RESUMO
FAT1, which encodes a protocadherin, is one of the most frequently mutated genes in human cancers1-5. However, the role and the molecular mechanisms by which FAT1 mutations control tumour initiation and progression are poorly understood. Here, using mouse models of skin squamous cell carcinoma and lung tumours, we found that deletion of Fat1 accelerates tumour initiation and malignant progression and promotes a hybrid epithelial-to-mesenchymal transition (EMT) phenotype. We also found this hybrid EMT state in FAT1-mutated human squamous cell carcinomas. Skin squamous cell carcinomas in which Fat1 was deleted presented increased tumour stemness and spontaneous metastasis. We performed transcriptional and chromatin profiling combined with proteomic analyses and mechanistic studies, which revealed that loss of function of FAT1 activates a CAMK2-CD44-SRC axis that promotes YAP1 nuclear translocation and ZEB1 expression that stimulates the mesenchymal state. This loss of function also inactivates EZH2, promoting SOX2 expression, which sustains the epithelial state. Our comprehensive analysis identified drug resistance and vulnerabilities in FAT1-deficient tumours, which have important implications for cancer therapy. Our studies reveal that, in mouse and human squamous cell carcinoma, loss of function of FAT1 promotes tumour initiation, progression, invasiveness, stemness and metastasis through the induction of a hybrid EMT state.
Assuntos
Caderinas/deficiência , Transição Epitelial-Mesenquimal/genética , Deleção de Genes , Metástase Neoplásica/genética , Neoplasias/genética , Neoplasias/patologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Caderinas/genética , Caderinas/metabolismo , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Progressão da Doença , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica , Humanos , Receptores de Hialuronatos/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Mesoderma/metabolismo , Mesoderma/patologia , Camundongos , Metástase Neoplásica/tratamento farmacológico , Neoplasias/tratamento farmacológico , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Fenótipo , Fosfoproteínas/análise , Fosfoproteínas/metabolismo , Proteômica , Fatores de Transcrição SOXB1/metabolismo , Transdução de Sinais , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia , Fatores de Transcrição/metabolismo , Proteínas de Sinalização YAP , Homeobox 1 de Ligação a E-box em Dedo de Zinco/metabolismo , Quinases da Família src/metabolismoRESUMO
BACKGROUND: Variants in PPP2R5D, affecting the regulatory B56δ subunit of protein phosphatase 2A (PP2A), have been identified in individuals with neurodevelopmental abnormalities. However, the molecular and clinical spectra remain incompletely understood. METHODS: Individuals with PPP2R5D variants were enrolled through Simons Variation in Individuals Project/Simons Searchlight. Data were collected from medical history interviews, medical record review, online validated instruments and neuroimaging review. Genetic variants were biochemically characterised. RESULTS: We studied 76 individuals with PPP2R5D variants, including 68 with pathogenic de novo variants, four with a variant of uncertain significance (VUS) and four siblings with a novel dominantly inherited pathogenic variant. Among 13 pathogenic variants, eight were novel and two (p.Glu198Lys and p.Glu200Lys) were highly recurrent. Functional analysis revealed impaired PP2A A/C-subunit binding, decreased short linear interaction motif-dependent substrate binding or both-with the most severe phenotypes associated with variants that completely retained one of these binding characteristics and lost the other-further supporting a dominant-negative disease mechanism. p.Glu198Lys showed the highest C-binding defect and a more severe clinical phenotype. The inherited p.Glu197Gly variant had a mild substrate binding defect, and three of four VUS had no biochemical impact. Common clinical phenotypes were language, intellectual or learning disabilities (80.6%), hypotonia (75.0%), macrocephaly (66.7%), seizures (45.8%) and autism spectrum disorder (26.4%). The mean composite Vineland score was 59.8, and most participants were in the 'moderate to low' and 'low' adaptive levels in all domains. CONCLUSION: Our study delineates the most common features of PPP2R5D-related neurodevelopmental disorders, expands the clinical and molecular spectrum and identifies genotype-phenotype correlations.
Assuntos
Deficiência Intelectual , Transtornos do Neurodesenvolvimento , Humanos , Transtorno do Espectro Autista/genética , Genótipo , Deficiência Intelectual/genética , Deficiência Intelectual/patologia , Transtornos do Neurodesenvolvimento/diagnóstico , Transtornos do Neurodesenvolvimento/genética , Fenótipo , Proteína Fosfatase 2/genéticaRESUMO
BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) has a low survival, its incidence is rising and little therapeutic improvements are expected in the near future. It has been observed that Epithelial-to-Mesenchymal transition (EMT) contributes (including in PDAC) to a more aggressive cancer phenotype. Additionally, largely unexplored, studies indicate a mechanistic interplay between Protein Phosphatase Type 2A (PP2A) enzymes and EMT that could offer treatment opportunities. The aim was to investigate the relation of a PP2A expression signature (encompassing all PP2A subunits, endogenous inhibitors and activators) with EMT and aggressive pancreatic cancer, and to discuss possible implications. METHODS: We retrieved different PDAC expression datasets from NCBI to capture the variation in patients, and analyzed these using datamining, survival analysis, differential gene and protein expression. We determined genes highly associated with aggressive PDAC. For in vitro evaluation, Panc-1 cells were treated with the pharmacologic PP2A inhibitor Okadaic Acid (OA). Additionally, two OA-resistant Panc-1 clones were developed and characterized. RESULTS: In patients, there is a strong correlation between EMT and aggressive PDAC, and between aggressive PDAC and PP2A, with a significant upregulation of PP2A inhibitor genes. Several PP2A genes significantly correlated with decreased survival. In vitro, short-term exposure to OA induced EMT in Panc-1 cells. This shift towards EMT was further pronounced in the OA-resistant Panc-1 clones, morphologically and by pathway analysis. Proteomic analysis and gene sequencing showed that the advanced OA-resistant model most resembles the clinical PDAC presentation (with EMT signature, and with several specific PP2A genes upregulated, and others downregulated). CONCLUSIONS: We demonstrated a strong association between EMT, altered PP2A expression and aggressive PDAC in patients. Also, in vitro, PP2A inhibition induces EMT. Overall, statistics suggests the mechanistic importance of PP2A dysregulation for PDAC progression. Translationally, our observations indicate that pharmacologic restoration of PP2A activity could be an attractive therapeutic strategy to block or reverse progression.
Assuntos
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Humanos , Proteômica , Proliferação de Células/genética , Neoplasias Pancreáticas/patologia , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patologia , Transição Epitelial-Mesenquimal/genética , Fosfoproteínas Fosfatases/genética , Fosfoproteínas Fosfatases/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Regulação Neoplásica da Expressão Gênica , Neoplasias PancreáticasRESUMO
Type 2A protein phosphatases (PP2As) are highly expressed in the brain and regulate neuronal signaling by catalyzing phospho-Ser/Thr dephosphorylations in diverse substrates. PP2A holoenzymes comprise catalytic C-, scaffolding A-, and regulatory B-type subunits, which determine substrate specificity and physiological function. Interestingly, de novo mutations in genes encoding A- and B-type subunits have recently been implicated in intellectual disability (ID) and developmental delay (DD). We now report 16 individuals with mild to profound ID and DD and a de novo mutation in PPP2CA, encoding the catalytic Cα subunit. Other frequently observed features were severe language delay (71%), hypotonia (69%), epilepsy (63%), and brain abnormalities such as ventriculomegaly and a small corpus callosum (67%). Behavioral problems, including autism spectrum disorders, were reported in 47% of individuals, and three individuals had a congenital heart defect. PPP2CA de novo mutations included a partial gene deletion, a frameshift, three nonsense mutations, a single amino acid duplication, a recurrent mutation, and eight non-recurrent missense mutations. Functional studies showed complete PP2A dysfunction in four individuals with seemingly milder ID, hinting at haploinsufficiency. Ten other individuals showed mutation-specific biochemical distortions, including poor expression, altered binding to the A subunit and specific B-type subunits, and impaired phosphatase activity and C-terminal methylation. Four were suspected to have a dominant-negative mechanism, which correlated with severe ID. Two missense variants affecting the same residue largely behaved as wild-type in our functional assays. Overall, we found that pathogenic PPP2CA variants impair PP2A-B56(δ) functionality, suggesting that PP2A-related neurodevelopmental disorders constitute functionally converging ID syndromes.
Assuntos
Deficiência Intelectual/genética , Mutação , Proteína Fosfatase 2/genética , Adolescente , Criança , Pré-Escolar , Análise Mutacional de DNA , Feminino , Células HEK293 , Haploinsuficiência/genética , Humanos , Masculino , Ligação Proteica/genética , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , SíndromeRESUMO
By removing Ser/Thr-specific phosphorylations in a multitude of protein substrates in diverse tissues, Protein Phosphatase type 2A (PP2A) enzymes play essential regulatory roles in cellular signalling and physiology, including in brain function and development. Here, we review current knowledge on PP2A gene mutations causally involved in neurodevelopmental disorders and intellectual disability, focusing on PPP2CA, PPP2R1A and PPP2R5D. We provide insights into the impact of these mutations on PP2A structure, substrate specificity and potential function in neurobiology and brain development.
Assuntos
Encéfalo/fisiologia , Deficiência Intelectual/genética , Isoenzimas/genética , Mutação , Transtornos do Neurodesenvolvimento/genética , Proteína Fosfatase 2/genética , Animais , Encéfalo/crescimento & desenvolvimento , Humanos , Deficiência Intelectual/enzimologia , Isoenzimas/metabolismo , Camundongos , Transtornos do Neurodesenvolvimento/enzimologia , Proteína Fosfatase 2/metabolismo , Especificidade por SubstratoRESUMO
We herein report the design, synthesis, and functional impact of an okadaic acid (OA) small analogue, ITH12680, which restores the activity of phosphoprotein phosphatase 2A (PP2A), whose deficient activity has been implicated in nicotine-mediated tumor progression and chemoresistance in non-small cell lung cancer (NSCLC). For its design, we paid attention to the structure of the PP2A-OA complex, where the C16-C38 OA fragment confers PP2A affinity and selectivity, but it is not involved in the inhibitory effect. Confirming this hypothesis, PP2A activity was not inhibited by ITH12680. By contrast, the compound partially restored OA-exerted PP2A inhibition in vitro. Moreover, flow cytometry and immunoblotting experiments revealed that ITH12680 reversed nicotine-induced cisplatin resistance in NSCLC cells, as it prevented nicotine-induced reduction of Bax expression and inhibited nicotine-mediated activation of cell survival and proliferation kinases, Akt and ERK1/2. Our findings suggest that the rescue of nicotine-inhibited PP2A activity could diminish the resistance to cisplatin treatment observed in NSCLC patients who continue smoking.
Assuntos
Antineoplásicos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Cisplatino/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Neoplasias Pulmonares/tratamento farmacológico , Ácido Okadáico/farmacologia , Proteína Fosfatase 2/metabolismo , Células A549 , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Ativação Enzimática/efeitos dos fármacos , Humanos , Neoplasias Pulmonares/metabolismo , Modelos Moleculares , Simulação de Acoplamento Molecular , Nicotina/efeitos adversos , Ácido Okadáico/análogos & derivadosRESUMO
Here, we identify the LIM protein lipoma-preferred partner (LPP) as a binding partner of a specific protein phosphatase 2A (PP2A) heterotrimer that is characterised by the regulatory PR130/Bâ³α1 subunit (encoded by PPP2R3A). The PR130 subunit interacts with the LIM domains of LPP through a conserved Zn²âº-finger-like motif in the differentially spliced N-terminus of PR130. Isolated LPP-associated PP2A complexes are catalytically active. PR130 colocalises with LPP at multiple locations within cells, including focal contacts, but is specifically excluded from mature focal adhesions, where LPP is still present. An LPP-PR130 fusion protein only localises to focal adhesions upon deletion of the domain of PR130 that binds to the PP2A catalytic subunit (PP2A/C), suggesting that PR130-LPP complex formation is dynamic and that permanent recruitment of PP2A activity might be unfavourable for focal adhesion maturation. Accordingly, siRNA-mediated knockdown of PR130 increases adhesion of HT1080 fibrosarcoma cells onto collagen I and decreases their migration in scratch wound and Transwell assays. Complex formation with LPP is mandatory for these PR130-PP2A functions, as neither phenotype can be rescued by re-expression of a PR130 mutant that no longer binds to LPP. Our data highlight the importance of specific, locally recruited PP2A complexes in cell adhesion and migration dynamics.
Assuntos
Proteínas do Citoesqueleto/metabolismo , Proteínas com Domínio LIM/metabolismo , Proteína Fosfatase 2/metabolismo , Domínio Catalítico/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Adesões Focais/genética , Humanos , Ligação Proteica , Proteína Fosfatase 2/genética , RNA Interferente Pequeno/genéticaRESUMO
Phosphoinositides, particularly phosphatidylinositol (3,4,5)-trisphosphate [PI(3,4,5)P3] and phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2], are recognized by SHIP2 (also known as INPPL1) a member of the inositol polyphosphate 5-phosphatase family. SHIP2 dephosphorylates PI(3,4,5)P3 to form PI(3,4)P2; the latter interacts with specific target proteins (e.g. lamellipodin). Although the preferred SHIP2 substrate is PI(3,4,5)P3, PI(4,5)P2 can also be dephosphorylated by this enzyme to phosphatidylinositol 4-phosphate (PI4P). Through depletion of SHIP2 in the glioblastoma cell line 1321 N1, we show that SHIP2 inhibits cell migration. In different glioblastoma cell lines and primary cultures, SHIP2 staining at the plasma membrane partly overlaps with PI(4,5)P2 immunoreactivity. PI(4,5)P2 was upregulated in SHIP2-deficient N1 cells as compared to control cells; in contrast, PI4P was very much decreased in SHIP2-deficient cells. Therefore, SHIP2 controls both PI(3,4,5)P3 and PI(4,5)P2 levels in intact cells. In 1321 N1 cells, the PI(4,5)P2-binding protein myosin-1c was identified as a new interactor of SHIP2. Regulation of PI(4,5)P2 and PI4P content by SHIP2 controls 1321 N1 cell migration through the organization of focal adhesions. Thus, our results reveal a new role of SHIP2 in the control of PI(4,5)P2, PI4P and cell migration in PTEN-deficient glioblastoma 1321 N1 cells.
Assuntos
Membrana Celular/metabolismo , Movimento Celular , Glioblastoma/enzimologia , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/metabolismo , Linhagem Celular Tumoral , Membrana Celular/genética , Adesões Focais/genética , Adesões Focais/metabolismo , Glioblastoma/genética , Glioblastoma/metabolismo , Glioblastoma/fisiopatologia , Humanos , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/genéticaRESUMO
The mammalian target of rapamycin (mTOR) pathway is activated by a variety of stimuli, including nutrients such as glucose and amino acids. The Ste20 family kinase MAP4K3 is regulated by amino acids and acts upstream of mTORC1. Here we investigate how MAP4K3 activity is regulated by amino acid sufficiency. We identify a transautophosphorylation site in the MAP4K3 kinase activation segment (Ser170) that is required for MAP4K3 activity and its activation of mTORC1 signaling. Following amino acid withdrawal, Ser170 is dephosphorylated via PP2A complexed to PR61 epsilon, a PP2A-targeting subunit. Inhibition of PR61 epsilon expression prevents MAP4K3 Ser170 dephosphorylation and impairs mTORC1 inhibition during amino acid withdrawal. We propose that during amino acid sufficiency Ser170-phosphorylated MAP4K3 activates mTORC1, but that upon amino acid restriction MAP4K3 preferentially interacts with PP2A(T61 epsilon), promoting dephosphorylation of Ser170, MAP4K3 inhibition, and, subsequently, inhibition of mTORC1 signaling.
Assuntos
Aminoácidos/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteína Fosfatase 2/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Proteínas Adaptadoras de Transdução de Sinal , Aminoácidos/deficiência , Linhagem Celular , Ativação Enzimática , Humanos , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Mutação , Fosforilação , Ligação Proteica , Proteína Fosfatase 2/genética , Proteínas Serina-Treonina Quinases/genética , Subunidades Proteicas , Proteínas/metabolismo , Interferência de RNA , Proteína Regulatória Associada a mTOR , Serina-Treonina Quinases TOR , TransfecçãoRESUMO
Type II endometrial carcinomas (ECs) are responsible for most endometrial cancer-related deaths due to their aggressive nature, late stage detection and high tolerance for standard therapies. However, there are no targeted therapies for type II ECs, and they are still treated the same way as the clinically indolent and easily treatable type I ECs. Therefore, type II ECs are in need of new treatment options. More recently, molecular analysis of endometrial cancer revealed phosphorylation-dependent oncogenic signalling in the phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) pathways to be most frequently altered in type II ECs. Consequently, clinical trials tested pharmacologic kinase inhibitors targeting these pathways, although mostly with rather disappointing results. In this review, we highlight the most common genetic alterations in type II ECs. Additionally, we reason why most clinical trials for ECs using targeted kinase inhibitors had unsatisfying results and what should be changed in future clinical trial setups. Furthermore, we argue that, besides kinases, phosphatases should no longer be ignored in clinical trials, particularly in type II ECs, where the tumour suppressive phosphatase protein phosphatase type 2A (PP2A) is frequently mutated. Lastly, we discuss the therapeutic potential of targeting PP2A for (re)activation, possibly in combination with pharmacologic kinase inhibitors.
Assuntos
Neoplasias do Endométrio/tratamento farmacológico , Inibidores de Proteínas Quinases/uso terapêutico , Neoplasias do Endométrio/genética , Neoplasias do Endométrio/patologia , Feminino , Humanos , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Inibidores de Proteínas Quinases/farmacologia , Proteína Fosfatase 2/antagonistas & inibidores , Proteína Fosfatase 2/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
To supply tissues with nutrients and oxygen, the cardiovascular system forms a seamless, hierarchically branched, network of lumenized tubes. Here, we show that maintenance of patent vessel lumens requires the Bα regulatory subunit of protein phosphatase 2A (PP2A). Deficiency of Bα in zebrafish precludes vascular lumen stabilization resulting in perfusion defects. Similarly, inactivation of PP2A-Bα in cultured ECs induces tubulogenesis failure due to alteration of cytoskeleton dynamics, actomyosin contractility and maturation of cell-extracellular matrix (ECM) contacts. Mechanistically, we show that PP2A-Bα controls the activity of HDAC7, an essential transcriptional regulator of vascular stability. In the absence of PP2A-Bα, transcriptional repression by HDAC7 is abrogated leading to enhanced expression of the cytoskeleton adaptor protein ArgBP2. ArgBP2 hyperactivates RhoA causing inadequate rearrangements of the EC actomyosin cytoskeleton. This study unravels the first specific role for a PP2A holoenzyme in development: the PP2A-Bα/HDAC7/ArgBP2 axis maintains vascular lumens by balancing endothelial cytoskeletal dynamics and cell-matrix adhesion.
Assuntos
Endotélio Vascular/fisiologia , Regulação da Expressão Gênica/fisiologia , Histona Desacetilases/metabolismo , Neovascularização Fisiológica/fisiologia , Proteína Fosfatase 2/metabolismo , Grau de Desobstrução Vascular/fisiologia , Proteínas Adaptadoras de Transdução de Sinal , Animais , Adesão Celular/fisiologia , Colágeno , Combinação de Medicamentos , Imunofluorescência , Regulação da Expressão Gênica/genética , Proteínas de Homeodomínio/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Processamento de Imagem Assistida por Computador , Laminina , Microscopia Confocal , Proteoglicanas , RNA Interferente Pequeno/genética , Proteínas de Ligação a RNA , Grau de Desobstrução Vascular/genética , Peixe-ZebraRESUMO
Calcium current through voltage-gated calcium channels (VGCC) controls gene expression. Here, we describe a novel signalling pathway in which the VGCC Cacnb4 subunit directly couples neuronal excitability to transcription. Electrical activity induces Cacnb4 association to Ppp2r5d, a regulatory subunit of PP2A phosphatase, followed by (i) nuclear translocation of Cacnb4/Ppp2r5d/PP2A, (ii) association with the tyrosine hydroxylase (TH) gene promoter through the nuclear transcription factor thyroid hormone receptor alpha (TRα), and (iii) histone binding through association of Cacnb4 with HP1γ concomitantly with Ser(10) histone H3 dephosphorylation by PP2A. This signalling cascade leads to TH gene repression by Cacnb4 and is controlled by the state of interaction between the SH3 and guanylate kinase (GK) modules of Cacnb4. The human R482X CACNB4 mutation, responsible for a form of juvenile myoclonic epilepsy, prevents association with Ppp2r5 and nuclear targeting of the complex by altering Cacnb4 conformation. These findings demonstrate that an intact VGCC subunit acts as a repressor recruiting platform to control neuronal gene expression.
Assuntos
Canais de Cálcio/biossíntese , Canais de Cálcio/genética , Epilepsias Mioclônicas/metabolismo , Regulação da Expressão Gênica , Transporte Ativo do Núcleo Celular , Animais , Biofísica/métodos , Canais de Cálcio/metabolismo , Eletrofisiologia/métodos , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Histonas/metabolismo , Humanos , Camundongos , Mutação , Proteína Fosfatase 2/metabolismo , Transdução de Sinais , Receptores alfa dos Hormônios Tireóideos/metabolismo , Transcrição GênicaRESUMO
Phosphoinositide (PI) phosphatases such as the SH2 domain-containing inositol 5-phosphatases 1/2 (SHIP1 and 2) are important signalling enzymes in human physiopathology. SHIP1/2 interact with a large number of immune and growth factor receptors. Tyrosine phosphorylation of SHIP1/2 has been considered to be the determining regulatory modification. However, here we present a hypothesis, based on recent key publications, highlighting the determining role of Ser/Thr phosphorylation in regulating several key properties of SHIP1/2. Since a subunit of the Ser/Thr phosphatase PP2A has been shown to interact with SHIP2, a putative mechanism for reversing SHIP2 Ser/Thr phosphorylation can be anticipated. PI phosphatases are potential target molecules in human diseases, particularly, but not exclusively, in cancer and diabetes. Therefore, this novel regulatory mechanism deserves further attention in the hunt for discovering novel or complementary therapeutic strategies. This mechanism may be more broadly involved in regulating PI signalling in the case of synaptojanin1 or the phosphatase, tensin homolog, deleted on chromosome TEN.
Assuntos
Monoéster Fosfórico Hidrolases/metabolismo , Serina/metabolismo , Transdução de Sinais , Treonina/metabolismo , Animais , Diferenciação Celular , Membrana Celular/metabolismo , Núcleo Celular/genética , Núcleo Celular/metabolismo , Ativação Enzimática , Humanos , Inositol Polifosfato 5-Fosfatases , Camundongos , Camundongos Knockout , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases , Monoéster Fosfórico Hidrolases/genética , Fosforilação , Mapeamento de Interação de Proteínas , Especificidade por Substrato , Tirosina/metabolismoRESUMO
Protein phosphatase 2A (PP2A) plays a major role in dephosphorylating the targets of the major mitotic kinase Cdk1 at mitotic exit, yet how it is regulated in mitotic progression is poorly understood. Here we show that mutations in either the catalytic or regulatory twins/B55 subunit of PP2A act as enhancers of gwl(Scant), a gain-of-function allele of the Greatwall kinase gene that leads to embryonic lethality in Drosophila when the maternal dosage of the mitotic kinase Polo is reduced. We also show that heterozygous mutant endos alleles suppress heterozygous gwl(Scant); many more embryos survive. Furthermore, heterozygous PP2A mutations make females heterozygous for the strong mutation polo(11) partially sterile, even in the absence of gwl(Scant). Heterozygosity for an endos mutation suppresses this PP2A/polo(11) sterility. Homozygous mutation or knockdown of endos leads to phenotypes suggestive of defects in maintaining the mitotic state. In accord with the genetic interactions shown by the gwl(Scant) dominant mutant, the mitotic defects of Endos knockdown in cultured cells can be suppressed by knockdown of either the catalytic or the Twins/B55 regulatory subunits of PP2A but not by the other three regulatory B subunits of Drosophila PP2A. Greatwall phosphorylates Endos at a single site, Ser68, and this is essential for Endos function. Together these interactions suggest that Greatwall and Endos act to promote the inactivation of PP2A-Twins/B55 in Drosophila. We discuss the involvement of Polo kinase in such a regulatory loop.
Assuntos
Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Mitose , Mutação , Peptídeos/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Animais , Células Cultivadas , Drosophila melanogaster/citologia , Feminino , Fertilidade/genética , Técnicas de Silenciamento de Genes , Redes Reguladoras de Genes , Estudos de Associação Genética , Larva/citologia , Larva/genética , Masculino , Microscopia de Fluorescência , Sistema Nervoso/citologia , Peptídeos/genética , Fosfoproteínas Fosfatases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Interferência de RNA , Imagem com Lapso de TempoRESUMO
Functional diversity of protein phosphatase 2A (PP2A) enzymes mainly results from their association with distinct regulatory subunits. To analyze the functions of one such holoenzyme in vivo, we generated mice lacking PR61/B'δ (B56δ), a subunit highly expressed in neural tissues. In PR61/B'δ-null mice the microtubule-associated protein tau becomes progressively phosphorylated at pathological epitopes in restricted brain areas, with marked immunoreactivity for the misfolded MC1-conformation but without neurofibrillary tangle formation. Behavioral tests indicated impaired sensorimotor but normal cognitive functions. These phenotypical characteristics were further underscored in PR61/B'δ-null mice mildly overexpressing human tau. PR61/B'δ-containing PP2A (PP2A(T61δ)) poorly dephosphorylates tau in vitro, arguing against a direct dephosphorylation defect. Rather, the activity of glycogen synthase kinase-3ß, a major tau kinase, was found increased, with decreased phosphorylation of Ser-9, a putative cyclin-dependent kinase 5 (CDK5) target. Accordingly, CDK5 activity is decreased, and its cellular activator p35, strikingly absent in the affected brain areas. As opposed to tau, p35 is an excellent PP2A(T61δ) substrate. Our data imply a nonredundant function for PR61/B'δ in phospho-tau homeostasis via an unexpected spatially restricted mechanism preventing p35 hyperphosphorylation and its subsequent degradation.
Assuntos
Encéfalo/enzimologia , Quinase 5 Dependente de Ciclina/metabolismo , Quinase 3 da Glicogênio Sintase/metabolismo , Dobramento de Proteína , Proteína Fosfatase 2/metabolismo , Tauopatias/enzimologia , Animais , Quinase 5 Dependente de Ciclina/genética , Quinase 3 da Glicogênio Sintase/genética , Glicogênio Sintase Quinase 3 beta , Camundongos , Camundongos Knockout , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Células PC12 , Fosforilação/genética , Proteína Fosfatase 2/genética , Ratos , Tauopatias/genética , Proteínas tau/genética , Proteínas tau/metabolismoRESUMO
Cellular signaling pathways rely on posttranslational modifications (PTMs) to finely regulate protein functions, particularly transcription factors. The Hedgehog (Hh) signaling cascade, crucial for embryonic development and tissue homeostasis, is susceptible to aberrations that lead to developmental anomalies and various cancers. At the core of Hh signaling are Gli proteins, whose dynamic balance between activator (GliA) and repressor (GliR) states shapes cellular outcomes. Phosphorylation, orchestrated by multiple kinases, is pivotal in regulating Gli activity. While kinases in this context have been extensively studied, the role of protein phosphatases, particularly Protein Phosphatase 2A (PP2A), remains less explored. This study unveils a novel role for the Bâ³gamma subunit of PP2A, PPP2R3C, in Hh signaling regulation. PPP2R3C interacts with Gli proteins, and its disruption reduces Hedgehog pathway activity as measured by reduced expression of Gli1/2 and Hh target genes upon Hh signaling activation, and reduced growth of a Hh signaling-dependent medulloblastoma cell line. Moreover, we establish an antagonistic connection between PPP2R3C and MEKK1 kinase in Gli protein phosphorylation, underscoring the intricate interplay between kinases and phosphatases in Hh signaling pathway. This study sheds light on the previously understudied role of protein phosphatases in Hh signaling and provides insights into their significance in cellular regulation.
RESUMO
PURPOSE: Uterine serous carcinoma (USC) is generally associated with poor prognosis due to a high recurrence rate and frequent treatment resistance; hence, there is a need for improved therapeutic strategies. Molecular analysis of USC identified several molecular markers, useful to improve current treatments or identify new druggable targets. PPP2R1A, encoding the Aα subunit of the tumor suppressive Ser/Thr phosphatase PP2A, is mutated in up to 40% of USCs. Here, we investigated the effect of the p.R183W PPP2R1A hotspot variant on treatment response to the nucleoside analogue clofarabine. METHODS AND RESULTS: USC cells stably expressing p.R183W Aα showed increased resistance to clofarabine treatment in vitro and, corroborated by decreased clofarabine-induced apoptosis, G1 phase arrest, DNA-damage (γH2AX) and activation of ATM and Chk1/2 kinases. Phenotypic rescue by pharmacologic PP2A inhibition or dicer-substrate siRNA (dsiRNA)-mediated B56δ subunit knockdown supported a gain-of-function mechanism of Aα p.R183W, promoting dephosphorylation and inactivation of deoxycytidine kinase (dCK), the cellular enzyme responsible for the conversion of clofarabine into its bioactive form. Therapeutic assessment of related nucleoside analogues (gemcitabine, cladribine) revealed similar effects, but in a cell line-dependent manner. Expression of two other PPP2R1A USC mutants (p.P179R or p.S256F) did not affect clofarabine response in our cell models, arguing for mutant-specific effects on treatment outcome as well. CONCLUSIONS: While our results call for PPP2R1A mutant and context-dependent effects upon clofarabine/nucleoside analogue monotherapy, combining clofarabine with a pharmacologic PP2A inhibitor proved synergistically in all tested conditions, highlighting a new generally applicable strategy to improve treatment outcome in USC.
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Liprin-α1 is a widely expressed scaffolding protein responsible for regulating cellular processes such as focal adhesion, cell motility, and synaptic transmission. Liprin-α1 interacts with many proteins including ELKS, GIT1, liprin-ß, and LAR-family receptor tyrosine protein phosphatase. Through these protein-protein interactions, liprin-α1 assembles large higher-order molecular complexes; however, the regulation of this complex assembly/disassembly is unknown. Liquid-liquid phase separation (LLPS) is a process that concentrates proteins within cellular nano-domains to facilitate efficient spatiotemporal signaling in response to signaling cascades. While there is no report that liprin-α1 spontaneously undergoes LLPS, we found that GFP-liprin-α1 expressed in HEK293 cells occasionally forms droplet-like condensates. MS-based interactomics identified Protein Phosphatase 2A (PP2A)/B56δ (PPP2R5D) trimers as specific interaction partners of liprin-α1 through a canonical Short Linear Interaction Motif (SLiM) in its N-terminal dimerization domain. Mutation of this SLiM nearly abolished PP2A interaction, and resulted in significantly increased LLPS. GFP-liprin-α1 showed significantly increased droplet formation in HEK293 cells devoid of B56δ (PPP2R5D knockout), suggesting that PPP2R5D/PP2A holoenzyme inhibits liprin-α1 LLPS. Guided by reported liprin-α1 Ser/Thr phosphorylation sites, we found liprin-α1 phospho-mimetic mutant at serine 763 (S763E) is sufficient to drive its LLPS. Domain mapping studies of liprin-α1 indicated that the intrinsically disordered region, the N-terminal dimerization domain, and the SAM domains are all necessary for liprin-α1 LLPS. Finally, expression of p.E420K, a human PPP2R5D variant causing Houge-Janssens Syndrome type 1 (also known as Jordan's Syndrome), significantly compromised suppression of liprin-α1 LLPS. Our work identified B56δ-PP2A holoenzyme as an inhibitor of liprin-α1 LLPS via regulation at multiple phosphorylation sites.
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Protein phosphatase 2A (PP2A), a major phospho-serine/threonine phosphatase, is conserved throughout eukaryotes. It dephosphorylates a plethora of cellular proteins, including kinases and other signaling molecules involved in cell division, gene regulation, protein synthesis and cytoskeleton organization. PP2A enzymes typically exist as heterotrimers comprising catalytic C-, structural A- and regulatory B-type subunits. The B-type subunits function as targeting and substrate-specificity factors; hence, holoenzyme assembly with the appropriate B-type subunit is crucial for PP2A specificity and regulation. Recently, several biochemical and structural determinants have been described that affect PP2A holoenzyme assembly. Moreover, the effects of specific post-translational modifications of the C-terminal tail of the catalytic subunit indicate that a 'code' might regulate dynamic exchange of regulatory B-type subunits, thus affecting the specificity of PP2A.