AC Dimer Research Articles

Abstract C080: Decoding the MYC-PDAC nexus: Unveiling the impact of PP2A-C subunit methylation on tumor onset and advancement

Abstract Introduction: Persistent disruptions in MYC regulation and the inactivation of the serine/threonine protein phosphatase 2A (PP2A) are prevalent among individuals diagnosed with pancreatic ductal adenocarcinoma (PDAC). While it is known that PP2A influences MYC activity by facilitating its degradation, the precise mechanism by which this occurs remains unclear. Small molecule activators PP2A (SMAPs) regulate the holoenzyme assembly consisting of a scaffolding-A subunit and a catalytic-C subunit, and over 40 distinct substrate directing regulatory-B subunits. PP2A heterotrimerization is regulated by post-translational modifications of the catalytic C subunit, notably methylation of L309, which impacts B subunit binding affinity to the AC dimer and thus the substrate selectivity. In this study, we aim to investigate whether the deregulation of MYC, attributed to the loss of PP2A-C subunit methylation, contributes to the initiation and progression of PDAC. Methods: We investigated the influence of SMAPs on MYC’s stability using clinical specimens, cellular, and mouse models. Clinical specimens consisted of tissue microarray (TMA) from patients with pancreatitis and PDAC. Cellular models involved ex-vivo acinar cell assays and proximity ligation assays. Mouse models included caerulein-induced pancreatitis, genetic mouse models like Ptf1a-Cre; LSL-KRASG12D (KC) and LSL-Kras G12D;P53 R172H/+;Pdx1-Cre (KPC), along with pharmacological inhibition studies with SMAPs. Results and discussion: Our TMA data revealed a consistent pattern of Methyl-PP2A-C (Methyl-C) loss and an increase in the stable and active phosphoform of MYC (p62MYC) throughout the sequential stages of PDAC progression, starting from normal tissue and advancing to acinar-to-ductal metaplasia, PanINs, and finally PDAC. In ex-vivo ADM assays, treatment with small molecule activators of PP2A (SMAPs), specifically DT061, significantly reduced the transformation of acinar cells to duct-like cells compared to untreated cells from both wild-type (WT) and KC mice. This suggests that PP2A plays a crucial role in the early events that drive the formation of pancreatic lesions. Moreover, SMAPs demonstrated the ability to alleviate caerulein-induced inflammation in normal mice, as well as suppress the development of pancreatic PanIN lesions and collagen deposition in caerulein-treated KC mice. These effects were associated with decreased levels of p62MYC and increased Methyl-C staining in both WT and KC mice treated with caerulein. Additionally, in KPC mice, treatment with DT061 resulted in reduced PDAC tumor growth, accompanied by decreased levels of p62MYC. Conclusion: Our results provide compelling evidence that small molecule activators of PP2A (SMAPs), through their ability to downregulate MYC, hold significant promise as a therapeutic strategy for individuals at high risk of developing PDAC, particularly those with chronic pancreatitis. This approach has the potential to effectively prevent PDAC development and mitigate its onset in this high-risk population. Citation Format: Vidhi Shah, Alexander Smith, Gabriel Cohn, Motoyuki Tsuda, Goutham Narla, Jonathan Brody, Brett Sheppard, Rosalie Sears. Decoding the MYC-PDAC nexus: Unveiling the impact of PP2A-C subunit methylation on tumor onset and advancement [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr C080.

Abstract PR002: Loss of protein phosphatase PP2A activity drives AR addiction in prostate cancer

Abstract Reversible protein phosphorylation plays a fundamental role in numerous biological processes and is regulated by a dynamic interplay between protein kinases and phosphatases. Disruption of this balance due to aberrant activation of kinases and inactivation of phosphatases is a hallmark of cancer. The highly conserved protein phosphatase 2A (PP2A) is a major serine/threonine phosphatase with a complex heterotrimeric composition involved in the regulation of numerous critical aspects of cellular function. PP2A is one of the most abundant enzymes, accounting for up to 1% of the total cellular protein in some tissues and represents the majority of serine/threonine phosphatase activities in many tissues. PP2A is comprised of a core dimer consisting of a scaffolding ‘‘A’’ subunit (PP2A-A), and a catalytic ‘‘C’’ subunit (PP2A-C), which form a heterotrimeric complex with one of at least 20 substrate-directing regulatory “B” subunits. PP2A heterotrimerization is regulated through Leucine309 methylation of catalytic C subunit, which influence the binding affinity of the B subunits to the AC dimer and thus the substrate specificity. Loss of the tumor suppressive activity of PP2A is associated with prostate cancer; however, the molecular basis for this loss is not well understood. Here, we report that the leucine carboxy methyltransferase (LCMT1), which methylates the L309 of the catalytic C subunit of the PP2A heterodimer, acts as a suppressor of androgen receptor (AR) addicted prostate cancer (PCa). In a series of tissue microarrays (TMAs) representing 17,747 localized PCa, low methyl-PP2A-C levels were negatively associated with critical parameters of disease progression including high Gleason score, and lymph node metastasis. Biologically, LCMT1 silencing leads to increased AR phosphorylation/activity and enhanced CRPC growth in vivo. Conversely, LCMT1 dependent methyl sensitive PP2A heterotrimers target AR and its critical coactivator MED1 for dephosphorylation through binding of the PP2A-B56α regulatory subunit to two conserved small linear motifs (SLiMs) on MED1, resulting in the eviction of the AR-MED1 complex from the chromatin and loss of target gene expression. Mechanistically, LCMT1 is regulated by PI3K-AKT-mTOR-S6K1-mediated phosphorylation-induced degradation requiring the β-TRCP E3 ubiquitin ligase. Targeted degradation of LCMT1 through this mechanism is associated with acquired resistance to second-generation anti-androgens such as enzalutamide, apalutamide and darolutamide resulting in restored AR-addiction in CRPC cells. Finally, feedforward stabilization of LCMT1 by a small molecule activator of phosphatase (SMAP) results in attenuation of AR signaling and tumor regression in enzalutamide refractory PCa xenografts. Collectively, these results demonstrate that decreased methyl-PP2A-C is a novel prognostic marker for PCa progression and that the loss of LCMT1 is a major determinant of AR-addicted advanced PCa, and support the therapeutic potential of selective small molecules modulators of PP2A for metastatic prostate cancer treatment. Citation Format: Irfan A. Asangani. Loss of protein phosphatase PP2A activity drives AR addiction in prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr PR002.

Overexpression of AtPTPA in Arabidopsis increases protein phosphatase 2A activity by promoting holoenzyme formation and ABA negatively affects holoenzyme formation

AtPTPA is a critical regulator for the holoenzyme assembling of protein phosphatase 2A (PP2A) in Arabidopsis. Characterization of AtPTPA improves our understanding of the function and regulation of PP2A in eukaryotes. Further analysis of AtPTPA-overexpressing plants indicates that AtPTPA increases PP2A activity by promoting PP2A's AC dimer formation, thereby holoenzyme assembling. Plant hormone abscisic acid (ABA) reduces PP2A enzyme activity by negatively affects PP2A's AC dimer formation. Therefore, AtPTPA is a positive factor that promotes PP2A holoenzyme assembly, and ABA is a negative factor that prevents PP2A holoenzyme assembly.

Polymer Soft-Landing Isolation of Acetylene on Polystyrene and Poly(vinylpyridine): A Novel Approach to Probing Hydrogen Bonding in Polymers.

Hydrogen-bonded complexes of acetylene (Ac) with the polymers polystyrene (PS), poly(4-vinylpyridine) (P4VP), and poly(2-vinylpyridine) (P2VP) have been characterized for the first time at 16 K in a "polymer soft-landing isolation" experiment which is being pioneered in our research laboratory. In particular, changes in vibrational modes of Ac provide ample evidence for hydrogen-bonded complexes between Ac and the phenyl groups of PS or the pyridyl groups of P4VP and P2VP. With PS, the proton on the top Ac molecule of the classic T-shaped Ac dimer interacts with the π cloud of the benzene (Bz) ring to form a C-H---π interaction, while the π cloud of the lower Ac forms a second C-H---π interaction with a proton on the Bz ring. An analogous (ring)1-(Ac)2 double interaction occurs between an Ac dimer and the pyridine (Pyr) rings on both P2VP and P4VP, yielding a C-H---N and C-H---π interaction. With P4VP and P2VP a second bridged (ring)2-(Ac)2 product is formed, with the Ac dimer forming nearly collinear C-H---N hydrogen bonds to adjacent Pyr rings. On P2VP this bridged product is the only one after extensive annealing. These complexes in which Ac acts as both proton donor and acceptor have not previously been observed in conventional matrix isolation experiments. This study is the second from our laboratory employing this method, which represents a slight modification of the traditional matrix isolation technique.

Abstract 802: The inactivation of PP2A caused by the replacement of its regulatory subunit plays a crucial role in the hemangiomagenesis

Abstract Hemangioma is the most frequent vascular tumor of early childhood. However, the mechanism leading to their development, progression and maintenance has remained unclear. In this study, we generated an endothelial-specific PyMT-expressing transgenic (Tg) mouse (Tie2-PyMT) that spontaneously develop hemangioma. Using Tie2-PyMT transgenic mice and bEnd.3 cells, an PyMT induced mouse brain endothelial cell line idicates that the direct expression of PyMT gene in vascular endothelial cells leads to rapid cell proliferation and hemangioma development. An interaction between the core AC dimer of protein phosphatase 2A (PP2A) and PyMT, but not B regulatory subunit was verified both in hemangioma endothelial cells of Tie2-PyMT Tg mice and bEnd.3 cells by immunoprecipitation. Whereas in endothelial cells of Tg negative mice and PyMT silenced bEnd.3 cells, the B regulatory subunit binds to the core AC dimer of PP2A. PyMT binding to PP2A led to the inactivation of PP2A phosphatases and subsequently activates its downstream ERK1/2 and AKT pathway, which may be responsible for the proliferative effect of PyMT on vascular endothelial cells. Moreover, the effect of PyMT on PP2A phosphatases activity could be rescued by Okadaic acid (OA), a specific inhibitor of PP2A. Treamtment of Tie2-PyMT transgenic mice with the PP2A activator, FTY720, significantly delayed hemangiomagenesis in this model. Furthermore, PP2A complex composition and its phosphatases activity were also examined in patient hemangioma specimens. Consisten with the animal model, a decreased PP2A phosphatases activity and the dissociation of B subunit from the PP2A core dimer were observed. These findings suggest that the inactivation of PP2A caused by the replacement of its regulatory subunit plays a crucial role in the hemangiomagenesis. Citation Format: Qin Xu, Furong Xie. The inactivation of PP2A caused by the replacement of its regulatory subunit plays a crucial role in the hemangiomagenesis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 802. doi:10.1158/1538-7445.AM2015-802

Disruption and inactivation of the PP2A complex promotes the proliferation and angiogenesis of hemangioma endothelial cells through activating AKT and ERK.

Hemangioma is a benign vascular neoplasm of unknown etiology. In this study, we generated an endothelial-specific PyMT gene-expressing transgenic mouse model that spontaneously develops hemangioma. Based on this transgenic model, a specific binding between PyMT and the core AC dimer of protein phosphatase 2A (PP2A) was verified in hemangioma vascular endothelial cells. The binding between PyMT and the PP2A AC dimer resulted in dissociation of the B subunit from the PP2A complex and inactivation of PP2A phosphatases, which in turn activated AKT and ERK signaling and promoted cell proliferation, migration and angiogenesis in vitro and tumorigenesis in vivo. Consistent with the in vitro findings, decreased PP2A phosphatase activity and disruption of the PP2A heterotrimeric complex were also observed in both primary transgene-positive TG(+) mouse hemangioma endothelial cells (TG(+) HEC cells) and human proliferating phase hemangioma endothelial (human HEC-P) cells, but not in transgene-negative TG(-) mouse normal vascular endothelial cells (TG(-) NEC cells) and human involuting phase hemangioma endothelial (human HEC-I) cells. Further, it was observed that in human hemangioma cells, endoglin could compete with the PP2A/A, C subunits for binding to the PP2A/B subunit, thereby resulting in dissociation of the B subunit from the PP2A complex. Treatment of Tie2/PyMT transgenic mice with the PP2A activator FTY720 significantly delayed the occurrence of hemangioma. Our data provide evidence of a previously unreported anti-proliferation and anti-angiogenesis effect of PP2A in vascular endothelial cells, and show the therapeutic value of PP2A activators in hemangioma.

Polyoma and SV40 proteins differentially regulate PP2A to activate distinct cellular signaling pathways involved in growth control

Binding of Src family kinases to membrane-associated polyoma virus middle T-antigen (PyMT) can result in the phosphorylation of PyMT tyrosine 250, which serves as a docking site for the binding of Shc and subsequent activation of the Raf-MEK-ERK (MAP) kinase cascade. In a screen for PyMT variants that could not activate the ARF tumor suppressor, we isolated a cytoplasmic nontransforming mutant (MTA) that encoded a C-terminal truncated form of the PyMT protein. Surprisingly, MTA was able to strongly activate the MAP kinase pathway in the absence of Src family kinase and Shc binding. Interestingly, the polyoma small T-antigen (PyST), which shares with MTA both partial amino acid sequence homology and cellular location, also activates the MAP kinase cascade. Activation of the MAP kinase cascade by both MTA and PyST has been demonstrated to be PP2A-dependent. Neither MTA nor PyST activate the phosphorylation of AKT. The SV40 small T-antigen, which is similar to PyST in containing a J domain and in binding to the PP2A AC dimer, does not activate the MAP kinase cascade, but does stimulate phosphorylation of AKT in a PP2A-dependent manner. These findings highlight a novel role of PP2A in stimulating the MAP kinase cascade and indicate that the similar polyoma and SV40 small T-antigens influence PP2A to activate discrete cellular signaling pathways involved in growth control.

Should activin βC be more than a fading snapshot in the activin/TGFβ family album?

The activin growth factors consist of dimeric proteins made up of activin β subunits and have been shown to be essential regulators of diverse systems in physiology. Four subunits are known to be expressed in mammalian cells: βA, βB, βC, and βE. Surprisingly, deletion of activin βC and βE subunits in vivo does not affect embryonic development or adult physiology which has led to the activin βC and βE subunits being regarded as non-essential and unimportant. The steady accumulation of circumstantial evidence to the contrary has led this lab to reassess the role of the activin βC subunit. Activin βC protein is expressed more widely than indicated by mRNA localisation. Experiments overexpressing activin βC subunit or adding exogenous Activin C in vitro are contradictory but suggest roles for activin βC in regulating Activin A action in apoptosis and homeostasis. Sequestration of βA subunits by dimerisation with βC subunits to form Activin AC represents an intracellular regulator of Activin A bioactivity. Activins play a pivotal role in normal physiology and carcinogenesis, so any molecule, such as the activin βC subunit, that can affect activin action is potentially significant. Advancing our understanding of the physiological role of the activin βC subunit requires new tools and reagents. Direct detection of the Activin AC dimer will be essential and will necessitate the purification of heteromeric Activin AC protein. In addition, there is a need for the development of an in vivo model of activin βC subunit overexpression. With development of these tools, research into activin action in development and physiology can expand to include the less well understood members of the activin family such as activin βC.

Modeling of Gαs and Gαi Regulation of Human Type V and VI Adenylyl Cyclase

We examined the kinetics of Galpha(s) and Galpha(i) regulation of human type V and type VI adenylyl cyclase (AC V and AC VI) in order to better model interactions between AC and its regulators. Activation of AC VI by Galpha(s) displayed classical Michaelis-Menten kinetics, whereas AC V activation by Galpha(s) was cooperative with a Hill coefficient of 1.4. The basal activity of human AC V, but not that of AC VI, was inhibited by Galpha(i). Both enzymes showed greater inhibition by Galpha(i) at low Galpha(s) concentrations; however, human AC V was activated by Galpha(i) at high Galpha(s) concentrations. Neither regulator had an effect on the K(m) for Mg-ATP. Mutations made within the Galpha(s) binding pocket of AC V (N1090D) and VI (F1078S) displayed 6- and 14-fold greater EC(50) values for Galpha(s) activation but had no effect on Galpha(i) inhibition of basal activity or K(m) for Mg-ATP. Galpha(s)-stimulated AC VI-F1078S was not significantly inhibited by Galpha(i), despite normal inhibition by Galpha(i) upon forskolin stimulation. Mechanistic models for Galpha(s) and Galpha(i) regulation of AC V and VI were derived to describe these results. Our models are consistent with previous studies, predicting a decrease in affinity of Galpha(i) in the presence of Galpha(s). For AC VI, Galpha(s) is required for inhibition but not binding by Galpha(i). For AC V, binding of two molecules of Galpha(s) and Galpha(i) to an AC dimer are required to fully describe the data. These models highlight the differences between AC V and VI and the complex interactions with two important regulators.

High Resolution Crystal Structures of Piscine Transthyretin Reveal Different Binding Modes for Triiodothyronine and Thyroxine

Transthyretin (TTR) is an extracellular transport protein involved in the distribution of thyroid hormones and vitamin A. So far, TTR has only been found in vertebrates, of which piscine TTR displays the lowest sequence identity with human TTR (47%). Human and piscine TTR bind both thyroid hormones 3,5,3'-triiodo-l-thyronine (T(3)) and 3,5,3',5'-tetraiodo-l-thyronine (thyroxine, T(4)). Human TTR has higher affinity for T(4) than T(3), whereas the reverse holds for piscine TTR. X-ray structures of Sparus aurata (sea bream) TTR have been determined as the apo-protein at 1.75 A resolution and bound to ligands T(3) and T(4), both at 1.9 A resolution. The apo structure is similar to human TTR with structural changes only at beta-strand D. This strand forms an extended loop conformation similar to the one in chicken TTR. The piscine TTR.T(4) complex shows the T(4)-binding site to be similar but not identical to human TTR, whereas the TTR.T(3) complex shows the I3' halogen situated at the site normally occupied by the hydroxyl group of T(4). The significantly wider entrance of the hormone-binding channel in sea bream TTR, in combination with its narrower cavity, provides a structural explanation for the different binding affinities of human and piscine TTR to T(3) and T(4).

Structure of Protein Phosphatase Methyltransferase 1 (PPM1), a Leucine Carboxyl Methyltransferase Involved in the Regulation of Protein Phosphatase 2A Activity

The important role of the serine/threonine protein phosphatase 2A (PP2A) in various cellular processes requires a precise and dynamic regulation of PP2A activity, localization, and substrate specificity. The regulation of the function of PP2A involves the reversible methylation of the COOH group of the C-terminal leucine of the catalytic subunit, which, in turn, controls the enzyme's heteromultimeric composition and confers different protein recognition and substrate specificity. We have determined the structure of PPM1, the yeast methyltransferase responsible for methylation of PP2A. The structure of PPM1 reveals a common S-adenosyl-l-methionine-dependent methyltransferase fold, with several insertions conferring the specific function and substrate recognition. The complexes with the S-adenosyl-l-methionine methyl donor and the S-adenosyl-l-homocysteine product and inhibitor unambiguously revealed the co-substrate binding site and provided a convincing hypothesis for the PP2A C-terminal peptide binding site. The structure of PPM1 in a second crystal form provides clues to the dynamic nature of the PPM1/PP2A interaction.

Identification of a Subunit of a Novel Kleisin-β/SMC Complex as a Potential Substrate of Protein Phosphatase 2A

Protein phosphatase 2A (PP2A) holoenzymes consist of a catalytic C subunit, a scaffolding A subunit, and one of several regulatory B subunits that recruit the AC dimer to substrates [1, 2]. PP2A is required for chromosome segregation [3–6], but PP2A's substrates in this process remain unknown. To identify PP2A substrates, we carried out a two-hybrid screen with the regulatory B/PR55 subunit. We isolated a human homolog of C. elegans HCP6, a protein distantly related to the condensin subunit hCAP-D2, and we named this homolog hHCP-6. Both C. elegans HCP-6 and condensin are required for chromosome organization and segregation [7–11]. HCP-6 binding partners are unknown, whereas condensin is composed of the structural maintenance of chromosomes proteins SMC2 and SMC4 and of three non-SMC subunits [12]. Here we show that hHCP-6 becomes phosphorylated during mitosis and that its dephosphorylation by PP2A in vitro depends on B/PR55, suggesting that hHCP-6 is a B/PR55-specific substrate of PP2A. Unlike condensin, hHCP-6 is localized in the nucleus in interphase, but similar to condensin, hHCP-6 associates with chromosomes during mitosis. hHCP-6 is part of a complex that contains SMC2, SMC4, kleisin-β, and the previously uncharacterized HEAT repeat protein FLJ20311. hHCP-6 is therefore part of a condensin-related complex that associates with chromosomes in mitosis and may be regulated by PP2A.

Inhibition of Src by direct interaction with protein phosphatase 2A

In this study, we report that Src kinase is inhibited by protein phosphatase 2A (PP2A), a serine/threonine phosphatase. We carried out experiments in vitro using purified PP2A (AC dimer) and full-length v-Src or truncated forms of v-Src. The inhibition of v-Src by PP2A is concentration- and time-dependent. Addition of okadaic acid, a PP2A phosphatase inhibitor, abolished the PP2A-dependent inhibition of v-Src. When experiments were carried out at 4°C under conditions where PP2A activity is inhibited, Src activity was unaffected by the presence of PP2A, suggesting that PP2A binding alone is insufficient to block Src activity. These results imply that PP2A activity is essential for inhibition of v-Src. We also demonstrate that PP2A binds to the catalytic and the regulatory domains of v-Src.

Regulation by Methylation

Posttranslational modifications are important mechanisms for regulating protein activity. The catalytic subunit (C) of the protein phosphatase PP2A is modified by phosphorylation and carboxyl methylation. The interactions of the C subunit with regulatory B subunits and a scaffold subunit (A) are believed to contribute to the control of target specificity of PP2A. In vitro studies showed that carboxyl methylation of the C subunit influences the interaction of the AC dimer with the B subunit. Wu et al. identified the genes for the yeast carboxyl methyltransferase ( PPM1 and PPM2 ) and the gene for the yeast methylesterase ( PPE1 ) and found that only PPM1 and PPE1 appear to have measurable activity toward PP2A. By using genetic analysis, the consequences of altered levels of methylation of the C subunit on PP2A activity and regulatory protein interactions were studied. Ppm1 mutant yeast exhibit defects similar to those of yeast with defective B subunits, suggesting that methylation may influence the interaction of the C subunit with the B subunits. Furthermore, loss of PPM1 activity resulted in an enhanced interaction with another regulatory partner called Tap42. Thus, if the level of methylation of the C subunit changes in response to stimuli or during the cell cycle, this may help to control the distribution of the C subunit among various regulatory partners and, so, influence the pathway regulated by PP2A at any given time. Wu, J., Tolstykh, T., Lee, J., Boyd, K., Stock, J.B., and Broach, J.R. (2000) Carboxyl methylation of the phosphoprotein phosphatase 2A catalytic subunit promotes its functional association with regulatory subunits in vivo . EMBO J. 19 : 5672-5681. [Abstract] [Full Text]

Mutation of Tyr307 and Leu309 in the protein phosphatase 2A catalytic subunit favors association with the alpha 4 subunit which promotes dephosphorylation of elongation factor-2.

The cellular location and substrate specificity of the catalytic subunit (C) of protein phosphatase 2A (PP2A) depend on its interaction with A and B subunits. The distribution of epitope-tagged wild-type or mutated C subunits was studied by transient expression in COS-7 cells. Wild-type tagged C expressed at low levels formed ABC trimer and AC dimer like the endogenous C. Single mutations of C at the site of phosphorylation (Y307F) or carboxymethylation (L309Q) resulted in recovery of only AC dimer. Double mutation of both residues resulted in association of C with alpha 4 protein (alpha 4), a novel subunit of PP2A, instead of with A and B subunits. Thus, the distribution of C between ABC trimer, AC dimer, and alpha 4C complexes can be affected by modifications of the C-terminal residues. The alpha 4 protein is a homologue of the yeast Tap42 protein that functions downstream of the TOR protein to regulate protein synthesis. Transient overexpression of FLAG-alpha 4 resulted in increased dephosphorylation of elongation factor 2, but had no effect on phosphorylation of either p70S6 kinase or PHAS-I (eIF4E-BP). Signals that affect phosphorylation or methylation of the C subunit of PP2A may promote subunit exchange and direct phosphatase activity to specific intracellular substrates.

Roles of the helicase and primase domain of the gene 4 protein of bacteriophage T7 in accessing the primase recognition site.

The 63 kDa gene 4 protein of bacteriophage T7 provides both helicase and primase activities. The C-terminal helicase domain of the gene 4 protein is responsible for DNA-dependent NTP hydrolysis and for hexamer formation, whereas the N-terminal primase domain contains the zinc motif that is, in part, responsible for template-directed oligoribonucleotide synthesis. In the presence of beta, gamma-methylene dTTP, the protein forms a hexamer that surrounds and binds tightly to single-stranded DNA and consequently is unable to translocate to primase recognition sites, 5'-GTC-3', or to dissociate from the molecule to which it is bound. Nonetheless, in the presence of beta,gamma-methylene dTTP, it catalyzes the synthesis of pppAC dimers at primase sites on M13 DNA. When bound to single-stranded DNA in the presence of beta,gamma-methylene dTTP, the primase can function at recognition sites on the same molecule to which it is bound provided that a sufficient distance exists between the recognition site and the site to which it is bound. Furthermore, the primase bound to one DNA strand can function at a primase site located on a second DNA strand. The results indicate that the primase domain resides on the outside of the hexameric ring, a location that enables it to access sites distal to its site of binding.

B cell receptor-associated protein alpha4 displays rapamycin-sensitive binding directly to the catalytic subunit of protein phosphatase 2A.

Recently, TAP42 was isolated as a high copy suppressor of sit4-, a yeast phosphatase related to protein phosphatase 2A (PP2A). TAP42 is related to the murine alpha4 protein, which was discovered independently by its association with Ig-alpha in the B cell receptor complex. Herein we show that a glutathione S-transferase (GST)-alpha4 fusion protein bound the catalytic subunit (C) of human PP2A from monomeric or multimeric preparations of PP2A in a "pull-down" assay. In an overlay assay, the GST-alpha4 protein bound to the phosphorylated and unphosphorylated forms of C that were separated in two-dimensional gels and immobilized on filters. The results show direct and exclusive binding of alpha4 to C. This is unusual because all known regulatory B subunits, or tumor virus antigens, bind stably only to the AC dimer of PP2A. The alpha4-C form of PP2A had an increased activity ratio compared with the AC form of PP2A when myelin basic protein phosphorylated by mitogen-activated protein kinase and phosphorylase a were used as substrates. Recombinant alpha4 cleaved from GST was phosphorylated by p56(lck) tyrosine kinase and protein kinase C. A FLAG-tagged alpha4 expressed in COS7 cells was recovered as a protein containing phosphoserine and coimmunoprecipitated with the C but not the A subunit of PP2A. Treatment of cells with rapamycin prevented the association of PP2A with FLAG-alpha4. The results reveal a novel heterodimer alpha4-C form of PP2A that may be involved in rapamycin-sensitive signaling pathways in mammalian cells.

NMR solution structure of the 32-kDa platelet factor 4 ELR-motif N-terminal chimera: a symmetric tetramer.

Native human platelet factor 4 (PF4) is a homotetrameric protein (70 residues/subunit) known for its anticoagulant heparin binding activity. 2D 15N--1H HSQC NMR experiments of native PF4 in solution show the presence of conformational heterogeneity consistent with the formation of asymmetric homo-tetramers as observed in the X-ray crystal structure of both human and bovine PF4. A chimeric mutant of PF4 (called PF4-M2) which substitutes the first 11 N-terminal residues for the first eight residues from homologous interleukin-8 forms symmetric homo-tetramers with essentially the same heparin binding activity as native PF4. The solution structure of PF4-M2 has been investigated by using two- and three-dimensional 1H- and 15N-NMR spectroscopy and NOE-restrained simulated annealing molecular dynamics. As with other members of the CXC chemokine family whose structures are known, the PF4-M2 subunit monomer consists of a mostly hydrophobic, triple-stranded antiparallel beta-sheet onto which is folded an amphipathic C-terminal helix and a less periodic N-terminal domain. Although N-terminal substitution with the less acidic interleukin-8 sequence most affects the quarternary structure relative to native PF4 at the AC and AD dimer interfaces, AB dimer stability is weakened as reflected in reduced equilibrium association binding constants.