RESUMEN
The inactivation of the p53 tumor suppressor pathway in many cancers often increases their resistance to anticancer therapy. Here we show that a previously proposed strategy directed to Wip1 inhibition could be ineffective in tumors lacking p53. On the contrary, Wip1 overexpression sensitized these tumors to chemotherapeutic agents. This effect was mediated through interaction between Wip1 and RUNX2 that resulted, in response to anticancer treatment, in RUNX2-dependent transcriptional induction of the proapoptotic Bax protein. The potentiating effects of Wip1 overexpression on chemotherapeutic agents were directed only to tumor cells lacking p53. The overexpression of Wip1 in normal tissues provided protection from cisplatin-induced apoptosis through decreased strength of upstream signaling to p53. Thus, Wip1 phosphatase promotes apoptosis in p53-negative tumors and protects normal tissues during treatment with anticancer agents.
Asunto(s)
Antineoplásicos/farmacología , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Regulación Neoplásica de la Expresión Génica/fisiología , Neoplasias/tratamiento farmacológico , Fosfoproteínas Fosfatasas/metabolismo , Proteína p53 Supresora de Tumor/deficiencia , Animales , Antineoplásicos/metabolismo , Antineoplásicos/uso terapéutico , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Western Blotting , Línea Celular , Cartilla de ADN/genética , Sinergismo Farmacológico , Femenino , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Inmunohistoquímica , Inmunoprecipitación , Ratones , Neoplasias/metabolismo , Plásmidos/genética , Proteína Fosfatasa 2C , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteína p53 Supresora de Tumor/metabolismo , Proteína X Asociada a bcl-2/metabolismoRESUMEN
The PPM phosphatases require millimolar concentrations of Mg²âº or Mn²âº to activate phosphatase activity in vitro. The human phosphatases PP2Cα (PPM1A) and Wip1 (PPM1D) differ in their physiological function, substrate specificity, and apparent metal affinity. A crystallographic structure of PP2Cα shows only two metal ions in the active site. However, recent structural studies of several bacterial PP2C phosphatases have indicated three metal ions in the active site. Two residues that coordinate the third metal ion are highly conserved, suggesting that human PP2C phosphatases may also bind a third ion. Here, isothermal titration calorimetry analysis of Mg²âº binding to PP2Cα distinguished binding of two ions to high affinity sites from the binding of a third ion with a millimolar affinity, similar to the apparent metal affinity required for catalytic activity. Mutational analysis indicated that Asp239 and either Asp146 or Asp243 was required for low-affinity binding of Mg²âº, but that both Asp146 and Asp239 were required for catalysis. Phosphatase activity assays in the presence of MgCl2, MnCl2, or mixtures of the two, demonstrate high phosphatase activity toward a phosphopeptide substrate when Mg²âº was bound to the low-affinity site, whether Mg²âº or Mn²âº ions were bound to the high affinity sites. Mutation of the corresponding putative third metal ion-coordinating residues of Wip1 affected catalytic activity similarly both in vitro and in human cells. These results suggest that phosphatase activity toward phosphopeptide substrates by PP2Cα and Wip1 requires the binding of a Mg²âº ion to the low-affinity site.
Asunto(s)
Dominio Catalítico , Magnesio/metabolismo , Fosfoproteínas Fosfatasas/metabolismo , Alanina/química , Secuencia de Aminoácidos , Ácido Aspártico/química , Catálisis , Células HEK293 , Humanos , Manganeso/metabolismo , Fosfopéptidos/metabolismo , Fosfoproteínas Fosfatasas/genética , Proteína Fosfatasa 2C , Alineación de SecuenciaRESUMEN
PPM1D (PP2Cδ or Wip1) was identified as a wild-type p53-induced Ser/Thr phosphatase that accumulates after DNA damage and classified into the PP2C family. It dephosphorylates and inactivates several proteins critical for cellular stress responses, including p38 MAPK, p53, and ATM. Furthermore, PPM1D is amplified and/or overexpressed in a number of human cancers. Thus, inhibition of its activity could constitute an important new strategy for therapeutic intervention to halt the progression of several different cancers. Previously, we reported the development of a cyclic thioether peptide with low micromolar inhibitory activity toward PPM1D. Here, we describe important improvements in the inhibitory activity of this class of cyclic peptides and also present a binding model based upon the results. We found that specific interaction of an aromatic ring at the X1 position and negative charge at the X5 and X6 positions significantly increased the inhibitory activity of the cyclic peptide, with the optimized molecule having a K(i) of 110 nM. To the best of our knowledge, this represents the highest inhibitory activity reported for an inhibitor of PPM1D. We further developed an inhibitor selective for PPM1D over PPM1A with a K(i) of 2.9 µM. Optimization of the cyclic peptide and mutagenesis experiments suggest that a highly basic loop unique to PPM1D is related to substrate specificity. We propose a new model for the catalytic site of PPM1D and inhibition by the cyclic peptides that will be useful both for the subsequent design of PPM1D inhibitors and for identification of new substrates.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Péptidos Cíclicos/farmacología , Fosfoproteínas Fosfatasas/antagonistas & inhibidores , Secuencia de Aminoácidos , Secuencia de Bases , Dicroismo Circular , Cartilla de ADN , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Fosfoproteínas Fosfatasas/química , Fosfoproteínas Fosfatasas/genética , Proteína Fosfatasa 2C , Homología de Secuencia de AminoácidoRESUMEN
The dual-specificity phosphatase (DUSP) 13 gene encodes two atypical DUSPs, DUSP13B/TMDP and DUSP13A/MDSP using alternative exons. DUSP13B protein is most highly expressed in testis, particularly in spermatocytes and round spermatids of the seminiferous tubules, while that of DUSP13A is restricted to skeletal muscle. Here, we show that DUSP13B inactivated MAPK activation in the order of selectivity, JNK = p38>ERK in cells, while DUSP13A did not show MAPK phosphatase activity. Reporter gene analysis showed that DUSP13B had significant inhibitory effect on AP-1-dependent gene expression, but DUSP13A did not. To our knowledge, DUSP13B is the first identified testis-specific phosphatase that inhibits stress-activated MAPKs. These data suggest an important role for DUSP13B in protection from external stress during spermatogenesis.
Asunto(s)
Fosfatasas de Especificidad Dual/fisiología , Regulación de la Expresión Génica/fisiología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Factor de Transcripción AP-1/fisiología , Animales , Línea Celular , Activación Enzimática , Exones , Humanos , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacologíaRESUMEN
Pig kidney Na/K-ATPase preparations showed a positive cooperative effect for pNPP in Na-pNPPase activity. Measurements of the Na-pNPPase activity, Na-ATPase activity and the accumulation of phosphoenzyme (EP) under conditions of pNPP saturation showed several different ATP affinities. The presence of pNPP reduced both the maximum amount of EP and Na-ATPase activity to half showing a value of 4 and a 3,700-fold reduced ATP affinity for EP formation, and a 7 and 1,300-fold reduced affinity for Na-ATPase activity. The presence of low concentrations of ATP in the phosphorylation induced a 2-fold enhancement in Na-pNPPase activity despite a reduction in available pNPP sites. However, higher concentrations of ATP inhibited the Na-pNPPase activity and a much higher concentration of ATP increased both the phosphorylation and Na-ATPase activity to the maximum levels. The maximum Na-pNPPase activity was 1.7 and 3.4-fold higher without and with ATP, respectively, than the maximum Na-ATPase activity. These data and the pNPP dependent reduction in both Na-ATPase activity and the amount of enzyme bound ATP provide new evidence to show that ATP, pNPP and ATP with pNPP, respectively, induce different subunit interactions resulting a difference in the maximum Na(+)-dependent catalytic activity in tetraprotomeric Na/K-ATPase.
Asunto(s)
4-Nitrofenilfosfatasa/química , Adenosina Trifosfato/química , Modelos Biológicos , ATPasa Intercambiadora de Sodio-Potasio/química , Animales , Dominio Catalítico , Activación Enzimática , Riñón/enzimología , Nitrofenoles/química , Compuestos Organofosforados/química , Fosforilación , PorcinosRESUMEN
We have identified a novel dual-specificity phosphatase (DSP), called LDP-2 (low-molecular-mass DSP-2), composed of 220 amino acid residues showing high sequence homology to VHR and LDP-1/TMDP, which belong to a family of DSPs with low molecular masses. The LDP-2 gene is ubiquitously expressed, and LDP-2 is localized in the cytoplasm. The main structural feature of LDP-2 is that the serine-156 residue located in the common active site sequence motif, HCXXGXXRS, for DSP is naturally substituted with an alanine residue. The recombinant LDP-2 protein showed extremely low phosphatase activity towards p-nitrophenyl phosphate (pNPP). Back-mutation of Ala-156 in LDP-2 to a serine (A156S mutation) conferred significant phosphatase activity towards pNPP. However, both LDP-2 and LDP-2 (A156S) exhibited substantial phosphatase activities towards both phospho-seryl/threonyl and -tyrosyl residues of myelin basic protein, with similar specific activities. Ala-156 of LDP-2 might be crucially involved in the recognition of a physiological substrate. We analyzed the effect of VHR and LDP-2 on mitogen-activated protein kinases (MAPKs) in vivo. We first found that VHR inhibits the activation of p38 as well as ERK and JNK, with similar efficiency. Under the conditions used, LDP-2 specifically suppressed JNK activation.
Asunto(s)
Proteínas Tirosina Fosfatasas/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Células COS , Clonación Molecular , ADN Complementario , Células HeLa , Humanos , Cinética , Datos de Secuencia Molecular , Peso Molecular , Mutagénesis , Fosforilación , Proteína Fosfatasa 2 , Proteínas Tirosina Fosfatasas/química , Proteínas Tirosina Fosfatasas/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Especificidad por SustratoRESUMEN
The Wip1 phosphatase is an oncogene that is overexpressed in a variety of primary human cancers. We were interested in identifying genetic variants that could change Wip1 activity. We identified 3 missense SNPs of the human Wip1 phosphatase, L120F, P322Q, and I496V confer a dominant-negative phenotype. On the other hand, in primary human cancers, PPM1D mutations commonly result in a gain-of-function phenotype, leading us to identify a hot-spot truncating mutation at position 525. Surprisingly, we also found a significant number of loss-of-function mutations of PPM1D in primary human cancers, both in the phosphatase domain and in the C terminus. Thus, PPM1D has evolved to generate genetic variants with lower activity, potentially providing a better fitness for the organism through suppression of multiple diseases. In cancer, however, the situation is more complex, and the presence of both activating and inhibiting mutations requires further investigation to understand their contribution to tumorigenesis.
Asunto(s)
Daño del ADN/genética , Evolución Molecular , Variación Genética , Modelos Moleculares , Neoplasias/genética , Fosfoproteínas Fosfatasas/genética , Secuencia de Aminoácidos , Western Blotting , Ensayo de Unidades Formadoras de Colonias , Técnica del Anticuerpo Fluorescente , Humanos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación/genética , Fosfoproteínas Fosfatasas/química , Proteína Fosfatasa 2CRESUMEN
Modification of histone proteins by lysine methylation is a principal chromatin regulatory mechanism (Shi, Y., and Whetstine, J. R. (2007) Mol. Cell 25, 1-14). Recently, lysine methylation has been shown also to play a role in regulating non-histone proteins, including the tumor suppressor protein p53 (Huang, J., and Berger, S. L. (2008) Curr. Opin. Genet. Dev. 18, 152-158). Here, we identify a novel p53 species that is dimethylated at lysine 382 (p53K382me2) and show that the tandem Tudor domain of the DNA damage response mediator 53BP1 acts as an "effector" for this mark. We demonstrate that the 53BP1 tandem Tudor domain recognizes p53K382me2 with a selectivity relative to several other protein lysine methylation sites and saturation states. p53K382me2 levels increase with DNA damage, and recognition of this modification by 53BP1 facilitates an interaction between p53 and 53BP1. The generation of p53K382me2 promotes the accumulation of p53 protein that occurs upon DNA damage, and this increase in p53 levels requires 53BP1. Taken together, our study identifies a novel p53 modification, demonstrates a new effector function for the 53BP1 tandem Tudor domain, and provides insight into how DNA damage signals are transduced to stabilize p53.