PP2A-C may be a promising candidate for postmortem interval estimation.

Determining the postmortem interval (PMI) is an important task in forensic pathology. However, a reliable means of determining the PMI between 24 h and approximately 7 days after death has not yet been established. A previous study demonstrated that subunit A of protein phosphatase 2A (PP2A-A) is a promising candidate to estimate the PMI during the first 96 h. However, more detailed work is still needed to investigate PP2A's function in PMI estimation. PP2A is a serine/threonine phosphatase consisting of three subunits (PP2A-A, PP2A-B, and PP2A-C), and its activation is reflected by Tyr-307 phosphorylation of the catalytic subunit (P-PP2A-C). In this study, we speculated that the other two subunits of PP2A and the activation of PP2A may play different roles in estimating the PMI. For this purpose, mice were euthanized and stored at different temperatures (4, 15, and 25 °C). At each temperature, the musculus vastus lateralis was collected at different time points (0, 24, 48, and 96 h) to investigate the degradation of PP2A-B, PP2A-C, and P-PP2A-C (Tyr-307). Homocysteine (Hcy) was used to establish a hyperhomocysteinemia animal model to explore the effects of plasma Hcy on PMI estimation. The data showed not only that PP2A-C was more stable than PP2A-B, but also that it was not affected by homocysteine (Hcy). These characteristics make PP2A-C a promising candidate for short-term (24 h to 48 h) PMI estimation.

Reduction of protein phosphatase 2A (PP2A) complexity reveals cellular functions and dephosphorylation motifs of the PP2A/B'δ holoenzyme.

Protein phosphatase 2A (PP2A) is a large enzyme family responsible for most cellular Ser/Thr dephosphorylation events. PP2A substrate specificity, localization, and regulation by second messengers rely on more than a dozen regulatory subunits (including B/R2, B'/R5, and B″/R3), which form the PP2A heterotrimeric holoenzyme by associating with a dimer comprising scaffolding (A) and catalytic (C) subunits. Because of partial redundancy and high endogenous expression of PP2A holoenzymes, traditional approaches of overexpressing, knocking down, or knocking out PP2A regulatory subunits have yielded only limited insights into their biological roles and substrates. To this end, here we sought to reduce the complexity of cellular PP2A holoenzymes. We used tetracycline-inducible expression of pairs of scaffolding and regulatory subunits with complementary charge-reversal substitutions in their interaction interfaces. For each of the three regulatory subunit families, we engineered A/B charge-swap variants that could bind to one another, but not to endogenous A and B subunits. Because endogenous Aα was targeted by a co-induced shRNA, endogenous B subunits were rapidly degraded, resulting in expression of predominantly a single PP2A heterotrimer composed of the A/B charge-swap pair and the endogenous catalytic subunit. Using B'δ/PPP2R5D, we show that PP2A complexity reduction, but not PP2A overexpression, reveals a role of this holoenzyme in suppression of extracellular signal-regulated kinase signaling and protein kinase A substrate dephosphorylation. When combined with global phosphoproteomics, the PP2A/B'δ reduction approach identified consensus dephosphorylation motifs in its substrates and suggested that residues surrounding the phosphorylation site play roles in PP2A substrate specificity.

To be or not to be: PP2A as a dual player in CNS functions, its role in neurodegeneration, and its interaction with brain insulin signaling.

Accumulating evidence has reached the consensus that the balance of phosphorylation state of signaling molecules is a pivotal point in the regulation of cell signaling. Therefore, characterizing elements (kinases-phosphatases) in the phosphorylation balance are at great importance. However, the role of phosphatase enzymes is less investigated than kinase enzymes. PP2A is a member of serine/threonine protein phosphatase that its imbalance has been reported in neurodegenerative diseases. Therefore, we reviewed the superfamily of phosphatases and more specifically PP2A, its regulation, and physiological functions participate in CNS. Thereafter, we discussed the latest findings about PP2A dysregulation in Alzheimer and Parkinson diseases and possible interplay between this phosphatase and insulin signaling pathways. Finally, activating/inhibitory modulators for PP2A activity as well as experimental methods for PP2A study have been reviewed.

Senkyunolide A protects neural cells against corticosterone-induced apoptosis by modulating protein phosphatase 2A and α-synuclein signaling.

BACKGROUND: Depression is characterized by a pathological injury to the hippocampal neurons. Senkyunolide A (SenA) is one of the major active components of Dan-zhi-xiao-yao-san, which is widely used in the treatment of depression-related disorders. MATERIALS AND METHODS: In the present study, it was hypothesized that the antidepressant effect of Dan-zhi-xiao-yao-san depended on the function of SenA and the authors attempted to reveal the molecular mechanism associated with the treatment. An in vitro depression model was induced using corticosterone (Cort), and the effect of SenA on the cell viability, apoptosis, and protein phosphatase 2A/α-synuclein (PP2A/α-syn) signaling was detected. To validate the mechanism driving the therapeutic effect of SenA, activity of PP2A and α-syn was modulated and the effect on neural cells was evaluated. RESULTS: The results showed that SenA protects Cort-induced cell apoptosis in PC12 cells. In addition, SenA increased Cort-induced reduction of PP2A activity, while it decreased the expression of p-PP2A, α-syn, and p-α-syn (Ser129). Further, modulation of PP2A activity with specific inhibitor okadaic acid (OA) increased Cort-induced cell apoptosis, while PP2A activator D-erythro-sphingosine (SPH) exhibited an opposite effect. The neuroprotective effects of SenA on neural cells also depended on inhibition of α-syn function, the regulation of which would influence the activity of PP2A in a negative loop. CONCLUSION: Collectively, the results suggested that the neuroprotective effects of SenA were exerted by modulating activities of PP2A activities and α-syn. The findings partially explained the mechanism associated with the neuroprotective effect of SenA.

Circulating Microvesicles from Pancreatic Cancer Accelerate the Migration and Proliferation of PANC-1 Cells.

Circulating microvesicles are able to mediate long-distance cell-cell communications. It is essential to understand how microvesicles from pancreatic cancer act on other cells in the body. In this work, serum-derived microvesicles were isolated from 10 patients with locally advanced pancreatic cancer and healthy controls. Using Cell Transwell and WST-1 reagents, we found that microvesicles from pancreatic cancer accelerated migration and proliferation of PANC-1 cells. Meanwhile, the proliferation of these cancer-microvesicle-treated cells (CMTCs) was affected less by 10 µM of gemcitabine relative to healthy microvesicle-treated cells (HMTCs). Next, we optimized the filter-aided sample preparation method to increase the recovery of protein samples and then applied it to the quantification of the proteome of CMTCs and HMTCs. The peptides were labeled and analyzed by liquid chromatography-tandem mass spectrometry. In total, 4102 proteins were identified, where 35 proteins were up-regulated with 27 down-regulated in CMTCs. We verified the quantitative results of three key proteins CD44, PPP2R1A, and TP53 by Western blot. The Ingenuity Pathway Analysis revealed pathways that cancer microvesicles might participate in to promote cell migration and proliferation. These findings may provide novel clues of treatment for tumorigenesis and metastasis.

Overexpression of phosphoprotein phosphatase 2A predicts worse prognosis in patients with breast cancer: a 15-year follow-up.

Breast cancer subtypes can be stratified by IHC expression of estrogen receptor, progesterone receptor, and human epidermal growth factor 2 (HER2). The signaling pathways mediated by these receptors are the dominant drivers of cell proliferation and survival in most human breast cancers. One of the most frequently overactivated pathways in breast cancer is the AKT signaling cascade. Protein phosphatase 2A (PP2A) acts as a switch to turn off signal transduction in the AKT pathway; however, it is frequently inactivated in many cancers by phosphorylation of Tyr-307 to form phosphoprotein phosphatase 2A (p-PP2A). This study aimed to investigate the clinical significance of p-PP2A and phospho-AKT (p-AKT) expression in 672 patients with breast cancer during a 15-year follow-up. The breast tissue microarray was evaluated for p-PP2A and p-AKT expression using IHC staining and scores. Analysis of IHC staining results revealed that p-PP2A expression was positively correlated with HER2, Ki-67, and p-AKT overexpression (P<.001, P=.003, and P=.001, respectively). At the time of diagnosis, breast cancer patients with higher p-PP2A expression had significantly shorter 15-year OS than patients with lower p-PP2A expression did (P=.017). Multivariate Cox regression analysis revealed that high p-PP2A expression was an independent prognostic factor for shorter OS (hazard ratio, 1.741; P=.012). Our data revealed that high p-PP2A expression is positively associated with HER2, Ki-67, and p-AKT expression. High p-PP2A expression correlates with poor clinical outcomes in breast cancer, especially in patients with TNBC.

The augmentation of BK channel activity by nitric oxide signaling in rat cerebral arteries involves co-localized regulatory elements.

Large conductance, Ca2+-activated K+ (BK) channels control cerebrovascular tone; however, the regulatory processes influencing these channels remain poorly understood. Here, we investigate the cellular mechanisms underlying the enhancement of BK current in rat cerebral arteries by nitric oxide (NO) signaling. In isolated cerebral myocytes, BK current magnitude was reversibly increased by sodium nitroprusside (SNP, 100 µM) and sensitive to the BK channel inhibitor, penitrem-A (100 nM). Fostriecin (30 nM), a protein phosphatase type 2A (PP2A) inhibitor, significantly prolonged the SNP-induced augmentation of BK current and a similar effect was produced by sildenafil (30 nM), a phosphodiesterase 5 (PDE5) inhibitor. Using proximity ligation assay (PLA)-based co-immunostaining, BK channels were observed to co-localize with PP2A, PDE5, and cGMP-dependent protein kinase (cGKI) (spatial restriction < 40 nm); cGKI co-localization increased following SNP exposure. SNP (10 µM) reversibly inhibited myogenic tone in cannulated cerebral arteries, which was augmented by either fostriecin or sildenafil and inhibited by penitrem-A. Collectively, these data suggest that (1) cGKI, PDE5, and PP2A are compartmentalized with cerebrovascular BK channels and determine the extent of BK current augmentation by NO/cGMP signaling, and (2) the dynamic regulation of BK activity by co-localized signaling enzymes modulates NO-evoked dilation of cerebral resistance arteries.

Protein Kinase C Controls Binding of Igo/ENSA Proteins to Protein Phosphatase 2A in Budding Yeast.

Protein phosphatase 2A (PP2A) plays important roles in controlling mitosis in all eukaryotic cells. The form of PP2A that controls mitosis is associated with a conserved regulatory subunit that is called B55 in vertebrates and Cdc55 in budding yeast. The activity of this form of PP2A can be inhibited by binding of conserved Igo/ENSA proteins. Although the mechanisms that activate Igo/ENSA to bind and inhibit PP2A are well understood, little is known about how Igo/Ensa are inactivated. Here, we have analyzed regulation of Igo/ENSA in the context of a checkpoint pathway that links mitotic entry to membrane growth in budding yeast. Protein kinase C (Pkc1) relays signals in the pathway by activating PP2ACdc55 We discovered that constitutively active Pkc1 can drive cells through a mitotic checkpoint arrest, which suggests that Pkc1-dependent activation of PP2ACdc55 plays a critical role in checkpoint signaling. We therefore used mass spectrometry to determine how Pkc1 modifies the PP2ACdc55 complex. This revealed that Pkc1 induces changes in the phosphorylation of multiple subunits of the complex, as well as dissociation of Igo/ENSA. Pkc1 directly phosphorylates Cdc55 and Igo/ENSA, and phosphorylation site mapping and mutagenesis indicate that phosphorylation of Cdc55 contributes to Igo/ENSA dissociation. Association of Igo2 with PP2ACdc55 is regulated during the cell cycle, yet mutation of Pkc1-dependent phosphorylation sites on Cdc55 and Igo2 did not cause defects in mitotic progression. Together, the data suggest that Pkc1 controls PP2ACdc55 by multiple overlapping mechanisms.

The three Type 2A protein phosphatases, PP2Ac, PP4c and PP6c, are differentially regulated by Alpha4.

Alpha4 is a non-canonical regulatory subunit of Type 2A protein phosphatases that interacts directly with the phosphatase catalytic subunits (PP2Ac, PP4c, and PP6c) and is upregulated in a variety of cancers. Alpha4 modulates phosphatase expression levels and activity, but the molecular mechanism of this regulation is unclear, and the extent to which the various Type 2A catalytic subunits associate with Alpha4 is also unknown. To determine the relative fractions of the Type 2A catalytic subunits associated with Alpha4, we conducted Alpha4 immunodepletion experiments in HEK293T cells and found that a significant fraction of total PP6c is associated with Alpha4, whereas a minimal fraction of total PP2Ac is associated with Alpha4. To facilitate studies of phosphatases in the presence of mutant or null Alpha4 alleles, we developed a facile and rapid method to simultaneously knockdown and rescue Alpha4 in tissue culture cells. This approach has the advantage that levels of endogenous Alpha4 are dramatically reduced by shRNA expression thereby simplifying interpretation of mutant phenotypes. We used this system to show that knockdown of Alpha4 preferentially impacts the expression of PP4c and PP6c compared to expression levels of PP2Ac.

Visualization of subunit interactions and ternary complexes of protein phosphatase 2A in mammalian cells.

Protein phosphatase 2A (PP2A) is a ubiquitous phospho-serine/threonine phosphatase that controls many diverse cellular functions. The predominant form of PP2A is a heterotrimeric holoenzyme consisting of a scaffolding A subunit, a variable regulatory B subunit, and a catalytic C subunit. The C subunit also associates with other interacting partners, such as α4, to form non-canonical PP2A complexes. We report visualization of PP2A complexes in mammalian cells. Bimolecular fluorescence complementation (BiFC) analysis of PP2A subunit interactions demonstrates that the B subunit plays a key role in directing the subcellular localization of PP2A, and confirms that the A subunit functions as a scaffold in recruiting the B and C subunits to form a heterotrimeric holoenzyme. BiFC analysis also reveals that α4 promotes formation of the AC core dimer. Furthermore, we demonstrate visualization of specific ABC holoenzymes in cells by combining BiFC and fluorescence resonance energy transfer (BiFC-FRET). Our studies not only provide direct imaging data to support previous biochemical observations on PP2A complexes, but also offer a promising approach for studying the spatiotemporal distribution of individual PP2A complexes in cells.

Protein phosphatase-2A is down-regulated in patients within clear cell renal cell carcinoma.

BACKGROUND: Protein phosphatase-2A (PP2A) is one of the major cellular serine-threonine phosphatases. It positively regulates apoptosis and negatively regulates the mitogenic pathway, suggesting that loss of it might be involved in cancer development. Recent studies found its association with breast, lung and colorectal cancer; however, its expression profile and its prognostic value in clear cell renal cell carcinoma (ccRCC) have not been investigated. METHODS: Real-time quantitative PCR (qRT-PCR) and Western blot were used to explore PP2A expression in ccRCC and normal renal tissues. Moreover immunohistochemistry (ICH) was used to detect the expression of PP2A in ccRCC. Spearman's rank correlation, Kaplan-Meier plots and Cox proportional hazards regression model were used to analyze the data. RESULTS: Down-regulated expression of PP2A mRNA and protein was observed in the majority of ccRCC by qRT-PCR and Western blot when compared with their paired normal renal tissues. Clinic pathological analysis was showed a significant correlation existed between the lower expression of PP2A protein with the histological grade, lymph node metastasis and tumor distant metastasis (P<0.05); Survival analysis by Kaplan-Meier survival curve and log-rank test demonstrated that reduced PP2A expression in cancer tissue predicted poorer overall survival (OS) compared with group in higher expression. Notably, multivariate analyses by Cox's proportional hazard model revealed that expression of PP2A was an independent prognostic factor in ccRCC. CONCLUSIONS: These results suggest that the aberrant expression of PP2A in human ccRCC is possibly involved with tumorigenesis and development, and the PP2A protein could act as a potential biomarker for prognosis assessment of renal cancer. Further studies on the cellular functions of PP2A need to address these issues.

The disturbance of hippocampal CaMKII/PKA/PKC phosphorylation in early experimental diabetes mellitus.

BACKGROUND: Defining the impact of diabetes and related risk factors on brain cognitive function is critically important for patients with diabetes. AIMS: To investigate the alterations in hippocampal serine/threonine kinases signaling in the early phase of type 1 and type 2 diabetic rats. METHODS: Early experimental diabetes mellitus was induced in rats with streptozotocin or streptozotocin/high fat. Changes in the phosphorylation of proteins were determined by immunoblotting and immunohistochemistry. RESULTS: Our data showed a pronounced decrease in the phosphorylation of Ca(2+) /calmodulin-dependent protein kinase II (CaMKII) in the hippocampi of both type 1 and type 2 diabetic rats compared with age-matched control rats. Unexpectedly, we found a significant increase in the phosphorylation of synapsin I (Ser 603) and GluR1 (Ser 831) in the same experiment. In addition, aberrant changes in hippocampal protein kinase C (PKC) and protein kinase A (PKA) signaling in type 1 and type 2 diabetic rats were also found. Moreover, PP1α and PP2A protein levels were decreased in the hippocampus of type 1 diabetic rats, but significantly up-regulated in type 2 diabetic rats. CONCLUSIONS: The disturbance of CaMKII/PKA/PKC phosphorylation in the hippocampus is an early change that may be associated with the development and progression of diabetes-related cognitive dysfunction.

Effects of hibernation on regulation of mammalian protein phosphatase type-2-A.

Hibernation is used by many small mammals for winter survival. Animals undergo cycles of deep torpor characterized by strong reductions in metabolic rate, body temperature, and all physiological activities. One prominent biochemical mechanism involved in regulating coordinated transitions to and from the hypometabolic state is reversible protein phosphorylation, a dynamic covalent modification maintained by the actions of protein kinases and protein phosphatases. The present study characterizes protein phosphatase type 2A (PP2A) from liver and skeletal muscle of Richardson's ground squirrels, Urocitellus richardsonii, to determine if the properties of this enzyme are modified between euthermic and hibernating states to provide differential control of the enzyme or its target proteins. Kinetic analysis showed a significant 1.5-fold increase in the Km for the synthetic phosphorylated peptide substrate (RRApTVA) for PP2A from skeletal muscle (but not liver) of torpid ground squirrels, as compared with euthermic controls, when measured at both 25 °C and 5 °C. This correlated with enhanced phosphorylation of PP2Aα Tyr-307 (a 2.3-fold increase) during hibernation in skeletal muscle (but not liver), a modification known to inhibit PP2A action. Hence, both kinetic and phosphorylation data suggest a suppression of PP2A function in the torpid state in skeletal muscle. However, total PP2A protein levels (assessed by immunoblotting) and maximal activities did not change in either tissue between euthermic and hibernating states. Liver PP2A from hibernating animals showed a broader pH profile with high activity retained at lower pH values (6.0-6.5) than the euthermic enzyme which might aid enzyme function in vivo under conditions of respiratory acidosis during torpor.

Inhibition equivalency factors for dinophysistoxin-1 and dinophysistoxin-2 in protein phosphatase assays: applicability to the analysis of shellfish samples and comparison with LC-MS/MS.

The protein phosphatase inhibition assay (PPIA) is a well-known strategy for the determination of diarrheic shellfish poisoning (DSP) lipophilic toxins, which deserves better characterization and understanding to be used as a routine screening tool in monitoring programs. In this work, the applicability of two PPIAs to the determination of okadaic acid (OA), dinophysistoxin-1 (DTX-1), dinophysistoxin-2 (DTX-2), and their acyl ester derivatives in shellfish has been investigated. The inhibitory potencies of the DSP toxins on a recombinant and a wild PP2A have been determined, allowing the establishment of inhibition equivalency factors (IEFs) (1.1 and 0.9 for DTX-1, and 0.4 and 0.6 for DTX-2, for recombinant and wild PP2A, respectively). The PPIAs have been applied to the determination of OA equivalent contents in spiked and naturally contaminated shellfish samples. Results have been compared to those obtained by LC-MS/MS analysis, after application of the IEFs, showing good agreements.

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Introducción La suplementación de la dieta con un 10% (p/v) de fructosa en el agua de bebida durante 14 días en ratas produce hipertrigliceridemia y esteatosis hepática como consecuencia de una reducción en la expresión y en la actividad transcripcional de (..) (AU)

Introduction The addition of fructose in drinking water (10% w/v) for two weeks to rats induces hypertriglyceridemia and fatty liver by reducing the expression and transcriptional activity of (..) (AU)

Human aqueous humor phosphatase activity in cataract and glaucoma.

PURPOSE: To investigate the presence and activity of protein phosphatase-2A (PPase2A), protein phosphatase-2C (PPase2C), and protein tyrosine phosphatases (PTPs) in the human aqueous humor (AH) of patients with primary open-angle glaucoma (POAG) and cataract and to study the correlation between these phosphatases and the redox state of the AH. METHODS: Eighty-six cataract patients and 29 POAG patients who were scheduled for cataract surgery with or without glaucoma surgery were enrolled in the study. PPase2A, PPase2C, and PTPs levels in AH were measured by enzyme-linked immunosorbent assays, Western blot analyses, and spectral METHODS: The redox state was measured by spectral and fluorescent methods. RESULTS: Phosphatase activity-positive results were significantly higher in AH samples from the POAG group (PP2A χ(2)(1) = 11.754, P < 0.01; PP2C χ(2)(1) = 8.754, P < 0.01; PTP χ(2)(1) = 11.073, P < 0.01). Western blot analysis revealed higher PP2C levels in the AH of glaucoma patients compared with PP2C levels in the AH of cataract patients (P = 0.012). Both oxidized/reduced glutathione ratios and superoxide dismutase levels in the AH were significantly higher in the glaucoma group than in the cataract group. Finally significant correlations were found between PP2A and PP2C, PP2A and PTP, and total antioxidant activity and PTP levels. CONCLUSIONS: There is a statistically significant difference between phosphatase levels in the AH of POAG patients and cataract patients. The phosphatase content of the AH represents tissue pathology, but their presence in the AH may be attributed to cell debris or to active signaling to other molecular events.

Molecular expression and characterization of a novel protein phosphatase 2A gene from Clonorchis sinensis.

Reversible phosphorylation of proteins is a critical mechanism involved in physiological function of organisms, including Clonorchis sinensis. In the present study, One cDNA clone encoding protein phosphatase 2A (CsPP2A) was isolated from a C. sinensis adult cDNA plasmid library. The open reading frame of the novel gene contains 924 bp and encoded a putative protein of 307 amino acids. A similarity analysis showed high homology with Schistosoma japonicum (76.3%) and Homo sapiens (84.4%), respectively. Recombinant CsPP2A (rCsPP2A) was expressed and purified from Escherichia coli BL21 using pET28a (+) as an expression vector. CsPP2A showed higher transcript level in adult worm but excysted metacercaria (P > 0.05), metacercaria (P < 0.05), and egg (P < 0.05) using real-time RT-PCR. Western blotting analysis showed that rCsPP2A could be identified by anti-rCsPP2A rat serum, C. sinensis-infected rat serum, and the serum from the rats immunized with excretory-secretory products of C. sinensis. Immunohistochemical assay showed that CsPP2A was deposited at the egg, the vitellarium of adult worm, and the excretory bladder of metacercaria. Collectively, the results of this study suggested that CsPP2A may be involved in the development of adult and metacercaria of C. sinensis.

Proteomics analysis of the cardiac myofilament subproteome reveals dynamic alterations in phosphatase subunit distribution.

Myofilament proteins are responsible for cardiac contraction. The myofilament subproteome, however, has not been comprehensively analyzed thus far. In the present study, cardiomyocytes were isolated from rodent hearts and stimulated with endothelin-1 and isoproterenol, potent inducers of myofilament protein phosphorylation. Subsequently, cardiomyocytes were "skinned," and the myofilament subproteome was analyzed using a high mass accuracy ion trap tandem mass spectrometer (LTQ Orbitrap XL) equipped with electron transfer dissociation. As expected, a small number of myofilament proteins constituted the majority of the total protein mass with several known phosphorylation sites confirmed by electron transfer dissociation. More than 600 additional proteins were identified in the cardiac myofilament subproteome, including kinases and phosphatase subunits. The proteomic comparison of myofilaments from control and treated cardiomyocytes suggested that isoproterenol treatment altered the subcellular localization of protein phosphatase 2A regulatory subunit B56alpha. Immunoblot analysis of myocyte fractions confirmed that beta-adrenergic stimulation by isoproterenol decreased the B56alpha content of the myofilament fraction in the absence of significant changes for the myosin phosphatase target subunit isoforms 1 and 2 (MYPT1 and MYPT2). Furthermore, immunolabeling and confocal microscopy revealed the spatial redistribution of these proteins with a loss of B56alpha from Z-disc and M-band regions but increased association of MYPT1/2 with A-band regions of the sarcomere following beta-adrenergic stimulation. In summary, we present the first comprehensive proteomics data set of skinned cardiomyocytes and demonstrate the potential of proteomics to unravel dynamic changes in protein composition that may contribute to the neurohormonal regulation of myofilament contraction.

Mapping of protein phosphatase-6 association with its SAPS domain regulatory subunit using a model of helical repeats.

BACKGROUND: Helical repeat motifs are common among regulatory subunits for type-1 and type-2A protein Ser/Thr phosphatases. Yeast Sit4 is a distinctive type-2A phosphatase that has dedicated regulatory subunits named Sit4-Associated Proteins (SAPS). These subunits are conserved, and three human SAPS-related proteins are known to associate with PP6 phosphatase, the Sit4 human homologue. RESULTS: Here we show that endogenous SAPS subunit PP6R3 co-precipitates half of PP6 in cell extracts, and the SAPS region of PP6R3 is sufficient for binding PP6. The SAPS domain of recombinant GST-PP6R3 is relatively resistant to trypsin despite having many K and R residues, and the purified SAPS domain (residues 1-513) has a circular dichroic spectrum indicative of mostly alpha helical structure. We used sequence alignments and 3D-jury methods to develop alternative models for the SAPS domain, based on available structures of other helical repeat proteins. The models were used to select sites for charge-reversal substitutions in the SAPS domain of PP6R3 that were tested by co-precipitation of endogenous PP6c with FLAG-tagged PP6R3 from mammalian cells. Mutations that reduced binding with PP6 suggest that SAPS adopts a helical repeat similar to the structure of p115 golgin, but distinct from the PP2A-A subunit. These mutations did not cause perturbations in overall PP6R3 conformation, evidenced by no change in kinetics or preferential cleavage by chymotrypsin. CONCLUSION: The conserved SAPS domain in PP6R3 forms helical repeats similar to those in golgin p115 and negatively charged residues in interhelical loops are used to associate specifically with PP6. The results advance understanding of how distinctive helical repeat subunits uniquely distribute and differentially regulate closely related Ser/Thr phosphatases.

Evidence for the involvement of FAM110C protein in cell spreading and migration.

A number of factors, including protein kinases, Rho GTPases and actin and microtubule cytoskeletons play a crucial role in cell migration and spreading. We have recently shown that ectopic expression of FAM110C can alter cellular morphology by mechanisms yet to be determined. In this study, a FAM110C antiserum has been developed and used to study endogenously expressed FAM110C. Our data show that FAM110C is expressed by different cell lines and it can be detected throughout the cell. Interestingly, depletion of FAM110C by short interfering RNA reduced integrin-mediated filopodia formation, hepatocyte growth factor-induced migration, and phosphorylation of the Akt1 kinase in the epithelial cell line HepG2. Furthermore, co-immunoprecipitation and co-localization studies show that both ectopically and endogenously expressed FAM110C interact, or is part of a protein complex, with the Akt1 kinase. This interaction is transient and follows the activation of Akt1. In addition, we show that alpha-tubulin co-precipitates with FAM110C which further supports an interaction with the microtubule cytoskeleton. Collectively, these findings suggest a new function for FAM110C in the regulation of cell spreading, migration and filopodia induction.