The biogenesis of active Protein Phosphatase 2A holoenzymes: a tightly regulated process creating phosphatase specificity

Protein Phosphatase type 2A (PP2A) enzymes comprise a large family of Ser/Thr phosphatases with multiple functions in cellular signaling and physiology. The composition of heterotrimeric PP2A holoenzymes, resulting from the combinatorial assembly of a catalytic C, structural A and regulatory B-type subunit, provides the essential determinants for substrate specificity, subcellular targeting and fine-tuning of phosphatase activity, largely explaining why PP2A is functionally involved in so many diverse physiological processes, sometimes in seemingly opposing ways. In this review, we highlight how PP2A holoenzyme biogenesis and enzymatic activity are controlled by a sophisticatedly coordinated network of five PP2A modulators, consisting of α4, PTPA, LCMT1, PME-1 and potentially TIPRL1, which serve to prevent promiscuous phosphatase activity until the holoenzyme is completely assembled. Likewise, these modulators may come into play when PP2A holoenzymes are disassembled following particular cellular stresses. Malfunctioning of these cellular control mechanisms contributes to human disease. The potential therapeutic benefits or pitfalls of interfering with these regulatory mechanisms will be briefly discussed.status: publishe

Structure, regulation and pharmacological modulation of PP2A phosphatases

Protein phosphatases of the type 2A family (PP2A) represent a major fraction of cellular Ser/Thr phosphatase activity in any given human tissue. In this review, we describe how the holoenzymic nature of PP2A and the existence of several distinct PP2A composing subunits allow for the generation of multiple structurally and functionally different PP2A complexes, explaining why PP2A is involved in the regulation of so many diverse cell biological and physiological processes. Moreover, in human disease, most notably in several cancers and Alzheimer's Disease, PP2A expression and/or activity have been found significantly decreased, underscoring its important functions as a major tumor suppressor and tau phosphatase. Hence, several recent preclinical studies have demonstrated that pharmacological restoration of PP2A activity, as well as pharmacological PP2A inhibition, under certain conditions, may be of significant future therapeutic value.status: publishe

Recurrent PPP2R1A mutations in uterine cancer act through a dominant-negative mechanism to promote malignant cell growth

Somatic missense mutations in the Ser/Thr protein phosphatase 2A (PP2A) Aα scaffold subunit gene PPP2R1A are among the few genomic alterations that occur frequently in serous endometrial carcinoma (EC) and carcinosarcoma, two clinically aggressive subtypes of uterine cancer with few therapeutic options. Previous studies reported that cancer-associated Aα mutants exhibit defects in binding to other PP2A subunits and contribute to cancer development by a mechanism of haploinsufficiency. Here we report on the functional significance of the most recurrent PPP2R1A mutations in human EC which cluster in Aα HEAT repeats 5 and 7. Beyond predicted loss-of-function effects on the formation of a subset of PP2A holoenzymes, we discovered that Aα mutants behave in a dominant-negative manner due to gain-of-function interactions with the PP2A inhibitor TIPRL1. Dominant-negative Aα mutants retain binding to specific subunits of the B56/B' family and form substrate trapping complexes with impaired phosphatase activity via increased recruitment of TIPRL1. Accordingly, overexpression of the Aα mutants in EC cells harboring wildtype PPP2R1A increased anchorage-independent growth and tumor formation, and triggered hyperphosphorylation of oncogenic PP2A-B56/B' substrates in the GSK3β, Akt and mTOR/p70S6K signaling pathways. TIPRL1 silencing restored GSK3β phosphorylation and rescued the EC cell growth advantage. Our results reveal how PPP2R1A mutations affect PP2A function and oncogenic signaling, illuminating the genetic basis for serous EC development and its potential control by rationally targeted therapies.status: publishe

Identification of PP2A/Set binding sites and design of interacting peptides with potential clinical applications

Protein phosphatase 2A (PP2A) is known to be a negative regulator of several survival and proliferating pathways that are frequently altered in cancer. In addition to chemical enzymatic inhibitors of the PP2A activity, the oncoprotein SET has been described as a physiological PP2A inhibitor by forming a complex with PP2A catalytic subunit (PP2Ac). Increased SET protein levels therefore directly reduce the tumor suppressor function of PP2A and promote tumor progression. We have used the PEP-Scan approach to identify the binding site between the serine/threonine phosphatase PP2A and the oncoprotein SET. For in vivo validation of the peptides, we have used chronic lymphocytic leukemia (CLL) xenograft models. In this manuscript we describe the identification of amino acid sequences involved in the complex formation, both at the PP2Ac and SET sides. The amino acid sequences of the binding sites were coupled to an optimized penetrating peptide in order to generate chimeras (Mut3DPT-PP2A and Mut3DPT-SET) able to target the PP2A/SET interaction. We demonstrate that these peptides have an in vitro apoptotic effect on breast and lung cancer cell lines, as well as an antitumoral effect on CLL and lymphoma xenograft models. The new generated chimeric peptides allow the modulation of the PP2Ac/SET interaction and might have a potential as a new therapeutic approach for cancer treatment.status: publishe

Glycolaldehyde-derived high-molecular-weight advanced glycation end-products induce cardiac dysfunction through structural and functional remodeling of cardiomyocytes

Background/Aims: High-molecular-weight advanced glycation end-products (HMW-AGEs) are abundantly present in our Western diet. There is growing evidence reporting that HMW-AGEs contribute to the development of cardiovascular dysfunction in vivo, next to the well-known low-molecular-weight AGEs. The goal of our study is to assess the ultrastructure and function of cardiomyocytes after chronic exposure to HMW-AGEs. A better understanding of underlying mechanisms is essential to create new opportunities for further research on the specific role of HMW-AGEs in the development and progression of cardiovascular diseases. Methods: Adult male rats were randomly assigned to daily intraperitoneal injection for six weeks with either HMW-AGEs (20 mg/kg/day) or a control solution. Hemodynamic measurements were performed at sacrifice. Single cardiomyocytes from the left ventricle were obtained by enzymatic dissociation through retrograde perfusion of the aorta. Unloaded cell shortening, time to peak and time to 50% relaxation were measured during field stimulation and normalized to diastolic length. L-type Ca2+ current density (ICaL) and steady-state inactivation of ICaL were measured during whole-cell ruptured patch clamp. Myofilament functional properties were measured in membrane-permeabilized cardiomyocytes. Ultrastructural examination of cardiac tissue was performed using electron microscopy. Results: Rats injected with HMW-AGEs displayed in vivo cardiac dysfunction, characterized by significant changes in left ventricular peak rate pressure rise and decline accompanied with an increased heart mass. Single cardiomyocytes isolated from the left ventricle revealed concentric hypertrophy, indicated by the increase in cellular width. Unloaded fractional cell shortening was significantly reduced in cells derived from the HMW-AGEs group and was associated with slower kinetics. Peak L-type Ca2+ current density was significantly decreased in the HMW-AGEs group. L-type Ca2+ channel availability was significantly shifted towards more negative potentials after HMW-AGEs injection. The impact of HMW-AGEs on myofilament function was measured in membrane-permeabilized cardiomyocytes showing a reduction in passive force, maximal Ca2+ activated force and rate of force development. Ultrastructural examination of cardiac tissue demonstrated adverse structural remodeling in HMW-AGEs group characterized by a disruption of the cyto-architecture, a decreased mitochondrial density and altered mitochondrial function. Conclusion: Our data indicate that HMW-AGEs induce structural and functional cellular remodeling via a different working mechanism as the well-known LMW-AGEs. Results of our research open the door for new strategies targeting HMW-AGEs to improve cardiac outcome

PP2A inactivation mediated by PPP2R4 haploinsufficiency promotes cancer development

Protein phosphatase 2A (PP2A) complexes counteract many oncogenic kinase pathways. In cancer cells, PP2A function can be compromised by several mechanisms, including sporadic mutations in its scaffolding A and regulatory B subunits or more frequently through overexpression of cellular PP2A inhibitors. Here we identify a novel genetic mechanism by which PP2A function is recurrently affected in human cancer, involving haploinsufficiency of PPP2R4, a gene encoding the cellular PP2A activator PTPA. Notably, up to 70% of cancer patients showed a heterozygous deletion or missense mutations in PPP2R4. Cancer-associated PTPA mutants exhibited decreased abilities to bind the PP2A-C subunit or activate PP2A and failed to reverse the tumorigenic phenotype induced by PTPA suppression, indicating they function as null alleles. In Ppp2r4 gene-trapped (gt) mice showing residual PTPA expression, total PP2A activity and methylation were reduced, selectively affecting specific PP2A holoenzymes. Both PTPAgt/gt and PTPA+/gt mice showed higher rates of spontaneous tumors, mainly hematologic malignancies and hepatocellular adenomas and carcinomas. These tumors exhibited increased c-Myc phosphorylation and increased Wnt or Hedgehog signaling. We observed a significant reduction in lifespan in PTPA+/gt mice compared to wildtype mice. Additionally, chemical induced skin carcinogenesis was accelerated in PTPA+/gt compared to wildtype mice. Our results provide evidence for PPP2R4 as a haploinsufficient tumor suppressor gene, defining a high-penetrance genetic mechanism for PP2A inhibition in human cancer.status: publishe