Holoenzyme Assembly Research Articles

Hypoxia modulates protein phosphatase 2A through HIF-1α dependent and independent mechanisms in human aortic smooth muscle cells and ventricular cardiomyocytes.

Although protein phosphatases regulate multiple cellular functions, their modulation under hypoxia remains unclear. We investigated expression of the protein phosphatase system under normoxic/hypoxic conditions and the mechanism by which hypoxia alters protein phosphatase 2A (PP2A) activity. Human cardiovascular cells were cultured in cell type specific media under normoxic or hypoxic conditions (1% O2 ). Effects on mRNA expression, phosphatase activity, post-translational modification, and involvement of hypoxia inducible factor 1α (HIF-1α) were assessed using RT-PCR, immunoblotting, an activity assay, and siRNA silencing. All components of the protein phosphatase system studied were expressed in each cell line. Hypoxia attenuated mRNA expression of the transcripts in a cell line- and time-dependent manner. In human aortic smooth muscle cells (HASMC) and AC16 cells, hypoxia decreased PP2Ac activity and mRNA expression without altering PP2Ac abundance. Hypoxia increased demethylated PP2Ac (DPP2Ac) and phosphatase methylesterase 1 (PME-1) abundance but decreased leucine carboxyl methyltransferase 1 (LCMT-1) abundance. HIF-1α siRNA prevented the hypoxia-mediated decrease in phosphatase activity and expression of the catalytic subunit of protein phosphatase 2A (PPP2CA), independently of altering pPP2Ac, DPP2Ac, LCMT-1, or PME-1 abundance. Cardiovascular cells express multiple components of the PP2A system. In HASMC and AC16 cells, hypoxia inhibits PP2A activity through HIF-1α-dependent and -independent mechanisms, with the latter being consistent with altered PP2A holoenzyme assembly. This indicates a complex inhibitory effect of hypoxia on the PP2A system, and highlights PP2A as a therapeutic target for diseases associated with dysregulated protein phosphorylation.

Structural basis for transcription initiation by bacterial ECF \u03c3 factors

Bacterial RNA polymerase employs extra-cytoplasmic function (ECF) σ factors to regulate context-specific gene expression programs. Despite being the most abundant and divergent σ factor class, the structural basis of ECF σ factor-mediated transcription initiation remains unknown. Here, we determine a crystal structure of Mycobacterium tuberculosis (Mtb) RNAP holoenzyme comprising an RNAP core enzyme and the ECF σ factor σH (σH-RNAP) at 2.7 Å, and solve another crystal structure of a transcription initiation complex of Mtb σH-RNAP (σH-RPo) comprising promoter DNA and an RNA primer at 2.8 Å. The two structures together reveal the interactions between σH and RNAP that are essential for σH-RNAP holoenzyme assembly as well as the interactions between σH-RNAP and promoter DNA responsible for stringent promoter recognition and for promoter unwinding. Our study establishes that ECF σ factors and primary σ factors employ distinct mechanisms for promoter recognition and for promoter unwinding.

Mitochondrial active and relaxed state respiration after heat shock mRNA response in the heart

Induction of Heat Shock Proteins results in cytoprotection. Beneficial effect results from transcription and translational cellular components’ involvement that defends metabolism and thus induce ischemic protection of the tissue. Mitochondrial respiration is also involved in stress- induced conditions. It is not a uniform process. Cytochrome c Oxidase (CytOx) representing complex IV of the Electron Transfer Chain (ETC) has a regulatory role for mitochondrial respiratory activity, which is tested in our study after hsp induction. Moreover, protein translation for mitochondrial components was probed by the detection of MT-CO1 for Subunit 1 of CytOx neosynthesis. Wistar rats were subjected to whole-body hyperthermia at 42.0–42.5 °C for 15 min followed by a normothermic recovery period. Heat shock response was monitored time dependent from LV biopsies of all control and heat treated animals with PCR-analysis for hsp 32, 60, 70.1, 70.2, 90 and MT-CO1 expression at 15, 30, 45, 60, 120 and 360 min recovery (n = 5 in each group), respectively. Enzymatic activity of CytOx were evaluated polarographically. High energy phosphates were detected by chromatographic analysis. The mRNA expression of MT-CO1 peaked at 60 min and was accompanied by hsp 32 (r = 0.457; p = 0.037) and hsp 70.2 (r = 0.615; p = 0.003) upregulation. With hsp induction, mitochondrial respiration was increased initially. Enzymatic activity reconciled from active into relaxed status wherein CytOx activity was completely inhibited by ATP. Myocardial ATP content increased from stress induced point i.e. < 1 µmol g−1 protein w/w to finally 1.5 ± 0.53 µmol g−1 protein w/w at 120 min recovery interval. Hyperthermic, myocardial hsp- induction goes along with increased CytOx activity representing an increased “active” mitochondrial respiration. In parallel, de -novo holoenzyme assembly of CytOx begins as shown by MT-CO1 upregulation at 60 min recovery time crossing with a final return to the physiological “relaxed” state and ATP -inhibited respiration.

151 - Rac1-induced ROS is implicated in the sustained activation of anti-apoptotic protein Bcl-2 in cancer cells

The anti-apoptotic protein Bcl-2 is strongly associated with the development of therapy resistance in a host of hematopoietic and other cancers. To that end, the post-translational modification of Bcl-2, in particular its mono- or multi-site phosphorylation, have been reported to impact its apoptosis inhibitory activity. While multi-site phosphorylation compromises its death inhibitory activity, our recent work has highlighted the critical involvement of serine 70 phosphorylation (S70pBcl-2) in stabilizing its anti-apoptotic activity. S70pBcl-2 is a function of either an amplification of the activities of various kinases involved in this post-translational modification or a loss of function of the phosphatase regulating this modification. The pleiotropic phosphatase, PP2A, is specifically involved in the dephosphorylation of Bcl-2 at S70. In our search to identify redox dependent pathways impacting the sustained activation of Bcl-2 via S70P, we provided evidence that redox modification of the B56δ sub-unit of PP2A resulted in the sustained S70P of Bcl-2, thereby promoting its death inhibitory activity in cancer cells. Interestingly, here we present substantial evidence for the existence of a similar redox-dependent regulation of Bcl-2 phosphorylation in cells expressing GTP-bound active Rac1. We show that active Rac1 promotes sustained S70pBcl2 levels in cancer cells via physical interaction with Bcl-2, thereby permitting Rac1-induced production of superoxide (O2–) in close proximity to inactivate PP2A holoenzyme assembly and specific activity. Corroborating this, pharmacological inhibition of the interaction with BH3 mimetics such as HA14-1 or ABT199 as well as scavengers of O2– such as Tiron reduced S70pBcl2. Importantly, the interaction between Rac1 and Bcl-2 to promote S70pBcl2 as well as the oxidative inhibition of PP2A are re-capitulated in primary cells derived from lymphoma patients. Collectively, our findings demonstrate a novel redox mechanism of active Rac1 in sustaining S70pBcl2 in cancer cells.

Cone Phosphodiesterase-6γ' Subunit Augments Cone PDE6 Holoenzyme Assembly and Stability in a Mouse Model Lacking Both Rod and Cone PDE6 Catalytic Subunits.

Rod and cone phosphodiesterase 6 (PDE6) are key effector enzymes of the vertebrate phototransduction pathway. Rod PDE6 consists of two catalytic subunits PDE6α and PDE6β and two identical inhibitory PDE6γ subunits, while cone PDE6 is composed of two identical PDE6α’ catalytic subunits and two identical cone-specific PDE6γ’ inhibitory subunits. Despite their prominent function in regulating cGMP levels and therefore rod and cone light response properties, it is not known how each subunit contributes to the functional differences between rods and cones. In this study, we generated an rd10/cpfl1 mouse model lacking rod PDE6β and cone PDE6α’ subunits. Both rod and cone photoreceptor cells are degenerated with age and all PDE6 subunits degrade in rd10/cpfl1 mice. We expressed cone PDE6α’ in both rods and cones of rd10/cpfl1 mice by adeno-associated virus (AAV)-mediated delivery driven by the ubiquitous, constitutive small chicken β-actin promoter. We show that expression of PDE6α’ rescues rod function in rd10/cpfl1 mice, and the restoration of rod light sensitivity is attained through restoration of endogenous rod PDE6γ and formation of a functional PDE6α’γ complex. However, improved photopic cone responses were achieved only after supplementation of both cone PDE6α’ and PDE6γ’ subunits but not by PDE6α’ treatment alone. We observed a two fold increase of PDE6α’ levels in the eyes injected with both PDE6α’ plus PDE6γ’ relative to eyes receiving PDE6α’ alone. Despite the presence of both PDE6γ’ and PDE6γ, the majority of PDE6α’ formed functional complexes with PDE6γ’, suggesting that PDE6α’ has a higher association affinity for PDE6γ’ than for PDE6γ. These results suggest that the presence of PDE6γ’ augments cone PDE6 assembly and enhances its stability. Our finding has important implication for gene therapy of PDE6α’-associated achromatopsia.

Aurora B opposes PP1 function in mitosis by phosphorylating the conserved PP1-binding RVxF motif in PP1 regulatory proteins.

Protein phosphatase 1 (PP1) is a highly conserved protein phosphatase that performs most of the serine- and threonine-dephosphorylation reactions in eukaryotes and opposes the actions of a diverse set of serine and threonine (Ser-Thr) protein kinases. PP1 gains substrate specificity through binding to a large number (>200) of regulatory proteins that control PP1 localization, activity, and interactions with substrates. PP1 recognizes the well-characterized RVxF binding motif that is present in many of these regulatory proteins, thus generating a multitude of distinct PP1 holoenzymes. We showed that a subset of the RVxF binding motifs, in which x is a phosphorylatable amino acid (RV[S/T]F), was phosphorylated specifically during mitosis and that this phosphorylation event abrogated the interaction of PP1 with the regulatory protein. We determined that this phosphorylation was primarily governed by the mitotic protein kinase Aurora B and that high phosphorylation site stoichiometry of these sites maintained the phosphorylation of PP1 substrates during mitosis by disrupting the assembly of PP1 holoenzymes. We generated an antibody that recognizes the phosphorylated form of the RV[S/T]F motif (RVp[S/T]F) and used it to identify known PP1 regulatory proteins (KNL1, CDCA2, and RIF1) and multiple proteins that could potentially act as PP1 binding partners (UBR5, ASPM, SEH1, and ELYS) governed by this mechanism. Together, these data suggest a general regulatory mechanism by which the coordinated activities of Aurora B and PP1 control mitotic progression.

P-32 - The tumor environment stabilizes c-Myc oncoprotein through its sustained phosphorylation at serine 62, which promotes its oncogenic activity

We have previously demonstrated that redox homeostasis is governed by two key ROS – superoxide anion (O2•-) and hydrogen peroxide (H2O2). Tilting the balance to O2•- contributes to engaging cancers onto a pro-survival trajectory. Here, we present a singular event wherein redox modulation of c-Myc attributes cancer cells with survival advantages and elevates their chemoresistance potential. Phosphorylation of c-Myc correlates with its stabilization in cancers. While S62 phosphorylation increases c-Myc stability, T58 phosphorylation destabilizes it. Of particular interest is phosphatase PP2A, as we have shown that it is amenable to redox insults. We have now elucidated that B56α regulatory subunit of PP2A is amenable to redox modification preventing interaction of c-Myc bound B56α with PP2A-AC core enzyme. As assembly of PP2A holoenzyme is disrupted, c-Myc remains phosphorylated at S62. Hyperphosphorylated c-Myc cannot be recognized and engaged by the proteasome leading to increased protein stability and a moderate rise in activation of c-Myc targets. Thus, we have established a novel mechanism wherein the balance between O2•-:H2O2 ratio stimulates a pro-survival milieu and reinforces chemoresistance through redox modification of PP2A-B56α, consequentially resulting in stabilization of c-Myc.

Rubisco Assembly in the Chloroplast.

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyzes the rate-limiting step in the Calvin-Benson cycle, which transforms atmospheric carbon into a biologically useful carbon source. The slow catalytic rate of Rubisco and low substrate specificity necessitate the production of high levels of this enzyme. In order to engineer a more efficient plant Rubisco, we need to better understand its folding and assembly process. Form I Rubisco, found in green algae and vascular plants, is a hexadecamer composed of 8 large subunits (RbcL), encoded by the chloroplast genome and 8 small, nuclear-encoded subunits (RbcS). Unlike its cyanobacterial homolog, which can be reconstituted in vitro or in E. coli, assisted by bacterial chaperonins (GroEL-GroES) and the RbcX chaperone, biogenesis of functional chloroplast Rubisco requires Cpn60-Cpn20, the chloroplast homologs of GroEL-GroES, and additional auxiliary factors, including Rubisco accumulation factor 1 (Raf1), Rubisco accumulation factor 2 (Raf2) and Bundle sheath defective 2 (Bsd2). The discovery and characterization of these factors paved the way for Arabidopsis Rubisco assembly in E. coli. In the present review, we discuss the uniqueness of hetero-oligomeric chaperonin complex for RbcL folding, as well as the sequential or concurrent actions of the post-chaperonin chaperones in holoenzyme assembly. The exact stages at which each assembly factor functions are yet to be determined. Expression of Arabidopsis Rubisco in E. coli provided some insight regarding the potential roles for Raf1 and RbcX in facilitating RbcL oligomerization, for Bsd2 in stabilizing the oligomeric core prior to holoenzyme assembly, and for Raf2 in interacting with both RbcL and RbcS. In the long term, functional characterization of each known factor along with the potential discovery and characterization of additional factors will set the stage for designing more efficient plants, with a greater biomass, for use in biofuels and sustenance.

Using Separation-of-Function Mutagenesis To Define the Full Spectrum of Activities Performed by the Est1 Telomerase Subunit in Vivo.

A leading objective in biology is to identify the complete set of activities that each gene performs in vivo In this study, we have asked whether a genetic approach can provide an efficient means of achieving this goal, through the identification and analysis of a comprehensive set of separation-of-function (sof-) mutations in a gene. Toward this goal, we have subjected the Saccharomyces cerevisiae EST1 gene, which encodes a regulatory subunit of telomerase, to intensive mutagenesis (with an average coverage of one mutation for every 4.5 residues), using strategies that eliminated those mutations that disrupted protein folding/stability. The resulting set of sof- mutations defined four biochemically distinct activities for the Est1 telomerase protein: two temporally separable steps in telomerase holoenzyme assembly, a telomerase recruitment activity, and a fourth newly discovered regulatory function. Although biochemically distinct, impairment of each of these four different activities nevertheless conferred a common phenotype (critically short telomeres) comparable to that of an est1-∆ null strain. This highlights the limitations of gene deletions, even for nonessential genes; we suggest that employing a representative set of sof- mutations for each gene in future high- and low-throughput investigations will provide deeper insights into how proteins interact inside the cell.

Methylation-regulated decommissioning of multimeric PP2A complexes

Dynamic assembly/disassembly of signaling complexes are crucial for cellular functions. Specialized latency and activation chaperones control the biogenesis of protein phosphatase 2A (PP2A) holoenzymes that contain a common scaffold and catalytic subunits and a variable regulatory subunit. Here we show that the butterfly-shaped TIPRL (TOR signaling pathway regulator) makes highly integrative multibranching contacts with the PP2A catalytic subunit, selective for the unmethylated tail and perturbing/inactivating the phosphatase active site. TIPRL also makes unusual wobble contacts with the scaffold subunit, allowing TIPRL, but not the overlapping regulatory subunits, to tolerate disease-associated PP2A mutations, resulting in reduced holoenzyme assembly and enhanced inactivation of mutant PP2A. Strikingly, TIPRL and the latency chaperone, α4, coordinate to disassemble active holoenzymes into latent PP2A, strictly controlled by methylation. Our study reveals a mechanism for methylation-responsive inactivation and holoenzyme disassembly, illustrating the complexity of regulation/signaling, dynamic complex disassembly, and disease mutations in cancer and intellectual disability.

Identification and characterization of a heterotrimeric archaeal DNA polymerase holoenzyme

Since their initial characterization over 30 years ago, it has been believed that the archaeal B-family DNA polymerases are single-subunit enzymes. This contrasts with the multi-subunit B-family replicative polymerases of eukaryotes. Here we reveal that the highly studied PolB1 from Sulfolobus solfataricus exists as a heterotrimeric complex in cell extracts. Two small subunits, PBP1 and PBP2, associate with distinct surfaces of the larger catalytic subunit and influence the enzymatic properties of the DNA polymerase. Thus, multi-subunit replicative DNA polymerase holoenzymes are present in all three domains of life. We reveal the architecture of the assembly by a combination of cross-linking coupled with mass spectrometry, X-ray crystallography and single-particle electron microscopy. The small subunits stabilize the holoenzyme assembly and the acidic tail of one small subunit mitigates the ability of the enzyme to perform strand-displacement synthesis, with important implications for lagging strand DNA synthesis.

Simple peptides derived from the ribosomal core potentiate RNA polymerase ribozyme function.

The emergence of functional interactions between nucleic acids and polypeptides was a key transition in the origin of life and remains at the heart of all biology. However, how and why simple non-coded peptides could have become critical for RNA function is unclear. Here, we show that putative ancient peptide segments from the cores of both ribosomal subunits enhance RNA polymerase ribozyme (RPR) function, as do derived homopolymeric peptides comprising lysine or the non-proteinogenic lysine analogues ornithine or, to a lesser extent, diaminobutyric acid, irrespective of chirality or chiral purity. Lysine decapeptides enhance RPR function by promoting holoenzyme assembly through primer-template docking, accelerate RPR evolution, and allow RPR-catalysed RNA synthesis at near physiological (≥1 mM) Mg2+ concentrations, enabling templated RNA synthesis within membranous protocells. Our results outline how compositionally simple, mixed-chirality peptides may have augmented the functional potential of early RNAs and promoted the emergence of the first protocells.

Protein Phosphatase 2A: a Double-Faced Phosphatase of Cellular System and Its Role in Neurodegenerative Disorders.

Protein phosphatase 2A (PP2A), a ubiquitously expressed serine/threonine phosphatase, is a vitally important phosphatase for the cellular system. Structurally, it is constituted of three different subunits, namely catalytic subunit (PP2Ac), structural scaffold subunit (PP2A-A), and regulatory subunit (PP2A-B). All subunits have various isoforms, and catalytic and scaffold subunits are ubiquitously expressed, whereas regulatory subunits are more specific to tissue and cell type. It is the numerous possibilities of PP2A holoenzyme assembly with varying isoform components that make it possess a dual nature of activator or the inhibitory character in different signaling pathways, namely neural developmental pathways, Akt/protein kinase B pathway, NF-kB pathway, MAPK pathway, apoptosis pathway, and cell cycle progression to name a few. Importantly, the expression of PP2A in the brain is highest among the serine phosphatases and is known to actively participate in the neural development process. However, the exact mechanism of action of PP2A is still debated and enunciating the holoenzyme components, especially the regulatory subunit of PP2A involved in regulating neural developmental process is still poorly understood. In this review, we try to throw some light on the involvement of various PP2A holoenzyme forms in the process of neurogenesis and progression of neurodegenerative diseases.

PKA: Dynamic Assembly and Regulation of Macromolecular Signaling Complexes

PKA Signaling: Linkers, Loops, and Intrinsically Disorder Regions Drive the Dynamic Assembly of Holoenzyme Complexes.The PKA catalytic (C) subunit crystallized in 1991, gave us our first glimpse of the fold that describes all protein kinases. The two lobes that comprise the core are flanked by N-and C-terminal tails, which wrap around both lobes. By capturing all stages of catalysis in crystal structures we appreciate the dynamic nature of these tails and how they undergo order-disorder transitions as part of the catalytic cycle. Ordering the activation loop by a critical phosphate is another fundamental order/disorder transition that is a conserved feature of most protein kinases. These dynamic features allow the kinases to function as highly regulated molecular switches. In the case of PKA, the C-subunit is packaged with regulatory (R) subunit dimers where each chain contains two C-terminal cAMP binding (CNB) domains. The CNB domains are highly dynamic allosteric signaling modules that have been conserved to translate a biological response to cAMP. Although the fundamental features of this domain were elucidated by structures of R-monomers and R:C heterodimers, it required full-length R2C2 tetramers to appreciate the importance of symmetry for PKA signaling. Each R-subunit contains an N-terminal dimerization domain that is also a docking site for scaffold proteins referred to as A Kinase Anchoring Proteins (AKAPs). This domain is joined by a flexible linker to the CNB domains, and embedded within the linker is an inhibitor site that docks to the active site of the C-subunit in the holoenzyme. The order/disorder transitions of the linker drive the assembly of the holoenzymes which each have distinct quaternary structures. The linkers, rich in biological information, are an essential feature of PKA regulation. (Funded by NIH GM34921 and DK54441.)

TORC1-dependent sumoylation of Rpc82 promotes RNA polymerase III assembly and activity

Maintaining cellular homeostasis under changing nutrient conditions is essential for the growth and development of all organisms. The mechanisms that maintain homeostasis upon loss of nutrient supply are not well understood. By mapping the SUMO proteome in Saccharomyces cerevisiae, we discovered a specific set of differentially sumoylated proteins mainly involved in transcription. RNA polymerase III (RNAPIII) components, including Rpc53, Rpc82, and Ret1, are particularly prominent nutrient-dependent SUMO targets. Nitrogen starvation, as well as direct inhibition of the master nutrient response regulator target of rapamycin complex 1 (TORC1), results in rapid desumoylation of these proteins, which is reflected by loss of SUMO at tRNA genes. TORC1-dependent sumoylation of Rpc82 in particular is required for robust tRNA transcription. Mechanistically, sumoylation of Rpc82 is important for assembly of the RNAPIII holoenzyme and recruitment of Rpc82 to tRNA genes. In conclusion, our data show that TORC1-dependent sumoylation of Rpc82 bolsters the transcriptional capacity of RNAPIII under optimal growth conditions.

Mutations in the accessory subunit NDUFB10 result in isolated complex I deficiency and illustrate the critical role of intermembrane space import for complex I holoenzyme assembly.

An infant presented with fatal infantile lactic acidosis and cardiomyopathy, and was found to have profoundly decreased activity of respiratory chain complex I in muscle, heart and liver. Exome sequencing revealed compound heterozygous mutations in NDUFB10, which encodes an accessory subunit located within the PD part of complex I. One mutation resulted in a premature stop codon and absent protein, while the second mutation replaced the highly conserved cysteine 107 with a serine residue. Protein expression of NDUFB10 was decreased in muscle and heart, and less so in the liver and fibroblasts, resulting in the perturbed assembly of the holoenzyme at the 830 kDa stage. NDUFB10 was identified together with three other complex I subunits as a substrate of the intermembrane space oxidoreductase CHCHD4 (also known as Mia40). We found that during its mitochondrial import and maturation NDUFB10 transiently interacts with CHCHD4 and acquires disulfide bonds. The mutation of cysteine residue 107 in NDUFB10 impaired oxidation and efficient mitochondrial accumulation of the protein and resulted in degradation of non-imported precursors. Our findings indicate that mutations in NDUFB10 are a novel cause of complex I deficiency associated with a late stage assembly defect and emphasize the role of intermembrane space proteins for the efficient assembly of complex I.

Role for the MED21-MED7 Hinge in Assembly of the Mediator-RNA Polymerase II Holoenzyme

Mediator plays an integral role in activation of RNA polymerase II (Pol II) transcription. A key step in activation is binding of Mediator to Pol II to form the Mediator-Pol II holoenzyme. Here, we exploit a combination of biochemistry and macromolecular EM to investigate holoenzyme assembly. We identify a subset of human Mediator head module subunits that bind Pol II independent of other subunits and thus probably contribute to a major Pol II binding site. In addition, we show that binding of human Mediator to Pol II depends on the integrity of a conserved "hinge" in the middle module MED21-MED7 heterodimer. Point mutations in the hinge region leave core Mediator intact but lead to increased disorder of the middle module and markedly reduced affinity for Pol II. These findings highlight the importance of Mediator conformation for holoenzyme assembly.

Leucine Carboxyl Methyltransferase 1 (LCMT-1) Methylates Protein Phosphatase 4 (PP4) and Protein Phosphatase 6 (PP6) and Differentially Regulates the Stable Formation of Different PP4 Holoenzymes

The protein phosphatase 2A (PP2A) subfamily of phosphatases, PP2A, PP4, and PP6, are multifunctional serine/threonine protein phosphatases involved in many cellular processes. Carboxyl methylation of the PP2A catalytic subunit (PP2Ac) C-terminal leucine is regulated by the opposing activities of leucine carboxyl methyltransferase 1 (LCMT-1) and protein phosphatase methylesterase 1 (PME-1) and regulates PP2A holoenzyme formation. The site of methylation on PP2Ac is conserved in the catalytic subunits of PP4 and PP6, and PP4 is also methylated on that site, but the identities of the methyltransferase enzyme for PP4 are not known. Whether PP6 is methylated is also not known. Here we use antibodies specific for the unmethylated phosphatases to show that PP6 is carboxyl-methylated and that LCMT-1 is the major methyltransferase for PP2A, PP4, and PP6 in mouse embryonic fibroblasts (MEFs). Analysis of PP2A and PP4 complexes by blue native polyacrylamide gel electrophoresis (BN-PAGE) indicates that PP4 holoenzyme complexes, like those of PP2A, are differentially regulated by LCMT-1, with the PP4 regulatory subunit 1 (PP4R1)-containing PP4 complex being the most dramatically affected by the LCMT-1 loss. MEFs derived from LCMT-1 knock-out mouse embryos have reduced levels of PP2A B regulatory subunit and PP4R1 relative to control MEFs, indicating that LCMT-1 is important for maintaining normal levels of these subunits. Finally, LCMT-1 homozygous knock-out MEFs exhibited hyperphosphorylation of HDAC3, a reported target of the methylation-dependent PP4R1-PP4c complex. Collectively, our data suggest that LCMT-1 coordinately regulates the carboxyl methylation of PP2A-related phosphatases and, consequently, their holoenzyme assembly and function.

Crystal structure of the human Tip41 orthologue, TIPRL, reveals a novel fold and a binding site for the PP2Ac C-terminus.

TOR signaling pathway regulator-like (TIPRL) is a regulatory protein which inhibits the catalytic subunits of Type 2A phosphatases. Several cellular contexts have been proposed for TIPRL, such as regulation of mTOR signaling, inhibition of apoptosis and biogenesis and recycling of PP2A, however, the underlying molecular mechanism is still poorly understood. We have solved the crystal structure of human TIPRL at 2.15 Å resolution. The structure is a novel fold organized around a central core of antiparallel beta-sheet, showing an N-terminal α/β region at one of its surfaces and a conserved cleft at the opposite surface. Inside this cleft, we found a peptide derived from TEV-mediated cleavage of the affinity tag. We show by mutagenesis, pulldown and hydrogen/deuterium exchange mass spectrometry that this peptide is a mimic for the conserved C-terminal tail of PP2A, an important region of the phosphatase which regulates holoenzyme assembly, and TIPRL preferentially binds the unmodified version of the PP2A-tail mimetic peptide DYFL compared to its tyrosine-phosphorylated version. A docking model of the TIPRL-PP2Ac complex suggests that TIPRL blocks the phosphatase’s active site, providing a structural framework for the function of TIPRL in PP2A inhibition.

Deciphering CaMKII Multimerization using Holoenzyme Assembly Mutants, FCS, and Concurrent Homo- and Hetero-FRET Analysis

Monitoring changes in molecular conformation is essential for studying protein interactions within and between complexes. Current methods such as FRET or FCS can only reveal limited aspects of these changes, which in many cases is insufficient for interpretation. Fluorescent Polarization and Fluctuation Analysis (FPFA), a time-correlated single-photon counting technique that combines homo-FRET and FCS was developed to address this problem. In FPFA, changes in mass and/or shape of a protein complex, the number of fluorescent subunits per complex, as well as subunit proximity (1 - 10 nm), are simultaneously detected. Here we have extended FPFA by incorporating simultaneous hetero-FRET analysis so that additional structural changes can be monitored concurrently. This multi-modal approach was then used to observe changes in CaMKII holoenzyme in response to activation and subsequent interactions with NR2B. Revised FPFA revealed that catalytic-domain pairing is the fundamental structural motif of the holoenzyme organization. A genetically engineered dimeric-CaMKII functioned like native holoenzyme (8-14 subunits) in terms of activity, T286 autophosphorylation, structural changes triggered by calcium-calmodulin, and those associated with interaction with NR2B.