We tested the hypothesis that cTnI N-terminal pseudo-phosphorylation is sufficient to increase power and stretch activation. Molecular sufficiency was investigated by measuring power and stretch activation after cTn exchange into rat permeabilized slow-twitch skeletal muscle fibers, since slow-skeletal (ss)TnI lacks N-terminal phosphorylation sites. Four different human cTnI N-terminal pseudo-phosphorylation states were tested: (i) non-phosphorylated WT, (ii) the canonical S22/23D PKA sites, (iii) the tyrosine kinase Y26E site, also known to modulate myofilament Ca2+ sensitivity, and (iv) the combinatorial S22/23D + Y26E cTnI. Following S22/23D cTnI exchange, fiber power increased ∼18% compared to WT cTn exchange where power decreased ∼3% (p < 0.05). Following Y26E cTnI exchange, power also increased (∼6%), albeit to a less degree than S22/23D. Exchange of combined S22/23D + Y26E cTnI resulted in a similar power increase (∼17%) as S22/23D alone, indicating S22/23D cTnI pseudo-phosphorylation is sufficient to regulate myofilament power. Exchange with S22/23D, Y26E, and S22/23D + Y26E cTnI all augmented stretch activation to a similar amount, implicating redundant site-specific stretch regulation. Finally, only the combinatorial S22/23D + Y26E cTnI increased stretch-activated rates of force development, implicating synergistic regulation of stretch-activated force kinetics. Overall, cTnI N-terminal pseudo-phosphorylation regulates myofilament power and stretch activation by a combination of dominant, redundant and synergistic biophysical mechanisms, which may complement each other to tune beat-to-beat myocardial output demand.

90 YEARS OF PROGESTERONE: Steroid receptors as MAPK signaling sensors in breast cancer: let the fates decide.

Steroid hormone receptors (SRs) are classically defined as ligand-activated transcription factors that function as master regulators of gene programs important for a wide range of processes governing adult physiology, development, and cell or tissue homeostasis. A second function of SRs includes the ability to activate cytoplasmic signaling pathways. Estrogen (ER), androgen (AR), and progesterone (PR) receptors bind directly to membrane-associated signaling molecules including mitogenic protein kinases (i.e. c-SRC and AKT), G-proteins, and ion channels to mediate context-dependent actions via rapid activation of downstream signaling pathways. In addition to making direct contact with diverse signaling molecules, SRs are further fully integrated with signaling pathways by virtue of their N-terminal phosphorylation sites that act as regulatory hot-spots capable of sensing the signaling milieu. In particular, ER, AR, PR, and closely related glucocorticoid receptors (GR) share the property of accepting (i.e. sensing) ligand-independent phosphorylation events by proline-directed kinases in the MAPK and CDK families. These signaling inputs act as a 'second ligand' that dramatically impacts cell fate. In the face of drugs that reliably target SR ligand-binding domains to block uncontrolled cancer growth, ligand-independent post-translational modifications guide changes in cell fate that confer increased survival, EMT, migration/invasion, stemness properties, and therapy resistance of non-proliferating SR+ cancer cell subpopulations. The focus of this review is on MAPK pathways in the regulation of SR+ cancer cell fate. MAPK-dependent phosphorylation of PR (Ser294) and GR (Ser134) will primarily be discussed in light of the need to target changes in breast cancer cell fate as part of modernized combination therapies.

Roles of N-terminal Annexin A2 phosphorylation sites and miR-206 in colonic adenocarcinoma

AimsAnnexin A2 (ANXA2) is closely associated with tumor malignancy and its N-terminus includes a vital domain for its function. The aims are to explore the correlation between the sites (Tyr23, Ser1, Ser11 and Ser25) in the domain and its roles. Main methodsWe re-expressed ANXA2 with mutated sites in ANXA2-deleted human colonic adenocarcinoma cell line caco2 (ANXA2-/-caco2). A series of analyses were used to determine the correlation of each site with ANXA2 activation, cell malignancy enhancement and motility-associated microstructural development. Bioinformatics and luciferase reporter assays were employed to validate ANXA2-targeted miRNAs. Key findingsThe in vitro results showed that all single and multiple mutations of the ANXA2 N-terminal sites inhibited ANXA2 phosphorylation at different levels and subsequently inhibited the proliferation, motility, and polymerization of F-actin and β-tubulin in caco2 cells. Motility-associated microstructures were significantly remodeled when these sites were mutated. The forced expression of miR-206 significantly suppressed the proliferation, motility and epithelial-mesenchymal transition (EMT) of caco2 cells. The in vivo results showed that all the ANXA2 N-terminal site mutations and forced expression of miR-206 significantly inhibited tumor growth. Overall, this study demonstrated that the sites of the ANXA2 N-terminus, especially Tyr23, play crucial roles in maintaining the high malignancy of colonic adenocarcinoma. Furthermore, miR-206 targets ANXA2 and plays a role as a cancer suppressor in colonic adenocarcinoma. SignificanceOur study provided evidence that further elucidates the molecular mechanism of ANXA2 and its roles in colonic adenocarcinoma and suggested potential targets of ANXA2 for colonic adenocarcinoma therapy by using miR-206 as a novel strategy.

Centrobin is essential for C-tubule assembly and flagellum development in Drosophila melanogaster spermatogenesis.

Centrobin homologues identified in different species localize on daughter centrioles. In Drosophila melanogaster sensory neurons, Centrobin (referred to as CNB in Drosophila) inhibits basal body function. These data open the question of CNB's role in spermatocytes, where daughter and mother centrioles become basal bodies. In this study, we report that in these cells, CNB localizes equally to mother and daughter centrioles and is essential for C-tubules to attain the right position and remain attached to B-tubules as well as for centrioles to grow in length. CNB appears to be dispensable for meiosis, but flagellum development is severely compromised in Cnb mutant males. Remarkably, three N-terminal POLO phosphorylation sites that are critical for CNB function in neuroblasts are dispensable for spermatogenesis. Our results underpin the multifunctional nature of CNB that plays different roles in different cell types in Drosophila, and they identify CNB as an essential component for C-tubule assembly and flagellum development in Drosophila spermatogenesis.

β-Catenin Mutations: Insights into the APC Pathway and the Power of Genetics.

See related article by Sparks et al., [Cancer Res 1998;58:1130–4][1] . Visit the Cancer Research 75th Anniversary [timeline][2]. In the mid 1990s, the human genome project was well underway, but it would still be more than 5 years before the first draft of the human genome was published and more

Properties of African Cassava Mosaic Virus Capsid Protein Expressed in Fission Yeast.

The capsid proteins (CPs) of geminiviruses combine multiple functions for packaging the single-stranded viral genome, insect transmission and shuttling between the nucleus and the cytoplasm. African cassava mosaic virus (ACMV) CP was expressed in fission yeast, and purified by SDS gel electrophoresis. After tryptic digestion of this protein, mass spectrometry covered 85% of the amino acid sequence and detected three N-terminal phosphorylation sites (threonine 12, serines 25 and 62). Differential centrifugation of cell extracts separated the CP into two fractions, the supernatant and pellet. Upon isopycnic centrifugation of the supernatant, most of the CP accumulated at densities typical for free proteins, whereas the CP in the pellet fraction showed a partial binding to nucleic acids. Size-exclusion chromatography of the supernatant CP indicated high order complexes. In DNA binding assays, supernatant CP accelerated the migration of ssDNA in agarose gels, which is a first hint for particle formation. Correspondingly, CP shifted ssDNA to the expected densities of virus particles upon isopycnic centrifugation. Nevertheless, electron microscopy did not reveal any twin particles, which are characteristic for geminiviruses.

New insights into the post-translational modification of multiple phosphoenolpyruvate carboxylase isoenzymes by phosphorylation and monoubiquitination during sorghum seed development and germination.

Phosphoenolpyruvate carboxylase (PEPC; E.C. 4.1.1.31) was characterized in developing and germinating sorghum seeds, focusing on the transcript and polypeptide abundance of multiple plant-type phosphoenolpyruvate carboxylase (PTPC) genes, and the post-translational modification of each isoenzyme by phosphorylation versus monoubiquitination during germination. We observed high levels of SbPPC4 (Sb07g014960) transcripts during early development (stage I), and extensive transcript abundance of SbPPC2 (Sb02g021090) and SbPPC3 (Sb04g008720) throughout the entire life cycle of the seed. Although tandem mass spectrometry (MS) analysis of immunopurified PTPC indicated that four different PTPC isoenzymes were expressed in the developing and germinating seeds, SbPPC3 was the most abundant isozyme of the developing seed, and of the embryo and the aleurone layer of germinating seeds. In vivo phosphorylation of the different PTPC isoenzymes at their conserved N-terminal seryl phosphorylation site during germination was also established by MS/MS analysis. Furthermore, three of the four isoenzymes were partially monoubiquitinated, with MS/MS pinpointing SbPPC2 and SbPPC3 monoubiquitination at the conserved Lys-630 and Lys-624 residues, respectively. Our results demonstrate that monoubiquitination and phosphorylation simultaneously occur in vivo with different PTPC isozymes during seed germination. In addition, we show that PTPC monoubiquitination in germinating sorghum seeds always increases at stage II (emergence of the radicle), is maintained during the aerobic period of rapid cell division and reserve mobilization, and remains relatively constant until stage IV-V when coleoptiles initiate the formation of the photosynthetic tissues.

Mutation of androgen receptor N-terminal phosphorylation site Tyr-267 leads to inhibition of nuclear translocation and DNA binding.

Reactivation of androgen receptor (AR) may drive recurrent prostate cancer in castrate patients. Ack1 tyrosine kinase is overexpressed in prostate cancer and promotes castrate resistant xenograft tumor growth and enhances androgen target gene expression and AR recruitment to enhancers. Ack1 phosphorylates AR at Tyr-267 and possibly Tyr-363, both in the N-terminal transactivation domain. In this study, the role of these phosphorylation sites was investigated by characterizing the phosphorylation site mutants in the context of full length and truncated AR lacking the ligand-binding domain. Y267F and Y363F mutants showed decreased transactivation of reporters. Expression of wild type full length and truncated AR in LNCaP cells increased cell proliferation in androgen-depleted conditions and increased colony formation. However, the Y267F mutant of full length and truncated AR was defective in stimulating cell proliferation. The Y363F mutant was less severely affected than the Y267F mutant. The full length AR Y267F mutant was defective in nuclear translocation induced by androgen or Ack1 kinase. The truncated AR was constitutively localized to the nucleus. Chromatin immunoprecipitation analysis showed that it was recruited to the target enhancers without androgen. The truncated Y267F AR mutant did not exhibit constitutive nuclear localization and androgen enhancer binding activity. These results support the concept that phosphorylation of Tyr-267, and to a lesser extent Tyr-363, is required for AR nuclear translocation and recruitment and DNA binding and provide a rationale for development of novel approaches to inhibit AR activity.

Mutation of Androgen Receptor N-Terminal Phosphorylation Site Tyr-267 Leads to Inhibition of Nuclear Translocation and DNA Binding

Mutation of Androgen Receptor N-Terminal Phosphorylation Site Tyr-267 Leads to Inhibition of Nuclear Translocation and DNA Binding

A study of the DNA damage checkpoint in Candida albicans: uncoupling of the functions of Rad53 in DNA repair, cell cycle regulation and genotoxic stress‐induced polarized growth

In response to genotoxic stress (GS), Candida albicans can undergo polarized growth and massive genome rearrangements including loss-of-heterozygosity (LOH) events. We evaluated the contribution of the CaRad53p and CaDun1p kinases of the DNA damage checkpoint (DDCP) in these processes. Characterization of C. albicans rad53ΔΔ and dun1ΔΔ mutants revealed that the two kinases were involved in the maintenance of heterozygosity. SNP-RFLP typing and whole-genome sequencing of rad53ΔΔ isolates having undergone a LOH revealed that, according to the chromosome on which LOH had occurred, these were predominantly due to break-induced replication/mitotic cross-over or chromosome loss. Loss of CaRAD53 also resulted in frequent aneuploidies. Deletion of CaDUN1 led to an increase in recombination-dependent LOH but did not trigger aneuploidies. It also increased GS sensitivity but did not impair GS-induced polarized growth contrary to CaRAD53 deletion. Characterization of CaRad53p site-directed mutants demonstrated that its kinase activity and N-terminal phosphorylation sites were crucial for its function in the resistance to GS, maintenance of heterozygosity, cell cycle regulation and polarized growth. Moreover, using phosphomimic mutants, we revealed an uncoupling of the functions of CaRad53p in these different processes, thus providing a novel understanding of how the DDCP may regulate downstream events in response to GS.

The N-Terminus of Vps74p Is Essential for the Retention of Glycosyltransferases in the Golgi but Not for the Modulation of Apical Polarized Growth in Saccharomyces cerevisiae

Vps74p is a member of the PtdIns(4)P-binding protein family. Vps74p interacts with Golgi-resident glycosyltransferases and the coat protein COPI complex to modulate Golgi retention of glycosyltransferases and with the PtdIns(4)P phosphatase Sac1p to modulate PtdIns(4)P homeostasis at the Golgi. Genetic analysis has shown that Vps74p is required for the formation of abnormal elongated buds in cdc34-2 cells. The C-terminal region of Vps74p is required for Vps74p multimerization, Golgi localization, and glycosyltransferase interactions; however, the functional significance of the N-terminal region and three putative phosphorylation sites of Vps74p have not been well characterized. In this study, we demonstrate that Vps74p executes multiple cellular functions using different domains. We found that the N-terminal 66 amino acids of Vps74p are dispensable for its Golgi localization and modulation of cell wall integrity but are required for glycosyltransferase retention and glycoprotein processing. Deletion of the N-terminal 90 amino acids, but not the 66 amino acids, of Vps74p impaired its ability to restore the elongated bud phenotype in cdc34-2/vps74Δ cells. Deletion of Sac1p and Arf1p also specifically reduced the abnormal elongated bud phenotype in cdc34-2 cells. Furthermore, we found that three N-terminal phosphorylation sites contribute to rapamycin hypersensitivity, although these phosphorylation residues are not involved in Vps74p localization, ability to modulate glycosyltransferase retention, or elongated bud formation in cdc34-2 cells. Thus, we propose that Vps74p may use different domains to interact with specific effectors thereby differentially modulating a variety of cellular functions.

Molecular Basis of the Essential S Phase Function of the Rad53 Checkpoint Kinase

The essential yeast kinases Mec1 and Rad53, or human ATR and Chk1, are crucial for checkpoint responses to exogenous genotoxic agents, but why they are also required for DNA replication in unperturbed cells remains poorly understood. Here we report that even in the absence of DNA-damaging agents, the rad53-4AQ mutant, lacking the N-terminal Mec1 phosphorylation site cluster, is synthetic lethal with a deletion of the RAD9 DNA damage checkpoint adaptor. This phenotype is caused by an inability of rad53-4AQ to activate the downstream kinase Dun1, which then leads to reduced basal deoxynucleoside triphosphate (dNTP) levels, spontaneous replication fork stalling, and constitutive activation of and dependence on S phase DNA damage checkpoints. Surprisingly, the kinase-deficient rad53-K227A mutant does not share these phenotypes but is rendered inviable by additional phosphosite mutations that prevent its binding to Dun1. The results demonstrate that ultralow Rad53 catalytic activity is sufficient for normal replication of undamaged chromosomes as long as it is targeted toward activation of the effector kinase Dun1. Our findings indicate that the essential S phase function of Rad53 is comprised by the combination of its role in regulating basal dNTP levels and its compensatory kinase function if dNTP levels are perturbed.

CK2 Phosphorylation Inactivates DNA Binding by the Papillomavirus E1 and E2 Proteins

Papillomaviruses have complex life cycles that are understood only superficially. Although it is well established that the viral E1 and E2 proteins play key roles in controlling viral transcription and DNA replication, how these factors are regulated is not well understood. Here, we demonstrate that phosphorylation by the protein kinase CK2 controls the biochemical activities of the bovine papillomavirus E1 and E2 proteins by modifying their DNA binding activity. Phosphorylation at multiple sites in the N-terminal domain in E1 results in the loss of sequence-specific DNA binding activity, a feature that is also conserved in human papillomavirus (HPV) E1 proteins. The bovine papillomavirus (BPV) E2 protein, when phosphorylated by CK2 on two specific sites in the hinge, also loses its site-specific DNA binding activity. Mutation of these sites in E2 results in greatly increased levels of latent viral DNA replication, indicating that CK2 phosphorylation of E2 is a negative regulator of viral DNA replication during latent viral replication. In contrast, mutation of the N-terminal phosphorylation sites in E1 has no effect on latent viral DNA replication. We propose that the phosphorylation of the N terminus of E1 plays a role only in vegetative viral DNA replication, and consistent with such a role, caspase 3 cleavage of E1, which has been shown to be necessary for vegetative viral DNA replication, restores the DNA binding activity to phosphorylated E1.

Self-Interaction of Human Pex11pβ during Peroxisomal Growth and Division

Pex11 proteins are involved in membrane elongation and division processes associated with the multiplication of peroxisomes. Human Pex11pβ has recently been linked to a new disorder affecting peroxisome morphology and dynamics. Here, we have analyzed the exact membrane topology of Pex11pβ. Studies with an epitope-specific antibody and protease protection assays show that Pex11pβ is an integral membrane protein with two transmembrane domains flanking an internal region exposed to the peroxisomal matrix and N- and C-termini facing the cytosol. A glycine-rich internal region within Pex11pβ is dispensable for peroxisome membrane elongation and division. However, we demonstrate that an amphipathic helix (Helix 2) within the first N-terminal 40 amino acids is crucial for membrane elongation and self-interaction of Pex11pβ. Interestingly, we find that Pex11pβ self-interaction strongly depends on the detergent used for solubilization. We also show that N-terminal cysteines are not essential for membrane elongation, and that putative N-terminal phosphorylation sites are dispensable for Pex11pβ function. We propose that self-interaction of Pex11pβ regulates its membrane deforming activity in conjunction with membrane lipids.

Genes involved in cell adhesion and signaling: A new repertoire of Retinoic Acid Receptors target genes in mouse embryonic fibroblasts

Nuclear retinoic acid (RA) receptors (RARα, β and γ) are ligand-dependent transcription factors that regulate the expression of a battery of genes involved in cell differentiation and proliferation. They are also phosphoproteins and we previously showed the importance of their phosphorylation in their transcriptional activity. In the study reported here, we conducted a genome-wide analysis of the genes that are regulated by RARs in mouse embryonic fibroblasts (MEFs) by comparing wild-type MEFs to MEFs lacking the three RARs. We found that in the absence of RA, RARs control the expression of several gene transcripts associated with cell adhesion. Consequently the knockout MEFs are unable to adhere and to spread on substrates and they display a disrupted network of actin filaments, compared with the WT cells. In contrast, in the presence of the ligand, RARs control the expression of other genes involved in signaling and in RA metabolism. Taking advantage of rescue cell lines expressing the RARα or RARγ subtypes (either wild-type or mutated at the N-terminal phosphorylation sites) in the null background, we found that the expression of RA-target genes can be controlled either by a specific single RAR or by a combination of RAR isotypes, depending on the gene. We also selected genes that require the phosphorylation of the receptors for their regulation by RA. Our results increase the repertoire of genes that are regulated by RARs and highlight the complexity and diversity of the transcriptional programs regulated by RARs, depending on the gene.

N-Terminal Phosphorylation Sites of Herpes Simplex Virus 1 ICP0 Differentially Regulate Its Activities and Enhance Viral Replication

The herpes simplex virus 1 (HSV-1) infected cell protein 0 (ICP0) is an immediate-early phosphoprotein that transactivates viral gene expression. Evidence suggests that phosphorylation regulates the functions of ICP0, and three regions (termed regions I, II, and III) in the protein are known to be phosphorylated. Mutation of the putative phosphorylation sites within region I, termed Phos 1, which lies in the N-terminal portion of ICP0, impairs the E3 ubiquitin (Ub) ligase and ND10-disrupting activities of ICP0 in cell culture and diminishes viral replication. To identify the specific phosphorylation site(s) or residues responsible for the phenotypes observed with Phos 1, individual residues within region I were mutated to alanine (S224A, T226A, T231A, and T232A) and one double mutant S224A/T226A was constructed. Tissue culture studies demonstrated that the S224A, S224A/T226A, T231A, and T232A mutants were unable to dissociate the cellular protein PML from ND10 and that the S224/T226A mutant was defective in its ability to dissociate the cellular protein Sp100 from ND10. Additionally, the transactivation activity of ICP0 was impaired in the S224A and S224A/T226A mutants. The S224A and S224A/T226A mutant forms were more stable than wild-type ICP0, suggesting that their ability to autoubiquitinate was limited. Moreover, one ICP0 ubiquitination target, USP-7, was also more stable after infection with these two mutants. Lastly, the replication of the S224A and S224A/T226A mutant viruses was reduced in cell culture and in vivo. Overall, our data suggest that specific phosphorylation sites within region I differentially regulate the activities of ICP0, which are required for efficient viral replication.