RESUMO
The formation of a reversible disulfide bond between the catalytic cysteine and a spatially neighboring cysteine (backdoor) in protein tyrosine phosphatases (PTPs) serves as a critical regulatory mechanism for maintaining the activity of protein tyrosine phosphatases. The failure of such protection results in the formation of irreversibly oxidized cysteines into sulfonic acid in a highly oxidative cellular environment in the presence of free radicals. Hence, it is important to develop methods to interconvert PTPs into reduced and oxidized forms to understand their catalytic function in vitro. Protein tyrosine phosphatase 4A type 1 (PTP4A1), a dual-specificity phosphatase, is catalytically active in the reduced form. Unexpectedly, also its oxidized form performs a key biological function in systemic sclerosis (SSc) by forming a kinase-phosphatase complex with Src kinases. Thus, we developed simple and efficient protocols for producing oxidized and reduced PTP4A1 to elucidate their biological function, which can be extended to study other protein tyrosine phosphatases and other recombinantly produced proteins.
Assuntos
Cisteína , Fosfatases de Especificidade Dupla , Catálise , Citoplasma , Domínios ProteicosRESUMO
Penicillin-binding proteins (PBPs) are essential for the formation of the bacterial cell wall. They are also the targets of ß-lactam antibiotics. In Enterococcus faecium, high levels of resistance to ß-lactams are associated with the expression of PBP5, with higher levels of resistance associated with distinct PBP5 variants. To define the molecular mechanism of PBP5-mediated resistance we leveraged biomolecular NMR spectroscopy of PBP5 - due to its size (>70 kDa) a challenging NMR target. Our data show that resistant PBP5 variants show significantly increased dynamics either alone or upon formation of the acyl-enzyme inhibitor complex. Furthermore, these variants also exhibit increased acyl-enzyme hydrolysis. Thus, reducing sidechain bulkiness and expanding surface loops results in increased dynamics that facilitates acyl-enzyme hydrolysis and, via increased ß-lactam antibiotic turnover, facilitates ß-lactam resistance. Together, these data provide the molecular basis of resistance of clinical E. faecium PBP5 variants, results that are likely applicable to the PBP family.
Assuntos
Antibacterianos , Hexosiltransferases , Proteínas de Ligação às Penicilinas/genética , Proteínas de Ligação às Penicilinas/metabolismo , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Resistência beta-Lactâmica/genética , Monobactamas , beta-Lactamas/farmacologia , Testes de Sensibilidade MicrobianaRESUMO
The serine/threonine protein phosphatase 1 (PP1) dephosphorylates hundreds of substrates by associating with >200 regulatory proteins to form specific holoenzymes. The major PP1 targeting protein in the nucleolus is RRP1B (ribosomal RNA processing 1B). In addition to selectively recruiting PP1ß/PP1γ to the nucleolus, RRP1B also has a key role in ribosome biogenesis, among other functions. How RRP1B binds PP1 and regulates nucleolar phosphorylation signaling is not yet known. Here, we show that RRP1B recruits PP1 via established (RVxF/SILK/ΦΦ) and non-canonical motifs. These atypical interaction sites, the PP1ß/γ specificity, and N-terminal AF-binding pockets rely on hydrophobic interactions that contribute to binding and, via phosphorylation, regulate complex formation. This work advances our understanding of PP1 isoform selectivity, reveals key roles of N-terminal PP1 residues in regulator binding, and suggests that additional PP1 interaction sites have yet to be identified, all of which are necessary for a systems biology understanding of PP1 function.
Assuntos
Nucléolo Celular , Processamento Pós-Transcricional do RNA , Proteína Fosfatase 1 , Holoenzimas , FosforilaçãoRESUMO
Systemic sclerosis (SSc) is a fibrotic autoimmune disease characterized by pathogenic activation of fibroblasts enhanced by local oxidative stress. The tyrosine phosphatase PTP4A1 was identified as a critical promoter of TGF-ß signaling in SSc. Oxidative stress is known to functionally inactivate tyrosine phosphatases. Here, we assessed whether oxidation of PTP4A1 modulates its profibrotic action and found that PTP4A1 forms a complex with the kinase SRC in scleroderma fibroblasts, but surprisingly, oxidative stress enhanced rather than reduced PTP4A1's association with SRC and its profibrotic action. Through structural assessment of the oxo-PTP4A1-SRC complex, we unraveled an unexpected mechanism whereby oxidation of a tyrosine phosphatase promotes its function through modification of its protein complex. Considering the importance of oxidative stress in the pathogenesis of SSc and fibrosis, our findings suggest routes for leveraging PTP4A1 oxidation as a potential strategy for developing antifibrotic agents.
Assuntos
Escleroderma Sistêmico , Fibroblastos/metabolismo , Fibrose , Humanos , Estresse Oxidativo , Escleroderma Sistêmico/patologia , Tirosina/metabolismoRESUMO
The sequence-specific backbone assignment of the mitogen-activated protein kinase (MAPK) binding domain of the dual-specificity phosphatase 1 (DUSP1) has been accomplished using a uniformly [13C, 15N]-labeled protein. These assignments will facilitate further studies of DUSP1 in the presence of inhibitors/ligands to target MAPK associated diseases and provide further insights into the function of dual-specificity phosphatase 1 in MAPK regulation.
Assuntos
Proteínas Quinases p38 Ativadas por MitógenoRESUMO
Mitogen-activated protein kinase (MAPK; p38, ERK, and JNK) cascades are evolutionarily conserved signaling pathways that regulate the cellular response to a variety of extracellular stimuli, such as growth factors and interleukins. The MAPK p38 is activated by its specific upstream MAPK kinases, MKK6 and MKK3. However, a comprehensive molecular understanding of how these cognate upstream kinases bind and activate p38 is still missing. Here, we combine NMR spectroscopy and isothermal titration calorimetry to define the binding interface between full-length MKK6 and p38. It was shown that p38 engages MKK6 not only via its hydrophobic docking groove, but also influences helix αF, a secondary structural element that plays a key role in organizing the kinase core. It was also shown that, unlike MAPK phosphatases, the p38 conserved docking (CD) site is much less affected by MKK6 binding. Finally, it was demonstrated that these interactions with p38 are conserved independent of the MKK6 activation state. Together, the results revealed differences between specificity markers of p38 regulation by upstream kinases, which do not effectively engage the CD site, and downstream phosphatases, which require the CD site for productive binding.
Assuntos
MAP Quinase Quinase 6/química , Proteínas Quinases p38 Ativadas por Mitógeno/química , Sítios de Ligação , Ativação Enzimática , Humanos , MAP Quinase Quinase 6/genética , MAP Quinase Quinase 6/metabolismo , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica em alfa-Hélice , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Transcription factor phosphorylation at specific sites often activates gene expression, but how environmental cues quantitatively control transcription is not well-understood. Activating protein 1 transcription factors are phosphorylated by mitogen-activated protein kinases (MAPK) in their transactivation domains (TAD) at so-called phosphoswitches, which are a hallmark in response to growth factors, cytokines or stress. We show that the ATF2 TAD is controlled by functionally distinct signaling pathways (JNK and p38) through structurally different MAPK binding sites. Moreover, JNK mediated phosphorylation at an evolutionarily more recent site diminishes p38 binding and made the phosphoswitch differently sensitive to JNK and p38 in vertebrates. Structures of MAPK-TAD complexes and mechanistic modeling of ATF2 TAD phosphorylation in cells suggest that kinase binding motifs and phosphorylation sites line up to maximize MAPK based co-regulation. This study shows how the activity of an ancient transcription controlling phosphoswitch became dependent on the relative flux of upstream signals.
Assuntos
Fator 2 Ativador da Transcrição/metabolismo , Regulação da Expressão Gênica , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Transcrição Gênica , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Fator 2 Ativador da Transcrição/química , Motivos de Aminoácidos , Sequência de Aminoácidos , Células HEK293 , Humanos , Luciferases/metabolismo , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Fosforilação , Ligação Proteica , Dedos de ZincoRESUMO
Protein tyrosine phosphatase 1B (PTP1B) is a validated therapeutic target for the treatment of diabetes and obesity. Ertiprotafib is a PTP1B inhibitor that reached the clinical trial stage for the treatment of diabetes. Interestingly, Ertiprotafib reduces the melting temperature of PTP1B in differential scanning fluorimetry (DSF) assays, different from most drugs that increase the stability of their target upon binding. No molecular data on how Ertiprotafib functions has been published. Thus, to gain molecular insights into the mode of action of Ertiprotafib, we used biomolecular NMR spectroscopy to characterize the molecular details of the PTP1B:Ertiprotafib interaction. Our results show that Ertiprotafib induces aggregation of PTP1B in a concentration dependent manner. This shows that the insufficient clinical efficacy and adverse effects caused by Ertiprotafib is due to its tendency to cause aggregation of PTP1B.
Assuntos
Inibidores Enzimáticos/farmacologia , Fenilpropionatos/farmacologia , Proteína Tirosina Fosfatase não Receptora Tipo 1/antagonistas & inibidores , Tiofenos/farmacologia , Domínio Catalítico , Humanos , Proteína Tirosina Fosfatase não Receptora Tipo 1/químicaRESUMO
Protein-tyrosine phosphatase 1B (PTP1B) is the canonical enzyme for investigating how distinct structural elements influence enzyme catalytic activity. Although it is recognized that dynamics are essential for PTP1B function, the data collected thus far have not resolved whether distinct elements are dynamically coordinated or, alternatively, whether they fulfill their respective functions independently. To answer this question, we performed a comprehensive 13C-methyl relaxation study of Ile, Leu, and Val (ILV) residues of PTP1B, which, because of its substantially increased sensitivity, provides a comprehensive understanding of the influence of protein motions on different time scales for enzyme function. We discovered that PTP1B exhibits dynamics at three distinct time scales. First, it undergoes a distinctive slow motion that allows for the dynamic binding and release of its two most N-terminal helices from the catalytic core. Second, we showed that PTP1B 13C-methyl group side chain fast time-scale dynamics and 15N backbone fast time-scale dynamics are fully consistent, demonstrating that fast fluctuations are essential for the allosteric control of PTP1B activity. Third, and most importantly, using 13C ILV constant-time Carr-Purcell-Meiboom-Gill relaxation measurements experiments, we demonstrated that all four catalytically important loops-the WPD, Q, E, and substrate-binding loops-work in dynamic unity throughout the catalytic cycle of PTP1B. Thus, these data show that PTP1B activity is not controlled by a single functional element, but instead all key elements are dynamically coordinated. Together, these data provide the first fully comprehensive picture on how the validated drug target PTP1B functions.
Assuntos
Simulação de Dinâmica Molecular , Proteína Tirosina Fosfatase não Receptora Tipo 1/química , Humanos , Domínios Proteicos , Estrutura Secundária de Proteína , Proteína Tirosina Fosfatase não Receptora Tipo 1/genéticaRESUMO
Mitogen-activated protein kinases (MAPKs) control essential eukaryotic signaling pathways. While much has been learned about MAPK activation, much less is known about substrate recruitment and specificity. MAPK substrates may be other kinases that are crucial to promote a further diversification of the signaling outcomes. Here, we used a variety of molecular and cellular tools to investigate the recruitment of two substrate kinases, RSK1 and MK2, to three MAPKs (ERK2, p38α, and ERK5). Unexpectedly, we identified that kinase heterodimers form structurally and functionally distinct complexes depending on the activation state of the MAPK. These may be incompatible with downstream signaling, but naturally they may also form structures that are compatible with the phosphorylation of the downstream kinase at the activation loop, or alternatively at other allosteric sites. Furthermore, we show that small-molecule inhibitors may affect the quaternary arrangement of kinase heterodimers and thus influence downstream signaling in a specific manner.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Quinases S6 Ribossômicas 90-kDa/química , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Ativação Enzimática , Células HEK293 , Humanos , Espectroscopia de Ressonância Magnética , Proteína Quinase 1 Ativada por Mitógeno/química , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 14 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 14 Ativada por Mitógeno/química , Proteína Quinase 14 Ativada por Mitógeno/genética , Proteína Quinase 14 Ativada por Mitógeno/metabolismo , Proteína Quinase 7 Ativada por Mitógeno/química , Proteína Quinase 7 Ativada por Mitógeno/genética , Proteína Quinase 7 Ativada por Mitógeno/metabolismo , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Fosforilação , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Multimerização Proteica , Estrutura Quaternária de Proteína , Espalhamento a Baixo Ângulo , Difração de Raios XRESUMO
PURPOSE: Growing solid tumors mostly outstrip blood supply and become hypoxic (low oxygen supply). To survive under this pathological milieu, tumors overexpress a potent oncogenic factor, hypoxia-inducible factor-1α (HIF-1α). HIF-1α up-regulate HIF-1 signaling pathways and subsequently activate genes that promote cancer growth even under hypoxia. Also, HIF-1 pathway activation leads to aggressive tumor growth, metastasis, therapy resistance and ultimately poor patient prognosis as evidential by several clinical studies. Hence, targeting HIF-1 pathway is regarded as a promising strategy to treat cancer. To date, several synthetic HIF-1 pathway inhibitors have been developed to treat hypoxic tumors; however, they are clinically ineffective due to off-target effects, low potency and high toxicity. Hence, there is an urgent need to explore safe and promising drugs to combat hypoxic tumors. RESULTS: This article extensively reviews the therapeutic potential of various herbal nutraceuticals against wide varieties of hypoxic tumors. The inhibitory effects of each herbal nutraceutical on the pathological consequences of HIF-1 signaling pathway and also their ability to improve the response of hypoxic cancer cells to conventional cancer therapies are discussed. Furthermore, we have provided new directions to overcome challenges behind conducting in vivo and preclinical hypoxia research and developing herbal nutraceuticals into pharmaceuticals to treat cancer. CONCLUSIONS: The present review strongly suggests that herbal nutraceuticals are highly effective in combating the oncogenic effects of the HIF-1 pathway in wide varieties of tumors. However, more in vivo studies using zebrafish as a model system and extensive clinical studies in cancer patients with elevated tumor HIF-1α levels are highly warranted to ascertain the effective utilization of herbal nutraceuticals as adjunct/ alternative medicine in clinical practice to treat cancer.
Assuntos
Hipóxia Celular/efeitos dos fármacos , Suplementos Nutricionais , Neoplasias/tratamento farmacológico , Animais , Humanos , Neoplasias/metabolismo , Neoplasias/patologia , Extratos Vegetais/efeitos adversos , Extratos Vegetais/farmacologiaRESUMO
Serine/threonine phosphatases such as PP1 lack substrate specificity and associate with a large array of targeting subunits to achieve the requisite selectivity. The tumour suppressor ASPP (apoptosis-stimulating protein of p53) proteins associate with PP1 catalytic subunits and are implicated in multiple functions from transcriptional regulation to cell junction remodelling. Here we show that Drosophila ASPP is part of a multiprotein PP1 complex and that PP1 association is necessary for several in vivo functions of Drosophila ASPP. We solve the crystal structure of the human ASPP2/PP1 complex and show that ASPP2 recruits PP1 using both its canonical RVxF motif, which binds the PP1 catalytic domain, and its SH3 domain, which engages the PP1 C-terminal tail. The ASPP2 SH3 domain can discriminate between PP1 isoforms using an acidic specificity pocket in the n-Src domain, providing an exquisite mechanism where multiple motifs are used combinatorially to tune binding affinity to PP1.
Assuntos
Domínio Catalítico/fisiologia , Proteínas de Drosophila/metabolismo , Proteína Fosfatase 1/química , Proteína Fosfatase 1/metabolismo , Sequência de Aminoácidos , Animais , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Sítios de Ligação , Domínio Catalítico/genética , Drosophila , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Humanos , Ligação Proteica , Proteína Fosfatase 1/genética , Especificidade por Substrato , Domínios de Homologia de src/genética , Domínios de Homologia de src/fisiologiaRESUMO
Phosphorylation is a ubiquitous posttranslational modification that is essential for the regulation of many cellular processes. The human genome consists of more than 200,000 phosphorylation sites, whose phosphorylation is tightly controlled by ≥500 kinases and ~200 phosphatases. Given the large number of phosphorylation sites and the key role phosphorylation plays in regulating cellular processes, it is essential to characterize the impact of phosphorylation on substrate structure, dynamics, and function. However, a major challenge is the large-scale production of phosphorylated proteins in vitro for these structural, functional, and dynamic studies. Here, we describe an efficient protocol used routinely in our laboratory for the production of phosphorylated proteins. We also describe the methods used for identifying, characterizing, and separating the resulting phosphorylated proteins for subsequent studies.
Assuntos
Antígeno Ki-67/metabolismo , MAP Quinase Quinase 6/metabolismo , Ressonância Magnética Nuclear Biomolecular/métodos , Processamento de Proteína Pós-Traducional , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Eletroforese em Gel de Poliacrilamida/métodos , Humanos , Antígeno Ki-67/genética , MAP Quinase Quinase 6/genética , Fosforilação , Especificidade por Substrato , Proteínas Quinases p38 Ativadas por Mitógeno/genéticaRESUMO
Glycogen is the primary storage form of glucose. Glycogen synthesis and breakdown are tightly controlled by glycogen synthase (GYS) and phosphorylase, respectively. The enzyme responsible for dephosphorylating GYS and phosphorylase, which results in their activation (GYS) or inactivation (phosphorylase) to robustly stimulate glycogen synthesis, is protein phosphatase 1 (PP1). However, our understanding of how PP1 recruits these substrates is limited. Here, we show how PP1, together with its muscle glycogen-targeting (GM) regulatory subunit, recruits and selectively dephosphorylates its substrates. Our molecular data reveal that the GM carbohydrate binding module (GM CBM21), which is amino-terminal to the GM PP1 binding domain, has a dual function in directing PP1 substrate specificity: It either directly recruits substrates (i.e., GYS) or recruits them indirectly by localization (via glycogen for phosphorylase). Our data provide the molecular basis for PP1 regulation by GM and reveal how PP1-mediated dephosphorylation is driven by scaffolding-based substrate recruitment.
Assuntos
Glicogênio Sintase/metabolismo , Glicogênio/metabolismo , Músculo Esquelético/enzimologia , Proteína Fosfatase 1/metabolismo , Animais , Glicogênio Sintase/química , Humanos , Fosforilação , Conformação Proteica , Proteína Fosfatase 1/química , Coelhos , Especificidade por SubstratoRESUMO
Mitogen-activated protein kinases, which include p38, are essential for cell differentiation and autophagy. The current model for p38 activation involves activation-loop phosphorylation with subsequent substrate binding leading to substrate phosphorylation. Despite extensive efforts, the molecular mechanism of activation remains unclear. Here, using NMR spectroscopy, we show how the modulation of protein dynamics across timescales activates p38. We find that activation-loop phosphorylation does not change the average conformation of p38; rather it quenches the loop ps-ns dynamics. We then show that substrate binding to nonphosphorylated and phosphorylated p38 results in uniform µs-ms backbone dynamics at catalytically essential regions and across the entire molecule, respectively. Together, these results show that phosphorylation and substrate binding flatten the energy landscape of the protein, making essential elements of allostery and activation dynamically accessible. The high degree of structural conservation among ser/thr kinases suggests that elements of this mechanism may be conserved across the kinase family.
Assuntos
Simulação de Dinâmica Molecular , Proteínas Quinases p38 Ativadas por Mitógeno/química , Regulação Alostérica/fisiologia , Ativação Enzimática/fisiologia , Humanos , Ressonância Magnética Nuclear Biomolecular , Fosforilação/fisiologia , Estrutura Secundária de Proteína , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Ki-67 and RepoMan have key roles during mitotic exit. Previously, we showed that Ki-67 organizes the mitotic chromosome periphery and recruits protein phosphatase 1 (PP1) to chromatin at anaphase onset, in a similar manner as RepoMan (Booth et al., 2014). Here we show how Ki-67 and RepoMan form mitotic exit phosphatases by recruiting PP1, how they distinguish between distinct PP1 isoforms and how the assembly of these two holoenzymes are dynamically regulated by Aurora B kinase during mitosis. Unexpectedly, our data also reveal that Ki-67 and RepoMan bind PP1 using an identical, yet novel mechanism, interacting with a PP1 pocket that is engaged only by these two PP1 regulators. These findings not only show how two distinct mitotic exit phosphatases are recruited to their substrates, but also provide immediate opportunities for the design of novel cancer therapeutics that selectively target the Ki-67:PP1 and RepoMan:PP1 holoenzymes.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/metabolismo , Antígeno Ki-67/metabolismo , Proteínas Nucleares/metabolismo , Multimerização Proteica , Proteína Fosfatase 1/metabolismo , Proteínas de Transporte/química , Ciclo Celular , Proteínas de Ciclo Celular/química , Cristalografia por Raios X , Humanos , Antígeno Ki-67/química , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Proteínas Nucleares/química , Conformação Proteica , Proteína Fosfatase 1/químicaRESUMO
The cyclic AMP response element-binding protein (CREB) is a signal-dependent transcription factor that exerts its positive effects on gene transcription of a broad range of genes by recruiting coactivators, including CREB-binding protein (CBP), its paralog, p300, and the family of CRTC (CREB-regulated transcriptional coactivators) proteins. Whereas recruitment of CBP/p300 is dependent on CREB phosphorylation at Ser133, recruitment of CRTCs is not. Here we describe how both mechanisms could concurrently drive transcription of CREB targets in a subset of head and neck cancers featuring chromosomal translocations that fuse portions of CRTC1 and CRTC3 genes with that of the Mastermind-like transcriptional coactivator MAML2. We show that a peptide derived from transactivation domain 1 (TAD1) of MAML2 binds to the CBP KIX domain with micromolar affinity. An â¼20-residue segment within this peptide, conserved in MAML2 orthologs and paralogs, binds directly to a KIX surface previously shown to bind to MLL1. The 20-residue MAML2 segment shares sequence similarity with MLL1, especially at those positions in direct contact with KIX, and like MLL1, the segment is characterized by the presence of an â¼10-residue helix. Because CRTC1/3-MAML2 fusion proteins are constitutively nuclear, like CREB, our results suggest constitutive recruitment of CBP/p300 to CREB targets that could be further enhanced by signals that cause CREB Ser133 phosphorylation.
Assuntos
AMP Cíclico/metabolismo , Transdução de Sinais/fisiologia , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Fatores de Transcrição de p300-CBP/metabolismo , Sequência de Aminoácidos , Animais , AMP Cíclico/química , Células HEK293 , Humanos , Camundongos , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Transativadores/fisiologia , Fatores de Transcrição/química , Fatores de Transcrição de p300-CBP/químicaRESUMO
The serine/threonine protein phosphatase 1 (PP1) dephosphorylates hundreds of key biological targets by associating with nearly 200 regulatory proteins to form highly specific holoenzymes. However, how these proteins direct PP1 specificity and the ability to predict how these PP1 interacting proteins bind PP1 from sequence alone is still missing. PP1 nuclear targeting subunit (PNUTS) is a PP1 targeting protein that, with PP1, plays a central role in the nucleus, where it regulates chromatin decondensation, RNA processing, and the phosphorylation state of fundamental cell cycle proteins, including the retinoblastoma protein (Rb), p53, and MDM2. The molecular function of PNUTS in these processes is completely unknown. Here, we show that PNUTS, which is intrinsically disordered in its free form, interacts strongly with PP1 in a highly extended manner. Unexpectedly, PNUTS blocks one of PP1's substrate binding grooves while leaving the active site accessible. This interaction site, which we have named the arginine site, allowed us to define unique PP1 binding motifs, which advances our ability to predict how more than a quarter of the known PP1 regulators bind PP1. Additionally, the structure shows how PNUTS inhibits the PP1-mediated dephosphorylation of critical substrates, especially Rb, by blocking their binding sites on PP1, insights that are providing strategies for selectively enhancing Rb activity.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Regulação Enzimológica da Expressão Gênica/genética , Modelos Moleculares , Proteínas Nucleares/metabolismo , Conformação Proteica , Proteína Fosfatase 1/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteína do Retinoblastoma/metabolismo , Sequência de Aminoácidos , Calorimetria , Montagem e Desmontagem da Cromatina/fisiologia , Clonagem Molecular , Biologia Computacional , Cristalização , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Humanos , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Proteínas Nucleares/química , Proteínas Nucleares/genética , Fosforilação , Domínios e Motivos de Interação entre Proteínas/genética , Proteína Fosfatase 1/química , Proteína Fosfatase 1/genética , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Alinhamento de Sequência , Especificidade por SubstratoRESUMO
Mitogen-activated protein kinases (MAPKs) fulfill essential biological functions and are key pharmaceutical targets. Regulation of MAPKs is achieved via a plethora of regulatory proteins including activating MAPKKs and an abundance of deactivating phosphatases. Although all regulatory proteins use an identical interaction site on MAPKs, the common docking and hydrophobic pocket, they use distinct kinase interaction motif (KIM or D-motif) sequences that are present in linear, peptide-like, or well folded protein domains. It has been recently shown that a KIM-containing MAPK-specific dual specificity phosphatase DUSP10 uses a unique binding mode to interact with p38α. Here we describe the interaction of the MAPK binding domain of DUSP16 with p38α and show that despite belonging to the same dual specificity phosphatase (DUSP) family, its interaction mode differs from that of DUSP10. Indeed, the DUSP16 MAPK binding domain uses an additional helix, α-helix 4, to further engage p38α. This leads to an additional interaction surface on p38α. Together, these structural and energetic differences in p38α engagement highlight the fine-tuning necessary to achieve MAPK specificity and regulation among multiple regulatory proteins.
Assuntos
Fosfatases de Especificidade Dupla/metabolismo , Fosfatases da Proteína Quinase Ativada por Mitógeno/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Sítios de Ligação , Calorimetria , Regulação Enzimológica da Expressão Gênica , Humanos , Espectroscopia de Ressonância Magnética , Ligação Proteica , Estrutura Terciária de Proteína , Transdução de SinaisRESUMO
The MAP kinase p38α is essential for neuronal signaling. To better understand the molecular regulation of p38α we used atomistic and molecular techniques to determine the structural basis of p38α regulation by the two neuronal tyrosine phosphatases, PTPSL/PTPBR7 (PTPRR) and STEP (PTPN5). We show that, despite the fact that PTPSL and STEP belong to the same family of regulatory proteins, they interact with p38α differently and their distinct molecular interactions explain their different catalytic activities. Although the interaction of PTPSL with p38α is similar to that of the previously described p38α:HePTP (PTPN7) complex, STEP binds and regulates p38α in an unexpected manner. Using NMR and small-angle X-ray scattering data, we generated a model of the p38α:STEP complex and define molecular differences between its resting and active states. Together, these results provide insights into molecular regulation of p38α by key regulatory proteins.