Substrate recruitment via eIF2γ enhances catalytic efficiency of a holophosphatase that terminates the integrated stress response.

Dephosphorylation of pSer51 of the α subunit of translation initiation factor 2 (eIF2αP) terminates signaling in the integrated stress response (ISR). A trimeric mammalian holophosphatase comprised of a protein phosphatase 1 (PP1) catalytic subunit, the conserved C-terminally located ~70 amino acid core of a substrate-specific regulatory subunit (PPP1R15A/GADD34 or PPP1R15B/CReP) and G-actin (an essential cofactor) efficiently dephosphorylate eIF2αP in vitro. Unlike their viral or invertebrate counterparts, with whom they share the conserved 70 residue core, the mammalian PPP1R15s are large proteins of more than 600 residues. Genetic and cellular observations point to a functional role for regions outside the conserved core of mammalian PPP1R15A in dephosphorylating its natural substrate, the eIF2 trimer. We have combined deep learning technology, all-atom molecular dynamics simulations, X-ray crystallography, and biochemistry to uncover binding of the γ subunit of eIF2 to a short helical peptide repeated four times in the functionally important N terminus of human PPP1R15A that extends past its conserved core. Binding entails insertion of Phe and Trp residues that project from one face of an α-helix formed by the conserved repeats of PPP1R15A into a hydrophobic groove exposed on the surface of eIF2γ in the eIF2 trimer. Replacing these conserved Phe and Trp residues with Ala compromises PPP1R15A function in cells and in vitro. These findings suggest mechanisms by which contacts between a distant subunit of eIF2 and elements of PPP1R15A distant to the holophosphatase active site contribute to dephosphorylation of eIF2αP by the core PPP1R15 holophosphatase and to efficient termination of the ISR in mammals.

A novel peptide PDHK1-241aa encoded by circPDHK1 promotes ccRCC progression via interacting with PPP1CA to inhibit AKT dephosphorylation and activate the AKT-mTOR signaling pathway.

BACKGROUND: Clear cell renal cell carcinoma (ccRCC) is the most prevalent kidney cancer with high aggressive phenotype and poor prognosis. Accumulating evidence suggests that circRNAs have been identified as pivotal mediators in cancers. However, the role of circRNAs in ccRCC progression remains elusive. METHODS: The differentially expressed circRNAs in 4 paired human ccRCC and adjacent noncancerous tissues ccRCC were screened using circRNA microarrays and the candidate target was selected based on circRNA expression level using weighted gene correlation network analysis (WGCNA) and the gene expression omnibus (GEO) database. CircPDHK1 expression in ccRCC and adjacent noncancerous tissues (n = 148) were evaluated along with clinically relevant information. RT-qPCR, RNase R digestion, and actinomycin D (ActD) stability test were conducted to identify the characteristics of circPDHK1. The subcellular distribution of circPDHK1 was analyzed by subcellular fractionation assay and fluorescence in situ hybridization (FISH). Immunoprecipitation-mass spectrometry (IP-MS) and immunofluorescence (IF) were employed to evaluate the protein-coding ability of circPDHK1. ccRCC cells were transfected with siRNAs, plasmids or lentivirus approach, and cell proliferation, migration and invasion, as well as tumorigenesis and metastasis in nude mice were assessed to clarify the functional roles of circPDHK1 and its encoded peptide PDHK1-241aa. RNA-sequencing, western blot analysis, immunoprecipitation (IP) and chromatin immunoprecipitation (ChIP) assays were further employed to identify the underlying mechanisms regulated by PDHK1-241aa. RESULTS: CircPDHK1 was upregulated in ccRCC tissues and closely related to WHO/ISUP stage, T stage, distant metastasis, VHL mutation and Ki-67 levels. CircPDHK1 had a functional internal ribosome entry site (IRES) and encoded a novel peptide PDHK1-241aa. Functionally, we confirmed that PDHK1-241aa and not the circPDHK1 promoted the proliferation, migration and invasion of ccRCC. Mechanistically, circPDHK1 was activated by HIF-2A at the transcriptional level. PDHK1-241aa was upregulated and interacted with PPP1CA, causing the relocation of PPP1CA to the nucleus. This thereby inhibited AKT dephosphorylation and activated the AKT-mTOR signaling pathway. CONCLUSIONS: Our data indicated that circPDHK1-encoded PDHK1-241aa promotes ccRCC progression by interacting with PPP1CA to inhibit AKT dephosphorylation. This study provides novel insights into the multiplicity of circRNAs and highlights the potential use of circPDHK1 or PDHK1-241aa as a therapeutic target for ccRCC.

Cell Cycle-Specific Protein Phosphatase 1 (PP1) Substrates Identification Using Genetically Modified Cell Lines.

The identification of protein phosphatase 1 (PP1) holoenzyme substrates has proven to be a challenging task. PP1 can form different holoenzyme complexes with a variety of regulatory subunits, and many of those are cell cycle regulated. Although several methods have been used to identify PP1 substrates, their cell cycle specificity is still an unmet need. Here, we present a new strategy to investigate PP1 substrates throughout the cell cycle using clustered regularly interspersed short palindromic repeats (CRISPR)-Cas9 genome editing and generate cell lines with endogenously tagged PP1 regulatory subunit (regulatory interactor of protein phosphatase one, RIPPO). RIPPOs are tagged with the auxin-inducible degron (AID) or ascorbate peroxidase 2 (APEX2) modules, and PP1 substrate identification is conducted by SILAC proteomic-based approaches. Proteins in close proximity to RIPPOs are first identified through mass spectrometry (MS) analyses using the APEX2 system; then a list of differentially phosphorylated proteins upon RIPPOs rapid degradation (achieved via the AID system) is compiled via SILAC phospho-mass spectrometry. The "in silico" overlap between the two proteomes will be enriched for PP1 putative substrates. Several methods including fluorescence resonance energy transfer (FRET), proximity ligation assays (PLA), and in vitro assays can be used as substrate validations approaches.

Protein phosphatase 1 regulatory subunit 15 A promotes translation initiation and induces G2M phase arrest during cuproptosis in cancers.

Copper ions play a crucial role as cofactors for essential enzymes in cellular processes. However, when the intracellular concentration of copper ions exceeds the homeostatic threshold, they become toxic to cells. In our study, we demonstrated that elesclomol, as a carrier of copper ions, caused an upregulation of protein phosphatase 1 regulatory subunit 15 A (PPP1R15A), which plays a role in regulating substrate selectivity of protein phosphatase 1 during cuproptosis. Mechanistically, we investigated that PPP1R15A activated translation initiation by dephosphorylating eukaryotic translation initiation factor 2 subunit alpha at the S51 residue through protein phosphatase 1 and phosphorylating eukaryotic translation initiation factor 4E binding protein 1 at the T70 residue. In addition, PPP1R15A reduced H3K4 methylation by altering the phosphorylation of histone methyltransferases, which led to the silencing of MYC and G2M phase arrest.

Human Betacoronavirus OC43 Interferes with the Integrated Stress Response Pathway in Infected Cells.

Viruses evolve many strategies to ensure the efficient synthesis of their proteins. One such strategy is the inhibition of the integrated stress response-the mechanism through which infected cells arrest translation through the phosphorylation of the alpha subunit of the eukaryotic translation initiation factor 2 (eIF2α). We have recently shown that the human common cold betacoronavirus OC43 actively inhibits eIF2α phosphorylation in response to sodium arsenite, a potent inducer of oxidative stress. In this work, we examined the modulation of integrated stress responses by OC43 and demonstrated that the negative feedback regulator of eIF2α phosphorylation GADD34 is strongly induced in infected cells. However, the upregulation of GADD34 expression induced by OC43 was independent from the activation of the integrated stress response and was not required for the inhibition of eIF2α phosphorylation in virus-infected cells. Our work reveals a complex interplay between the common cold coronavirus and the integrated stress response, in which efficient viral protein synthesis is ensured by the inhibition of eIF2α phosphorylation but the GADD34 negative feedback loop is disrupted.

Crosstalk of HDAC4, PP1, and GSDMD in controlling pyroptosis.

Gasdermin D (GSDMD) functions as a pivotal executor of pyroptosis, eliciting cytokine secretion following cleavage by inflammatory caspases. However, the role of posttranslational modifications (PTMs) in GSDMD-mediated pyroptosis remains largely unexplored. In this study, we demonstrate that GSDMD can undergo acetylation at the Lysine 248 residue, and this acetylation enhances pyroptosis. We identify histone deacetylase 4 (HDAC4) as the specific deacetylase responsible for mediating GSDMD deacetylation, leading to the inhibition of pyroptosis both in vitro and in vivo. Deacetylation of GSDMD impairs its ubiquitination, resulting in the inhibition of pyroptosis. Intriguingly, phosphorylation of HDAC4 emerges as a critical regulatory mechanism promoting its ability to deacetylate GSDMD and suppress GSDMD-mediated pyroptosis. Additionally, we implicate Protein phosphatase 1 (PP1) catalytic subunits (PP1α and PP1γ) in the dephosphorylation of HDAC4, thereby nullifying its deacetylase activity on GSDMD. This study reveals a complex regulatory network involving HDAC4, PP1, and GSDMD. These findings provide valuable insights into the interplay among acetylation, ubiquitination, and phosphorylation in the regulation of pyroptosis, offering potential targets for further investigation in the field of inflammatory cell death.

mTORC1 controls murine postprandial hepatic glycogen synthesis via Ppp1r3b.

In response to a meal, insulin drives hepatic glycogen synthesis to help regulate systemic glucose homeostasis. The mechanistic target of rapamycin complex 1 (mTORC1) is a well-established insulin target and contributes to the postprandial control of liver lipid metabolism, autophagy, and protein synthesis. However, its role in hepatic glucose metabolism is less understood. Here, we used metabolomics, isotope tracing, and mouse genetics to define a role for liver mTORC1 signaling in the control of postprandial glycolytic intermediates and glycogen deposition. We show that mTORC1 is required for glycogen synthase activity and glycogenesis. Mechanistically, hepatic mTORC1 activity promotes the feeding-dependent induction of Ppp1r3b, a gene encoding a phosphatase important for glycogen synthase activity whose polymorphisms are linked to human diabetes. Reexpression of Ppp1r3b in livers lacking mTORC1 signaling enhances glycogen synthase activity and restores postprandial glycogen content. mTORC1-dependent transcriptional control of Ppp1r3b is facilitated by FOXO1, a well characterized transcriptional regulator involved in the hepatic response to nutrient intake. Collectively, we identify a role for mTORC1 signaling in the transcriptional regulation of Ppp1r3b and the subsequent induction of postprandial hepatic glycogen synthesis.

Inhibition of PPP1R15A alleviates osteoporosis via suppressing RANKL-induced osteoclastogenesis.

Osteoporosis results from overactivation of osteoclasts. There are currently few drug options for treatment of this disease. Since the successful development of allosteric inhibitors, phosphatases have become attractive therapeutic targets. Protein phosphatase 1, regulatory subunit 15 A (PPP1R15A), is a stress-responsive protein, which promotes the UPR (unfolded protein response) and restores protein homeostasis. In this study we investigated the role of PPP1R15A in osteoporosis and osteoclastogenesis. Ovariectomy (OVX)-induced osteoporosis mouse model was established, osteoporosis was evaluated in the left femurs using micro-CT. RANKL-stimulated osteoclastogenesis was used as in vitro models. We showed that PPP1R15A expression was markedly increased in BMMs derived from OVX mice and during RANKL-induced osteoclastogenesis in vitro. Knockdown of PPP1R15A or application of Sephin1 (a PPP1R15A allosteric inhibitor in a phase II clinical trial) significantly inhibited osteoclastogenesis in vitro. Sephin1 (0.78, 3.125 and 12.5 µM) dose-dependently mitigated the changes in NF-κB, MAPK, and c-FOS and the subsequent nuclear factor of activated T cells 1 (NFATc1) translocation in RANKL-stimulated BMMs. Both Sephin1 and PPP1R15A knockdown increased the phosphorylated form of eukaryotic initiation factor 2α (eIF2α); knockdown of eIF2α reduced the inhibitory effects of Sephin1 on NFATc1-luc transcription and osteoclast formation. Furthermore, Sephin1 or PPP1R15A knockdown suppressed osteoclastogenesis in CD14+ monocytes from osteoporosis patients. In OVX mice, injection of Sephin1 (4, 8 mg/kg, i.p.) every two days for 6 weeks significantly inhibited bone loss, and restored bone destruction and decreased TRAP-positive cells. This study has identified PPP1R15A as a novel target for osteoclast differentiation, and genetic inhibition or allosteric inhibitors of PPP1R15A, such as Sephin1, can be used to treat osteoporosis. This study revealed that PPP1R15A expression was increased in osteoporosis in both human and mice. Inhibition of PPP1R15A by specific knockdown or an allosteric inhibitor Sephin1 mitigated murine osteoclast formation in vitro and attenuated ovariectomy-induced osteoporosis in vivo. PPP1R15A inhibition also suppressed pathogenic osteoclastogenesis in CD14+ monocytes from osteoporosis patients. These results identify PPP1R15A as a novel regulator of osteoclastogenesis and a valuable therapeutic target for osteoporosis.

Arginine vasopressin regulates the renal Na+-Cl- and Na+-K+-Cl- cotransporters through with-no-lysine kinase 4 and inhibitor 1 phosphorylation.

Vasopressin regulates water homeostasis via the V2 receptor in the kidney at least in part through protein kinase A (PKA) activation. Vasopressin, through an unknown pathway, upregulates the activity and phosphorylation of Na+-Cl- cotransporter (NCC) and Na+-K+-2Cl- cotransporter 2 (NKCC2) by Ste20-related proline/alanine-rich kinase (SPAK) and oxidative stress-responsive kinase 1 (OSR1), which are regulated by the with-no-lysine kinase (WNK) family. Phosphorylation of WNK4 at PKA consensus motifs may be involved. Inhibitor 1 (I1), a protein phosphatase 1 (PP1) inhibitor, may also play a role. In human embryonic kidney (HEK)-293 cells, we assessed the phosphorylation of WNK4, SPAK, NCC, or NKCC2 in response to forskolin or desmopressin. WNK4 and cotransporter phosphorylation were studied in desmopressin-infused WNK4-/- mice and in tubule suspensions. In HEK-293 cells, only wild-type WNK4 but not WNK1, WNK3, or a WNK4 mutant lacking PKA phosphorylation motifs could upregulate SPAK or cotransporter phosphorylation in response to forskolin or desmopressin. I1 transfection maximized SPAK phosphorylation in response to forskolin in the presence of WNK4 but not of mutant WNK4 lacking PP1 regulation. We observed direct PP1 regulation of NKCC2 dephosphorylation but not of NCC or SPAK in the absence of WNK4. WNK4-/- mice with desmopressin treatment did not increase SPAK/OSR1, NCC, or NKCC2 phosphorylation. In stimulated tubule suspensions from WNK4-/- mice, upregulation of pNKCC2 was reduced, whereas upregulation of SPAK phosphorylation was absent. These findings suggest that WNK4 is a central node in which kinase and phosphatase signaling converge to connect cAMP signaling to the SPAK/OSR1-NCC/NKCC2 pathway.NEW & NOTEWORTHY With-no-lysine kinases regulate the phosphorylation and activity of the Na+-Cl- and Na+-K+-2Cl- cotransporters. This pathway is modulated by arginine vasopressin (AVP). However, the link between AVP and WNK signaling remains unknown. Here, we show that AVP activates WNK4 through increased phosphorylation at putative protein kinase A-regulated sites and decreases its dephosphorylation by protein phosphatase 1. This work increases our understanding of the signaling pathways mediating AVP actions in the kidney.

Recruitment of trimeric eIF2 by phosphatase non-catalytic subunit PPP1R15B.

Regulated protein phosphorylation controls most cellular processes. The protein phosphatase PP1 is the catalytic subunit of many holoenzymes that dephosphorylate serine/threonine residues. How these enzymes recruit their substrates is largely unknown. Here, we integrated diverse approaches to elucidate how the PP1 non-catalytic subunit PPP1R15B (R15B) captures its full trimeric eIF2 substrate. We found that the substrate-recruitment module of R15B is largely disordered with three short helical elements, H1, H2, and H3. H1 and H2 form a clamp that grasps the substrate in a region remote from the phosphorylated residue. A homozygous N423D variant, adjacent to H1, reducing substrate binding and dephosphorylation was discovered in a rare syndrome with microcephaly, developmental delay, and intellectual disability. These findings explain how R15B captures its 125 kDa substrate by binding the far end of the complex relative to the phosphosite to present it for dephosphorylation by PP1, a paradigm of broad relevance.

CircGPRC5A enhances colorectal cancer progress by stabilizing PPP1CA and inducing YAP dephosphorylation.

BACKGROUND: With the advancements in bioinformatic technology, an increasing number of circular RNAs (circRNAs) have been discovered and their crucial roles in the development and progression of various malignancies have been confirmed through multiple pathways. However, the specific mechanisms involving protein-binding circRNAs in colorectal cancer (CRC) remain largely unexplored. METHODS: Differential circRNA expression was assessed using a human circRNA microarray in five CRC tissue and paired normal samples. CircGPRC5A expression was then confirmed in the CRC tissues and paired normal samples using qRT-PCR. The biological function of circGPRC5A in CRC were studied in vitro and in vivo. Western blotting, fluorescence in situ hybridization, immunofluorescence, RNA pulldown, mass spectrometry, immunoprecipitation, quantitative phosphoproteomics, and RNA-binding protein immunoprecipitation assays were used to study circGPRC5A. RESULTS: Our analysis revealed that circGPRC5A expression was higher in CRC tissues compared to normal tissues and was associated with tumor size, tumor stage and lymph node status. CircGPRC5A promoted CRC cell proliferation, migration, and metastasis in vitro and in vivo. CircGPRC5A could stabilize PPP1CA protein by inhibiting the binding between UBA1 and PPP1CA, and increasing YAP dephosphorylation. CONCLUSIONS: Our study revealed that circGPRC5A plays an essential function in CRC progression by stabilizing PPP1CA protein and enhancing YAP dephosphorylation. CircGPRC5A could act as a novel and potential target for CRC.

TIMAP, a Regulatory Subunit of Protein Phosphatase 1, Inhibits In Vitro Neuronal Differentiation.

TIMAP (TGF-ß-inhibited membrane associated protein) is abundant in endothelial cells, and it has been regarded as a member of the myosin phosphatase targeting protein (MYPT) family. Our workgroup previously identified several interacting protein partners of TIMAP and proved its regulatory subunit role for protein phosphatase 1 catalytic subunit (PP1c). TIMAP is also expressed in neuronal cells, but details of its function have not been studied yet. Therefore, we aimed to explore the role of TIMAP in neuronal cells, especially during differentiation. Expression of TIMAP was proved both at mRNA and protein levels in SH-SY5Y human neuroblastoma cells. Differentiation of SH-SY5Y cells was optimized and proved by the detection of neuronal differentiation markers, such as ß3-tubulin, nestin and inhibitor of differentiation 1 (ID1) using qPCR and Western blot. We found downregulation of TIMAP during differentiation. In accordance with this, overexpression of recombinant TIMAP attenuated the differentiation of neuronal cells. Moreover, the subcellular localization of TIMAP has changed during differentiation as it translocated from the plasma membrane into the nucleus. The nuclear interactome of TIMAP revealed more than 50 proteins, offering the possibility to further investigate the role of TIMAP in several key physiological pathways of neuronal cells.

The PPP1R15 Family of eIF2-alpha Phosphatase Targeting Subunits (GADD34 and CReP).

The vertebrate PPP1R15 family consists of the proteins GADD34 (growth arrest and DNA damage-inducible protein 34, the product of the PPP1R15A gene) and CReP (constitutive repressor of eIF2α phosphorylation, the product of the PPP1R15B gene), both of which function as targeting/regulatory subunits for protein phosphatase 1 (PP1) by regulating subcellular localization, modulating substrate specificity and assembling complexes with target proteins. The primary cellular function of these proteins is to facilitate the dephosphorylation of eukaryotic initiation factor 2-alpha (eIF2α) by PP1 during cell stress. In this review, we will provide a comprehensive overview of the cellular function, biochemistry and pharmacology of GADD34 and CReP, starting with a brief introduction of eIF2α phosphorylation via the integrated protein response (ISR). We discuss the roles GADD34 and CReP play as feedback inhibitors of the unfolded protein response (UPR) and highlight the critical function they serve as inhibitors of the PERK-dependent branch, which is particularly important since it can mediate cell survival or cell death, depending on how long the stressful stimuli lasts, and GADD34 and CReP play key roles in fine-tuning this cellular decision. We briefly discuss the roles of GADD34 and CReP homologs in model systems and then focus on what we have learned about their function from knockout mice and human patients, followed by a brief review of several diseases in which GADD34 and CReP have been implicated, including cancer, diabetes and especially neurodegenerative disease. Because of the potential importance of GADD34 and CReP in aspects of human health and disease, we will discuss several pharmacological inhibitors of GADD34 and/or CReP that show promise as treatments and the controversies as to their mechanism of action. This review will finish with a discussion of the biochemical properties of GADD34 and CReP, their regulation and the additional interacting partners that may provide insight into the roles these proteins may play in other cellular pathways. We will conclude with a brief outline of critical areas for future study.

Protein phosphatase 1 regulates core PCP signaling.

Planar cell polarity (PCP) signaling polarizes epithelial cells within the plane of an epithelium. Core PCP signaling components adopt asymmetric subcellular localizations within cells to both polarize and coordinate polarity between cells. Achieving subcellular asymmetry requires additional effectors, including some mediating post-translational modifications of core components. Identification of such proteins is challenging due to pleiotropy. We used mass spectrometry-based proximity labeling proteomics to identify such regulators in the Drosophila wing. We identified the catalytic subunit of protein phosphatase1, Pp1-87B, and show that it regulates core protein polarization. Pp1-87B interacts with the core protein Van Gogh and at least one serine/threonine kinase, Dco/CKIε, that is known to regulate PCP. Pp1-87B modulates Van Gogh subcellular localization and directs its dephosphorylation in vivo. PNUTS, a Pp1 regulatory subunit, also modulates PCP. While the direct substrate(s) of Pp1-87B in control of PCP is not known, our data support the model that cycling between phosphorylated and unphosphorylated forms of one or more core PCP components may regulate acquisition of asymmetry. Finally, our screen serves as a resource for identifying additional regulators of PCP signaling.

Leishmania PNUTS discriminates between PP1 catalytic subunits through an RVxF-ΦΦ-F motif and polymorphisms in the PP1 C-tail and catalytic domain.

Phosphoprotein phosphatase 1 (PP1) associates with specific regulatory subunits to achieve, among other functions, substrate selectivity. Among the eight PP1 isotypes in Leishmania, PP1-8e associates with the regulatory protein PNUTS along with the structural factors JBP3 and Wdr82 in the PJW/PP1 complex that modulates RNA polymerase II (pol II) phosphorylation and transcription termination. Little is known regarding interactions involved in PJW/PP1 complex formation, including how PP1-8e is the selective isotype associated with PNUTS. Here, we show that PNUTS uses an established RVxF-ΦΦ-F motif to bind the PP1 catalytic domain with similar interfacial interactions as mammalian PP1-PNUTS and noncanonical motifs. These atypical interactions involve residues within the PP1-8e catalytic domain and N and C terminus for isoform-specific regulator binding. This work advances our understanding of PP1 isoform selectivity and reveals key roles of PP1 residues in regulator binding. We also explore the role of PNUTS as a scaffold protein for the complex by identifying the C-terminal region involved in binding JBP3 and Wdr82 and impact of PNUTS on the stability of complex components and function in pol II transcription in vivo. Taken together, these studies provide a potential mechanism where multiple motifs within PNUTS are used combinatorially to tune binding affinity to PP1, and the C terminus for JBP3 and Wdr82 association, in the Leishmania PJW/PP1 complex. Overall, our data provide insights in the formation of the PJW/PP1 complex involved in regulating pol II transcription in divergent protozoans where little is understood.

Opposing Roles for the α Isoform of the Catalytic Subunit of Protein Phosphatase 1 in Inside-Out and Outside-In Integrin Signaling in Murine Platelets.

Platelet activation during hemostasis and thrombosis is facilitated by agonist-induced inside-out and integrin αIIbß3-initiated outside-in signaling via protein kinases and phosphatases. Pharmacological inhibitor studies suggest that the serine/threonine protein phosphatase 1 (PP1) promotes platelet activation. However, since phosphatase inhibitors block all the isoforms of the catalytic subunit of PP1 (PP1c), the role of specific PP1c isoform in platelet signaling remains unclear. Here, we employed a platelet-specific PP1cα-/- mice to explore the contribution of a major PP1 isoform in platelet functions. Loss of PP1cα moderately decreased activation of integrin αIIbß3, binding of soluble fibrinogen, and aggregation to low-dose thrombin, ADP, and collagen. In contrast, PP1cα-/- platelets displayed increased adhesion to immobilized fibrinogen, fibrin clot retraction, and thrombus formation on immobilized collagen. Mechanistically, post-fibrinogen engagement potentiated p38 mitogen-activated protein kinase (MAPK) activation in PP1cα-/- platelets and the p38 inhibitor blocked the increased integrin-mediated outside-in signaling function. Tail bleeding time and light-dye injury-induced microvascular thrombosis in the cremaster venules and arterioles were not altered in PP1cα-/- mice. Thus, PP1cα displays pleiotropic signaling in platelets as it amplifies agonist-induced signaling and attenuates integrin-mediated signaling with no impact on hemostasis and thrombosis.

PPP1R12C Promotes Atrial Hypocontractility in Atrial Fibrillation.

BACKGROUND: Atrial fibrillation (AF)-the most common sustained cardiac arrhythmia-increases thromboembolic stroke risk 5-fold. Although atrial hypocontractility contributes to stroke risk in AF, the molecular mechanisms reducing myofilament contractile function remain unknown. We tested the hypothesis that increased expression of PPP1R12C (protein phosphatase 1 regulatory subunit 12C)-the PP1 (protein phosphatase 1) regulatory subunit targeting MLC2a (atrial myosin light chain 2)-causes hypophosphorylation of MLC2a and results in atrial hypocontractility. METHODS: Right atrial appendage tissues were isolated from human patients with AF versus sinus rhythm controls. Western blots, coimmunoprecipitation, and phosphorylation studies were performed to examine how the PP1c (PP1 catalytic subunit)-PPP1R12C interaction causes MLC2a dephosphorylation. In vitro studies of pharmacological MRCK (myotonic dystrophy kinase-related Cdc42-binding kinase) inhibitor (BDP5290) in atrial HL-1 cells were performed to evaluate PP1 holoenzyme activity on MLC2a. Cardiac-specific lentiviral PPP1R12C overexpression was performed in mice to evaluate atrial remodeling with atrial cell shortening assays, echocardiography, and AF inducibility with electrophysiology studies. RESULTS: In human patients with AF, PPP1R12C expression was increased 2-fold versus sinus rhythm controls (P=2.0×10-2; n=12 and 12 in each group) with >40% reduction in MLC2a phosphorylation (P=1.4×10-6; n=12 and 12 in each group). PPP1R12C-PP1c binding and PPP1R12C-MLC2a binding were significantly increased in AF (P=2.9×10-2 and 6.7×10-3, respectively; n=8 and 8 in each group). In vitro studies utilizing drug BDP5290, which inhibits T560-PPP1R12C phosphorylation, demonstrated increased PPP1R12C binding with both PP1c and MLC2a and dephosphorylation of MLC2a. Mice treated with lentiviral PPP1R12C vector demonstrated a 150% increase in left atrial size versus controls (P=5.0×10-6; n=12, 8, and 12), with reduced atrial strain and atrial ejection fraction. Pacing-induced AF in mice treated with lentiviral PPP1R12C vector was significantly higher than in controls (P=1.8×10-2 and 4.1×10-2, respectively; n=6, 6, and 5). CONCLUSIONS: Patients with AF exhibit increased levels of PPP1R12C protein compared with controls. PPP1R12C overexpression in mice increases PP1c targeting to MLC2a and causes MLC2a dephosphorylation, which reduces atrial contractility and increases AF inducibility. These findings suggest that PP1 regulation of sarcomere function at MLC2a is a key determinant of atrial contractility in AF.

A split-luciferase lysate-based approach to identify small-molecule modulators of phosphatase subunit interactions.

An emerging strategy for the therapeutic targeting of protein phosphatases involves the use of compounds that interfere with the binding of regulatory subunits or substrates. However, high-throughput screening strategies for such interfering molecules are scarce. Here, we report on the conversion of the NanoBiT split-luciferase system into a robust assay for the quantification of phosphatase subunit and substrate interactions in cell lysates. The assay is suitable to screen small-molecule libraries for interfering compounds. We designed and validated split-luciferase sensors for a broad range of PP1 and PP2A holoenzymes, including sensors that selectively report on weak interaction sites. To facilitate efficient hit triaging in large-scale screening campaigns, deselection procedures were developed to eliminate assay-interfering molecules with high fidelity. As a proof-of-principle, we successfully applied the split-luciferase screening tool to identify small-molecule disruptors of the interaction between the C-terminus of PP1ß and the ankyrin-repeat domain of the myosin-phosphatase targeting subunit MYPT1.

FAT10 differentially stabilizes MYPT2 isoforms.

Myosin phosphatase (MP) is an enzyme complex that regulates muscle contraction and plays important roles in various physiological and pathological conditions. Myosin phosphatase targeting subunit (MYPT) 2, a subunit of MP, interacts with protein phosphatase 1c to regulate its phosphatase activity. MYPT2 exists in various isoforms that differ in the composition of essential motifs that contribute to its function. However, regulatory mechanisms underlying these isoforms are poorly understood. Human leukocyte antigen-F adjacent transcript 10 (FAT10) is a ubiquitin-like modifier that not only targets proteins for proteasomal degradation but also stabilizes its interacting proteins. In this study, we investigated the effect of the interaction between FAT10 and MYPT2 isoform a (the canonical full-length form of MYPT2) or MYPT2 isoform f (the natural truncated form of MYPT2). FAT10 interacted with both MYPT2 isoforms a and f; however, only MYPT2 isoform f was increased by FAT10, whereas MYPT2 isoform a remained unaffected by FAT10. We further confirmed that, in contrast to MYPT2 isoform a, MYPT2 isoform f undergoes rapid degradation via the ubiquitin-proteasome pathway and that FAT10 stabilizes MYPT2 isoform f by inhibiting its ubiquitination. Therefore, our findings suggest that the interaction between FAT10 and MYPT2 isoforms leads to distinct stabilization effects on each isoform, potentially modulating MP activity.

The protein phosphatase PC1 dephosphorylates and deactivates CatC to negatively regulate H2O2 homeostasis and salt tolerance in rice.

Catalase (CAT) is often phosphorylated and activated by protein kinases to maintain hydrogen peroxide (H2O2) homeostasis and protect cells against stresses, but whether and how CAT is switched off by protein phosphatases remains inconclusive. Here, we identified a manganese (Mn2+)-dependent protein phosphatase, which we named PHOSPHATASE OF CATALASE 1 (PC1), from rice (Oryza sativa L.) that negatively regulates salt and oxidative stress tolerance. PC1 specifically dephosphorylates CatC at Ser-9 to inhibit its tetramerization and thus activity in the peroxisome. PC1 overexpressing lines exhibited hypersensitivity to salt and oxidative stresses with a lower phospho-serine level of CATs. Phosphatase activity and seminal root growth assays indicated that PC1 promotes growth and plays a vital role during the transition from salt stress to normal growth conditions. Our findings demonstrate that PC1 acts as a molecular switch to dephosphorylate and deactivate CatC and negatively regulate H2O2 homeostasis and salt tolerance in rice. Moreover, knockout of PC1 not only improved H2O2-scavenging capacity and salt tolerance but also limited rice grain yield loss under salt stress conditions. Together, these results shed light on the mechanisms that switch off CAT and provide a strategy for breeding highly salt-tolerant rice.