Blockade of NFAT Activation by the Second Calcineurin Binding Site

Martı́nez-Martı́nez, Sara; Rodrı́guez, Antonio; López-Maderuelo, María Dolores; Ortega-Pérez, Inmaculada; Vázquez, Jesús; Redondo, Juan Miguel

doi:10.1074/jbc.m513885200

Cited by 65 publications

(90 citation statements)

References 44 publications

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“…To confirm that the CsA-mediated reduction of Adamts-1 expression by VEGF was not the result of off-target effects of CsA, we transduced ECs and VSMCs with lentiviral vectors encoding LXVP, a peptide that inhibits CN phosphatase activity and blocks its docking site for NFAT (28). Consistent with the data obtained with CsA, LXVP expression in vascular cells abrogated Adamts-1 gene upregulation in response to VEGF, but not in response to Ang-II ( Fig.…”

Section: Different Signal Transduction Pathways Regulate Vegf-andsupporting

confidence: 72%

C/EBPβ and Nuclear Factor of Activated T Cells Differentially Regulate Adamts-1 Induction by Stimuli Associated with Vascular Remodeling

Oller

Alfranca

Villahoz

et al. 2015

Molecular and Cellular Biology

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Section: Different Signal Transduction Pathways Regulate Vegf-andsupporting

confidence: 72%

C/EBPβ and Nuclear Factor of Activated T Cells Differentially Regulate Adamts-1 Induction by Stimuli Associated with Vascular Remodeling

Oller

Alfranca

Villahoz

et al. 2015

Molecular and Cellular Biology

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“…Other proteins that interact with calcineurin and repress its activity include RCAN1.4, cyclophilin (complexed with CsA) (38), FKBP12 (complexed with FK506) (38), AKAP79 (7), Cain (33), Bcl-2 (57), and calsarcin (16). Interaction between NFAT and calcineurin depends on 2 conserved sites within the NFAT sequence, viz., PxIxIT and LxVP (42). AKAP79 and Cain similarly inhibit calcineurin through their PxIxIT motif (58).…”

Section: Discussionmentioning

confidence: 99%

FHL2 Binds Calcineurin and Represses Pathological Cardiac Growth

Hojayev

Rothermel²,

Gillette³

et al. 2012

Molecular and Cellular Biology

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Stress-induced hypertrophic growth of the heart predisposes the heart to arrhythmia, contractile dysfunction, and clinical heart failure. FHL2 (four-and-a-half LIM domain protein 2) is expressed predominantly in the heart, and inactivation of the gene coding for FHL2 leads to exaggerated responsiveness to adrenergic stress. Activation of calcineurin occurs downstream of ␤-adrenergic signaling and is required for isoproterenol-induced myocardial hypertrophy. Based on these facts, we hypothesized that FHL2 suppresses stress-induced activation of calcineurin. FHL2 is upregulated in mouse hearts exposed to isoproterenol, a ␤-adrenergic agonist, and isoproterenol-induced increases in the NFAT target genes RCAN1.4 and BNP were amplified significantly in FHL2 knockout (FHL2 ؊/؊ ) mice compared with levels in wild-type (WT) mice. To determine whether the effect of FHL2 on NFAT target gene transcript levels occurred at the level of transcription, HEK 293 cells and neonatal rat ventricular myocytes (NRVMs) were transfected with a luciferase reporter construct harboring the NFAT-dependent promoters of either RCAN1 or interleukin 2 (IL-2). Consistent with the in vivo data, small interfering RNA (siRNA) knockdown of FHL2 led to increased activation of these promoters by constitutively active calcineurin or the calcium ionophore ionomycin. Importantly, activation of the RCAN1 promoter by ionomycin, in control and FHL2 knockdown cells, was abolished by the calcineurin inhibitor cyclosporine, confirming the calcineurin dependence of the response. Overexpression of FHL2 inhibited activation of both NFAT reporter constructs. Furthermore, NRVMs overexpressing FHL2 exhibited reduced hypertrophic growth in response to constitutively active calcineurin, as measured by cell cross-sectional area and fetal gene expression. Finally, immunostaining in isolated adult cardiomyocytes revealed colocalization of FHL2 and calcineurin predominantly at the sarcomere and activation of calcineurin by endothelin-1-facilitated interaction between FHL2 and calcineurin. FHL2 is an endogenous, agonist-dependent suppressor of calcineurin. E pidemiological evidence links left ventricular (LV) hypertrophy with adverse cardiovascular events, including heart failure and death (1,13,35,36). Consistent with this, therapies that improve clinical outcomes are often associated with regression of ventricular hypertrophy (11,19,46). However, whereas significant strides have been made in elucidating the molecular circuitry governing pathological cardiac remodeling (23), few therapies in clinical use target cell growth mechanisms directly.Hypertrophic growth of the heart in response to a variety of pathological stresses is an initially adaptive response that, left unchecked, often progresses to heart failure (25). In many instances, the intracellular protein phosphatase calcineurin is a major mediator of stress-induced cardiac hypertrophy. Upon activation, calcineurin dephosphorylates NFAT (nuclear factor of activated T cells), which, in turn, translocates into the n...

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“…Here we report three findings that support this hypothesis: (i) mCherry-Cts1 and GFP-Cna1 colocalize specifically during thermal stress at 37°C; (ii) Cts1 is hyperphosphorylated in the presence of the calcineurin inhibitor FK506 at 37°C, which suggests that Cts1 is dephosphorylated in a calcineurin-dependent manner; and (iii) mCherryCts1 and GFP-Cna1 coimmunoprecipitate, with stronger complex formation at 37°C than at 24°C. It is important that Cts1 also contains the LAVP calcineurin-binding motif found in NFATc1 and NFATc4, as well as the LAPP calcineurinbinding motif found in the calcineurin regulatory protein RCAN1 (25,27,29,37,43) (Fig. 1A).…”

Section: Discussionmentioning

confidence: 99%

The C2 Domain Protein Cts1 Functions in the Calcineurin Signaling Circuit during High-Temperature Stress Responses in Cryptococcus neoformans

et al. 2011

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Calcineurin is a conserved calcium/calmodulin-dependent serine/threonine-specific protein phosphatase that acts in cell stress responses. Calcineurin is essential for growth at 37°C and for virulence of the human fungal pathogen Cryptococcus neoformans, but its substrates remain unknown. The C2 domain-containing, phospholipid-binding protein Cts1 was previously identified as a multicopy suppressor of a calcineurin mutation in C. neoformans. Here we further characterize the function of Cts1 and the links between Cts1 and calcineurin. GFP-Cts1 localizes to cytoplasmic puncta and colocalizes with the endosomal marker FM4-64. The cts1⌬ mutant shows a distinct FM4-64 staining pattern, suggesting involvement of Cts1 in endocytic trafficking. In large budded cells, GFP-Cts1 localizes transiently at the mother bud neck, as a single ring that undergoes contraction. mCherry-Cts1 colocalizes with the GFP-tagged calcineurin catalytic subunit Cna1 at sites of mRNA processing at 37°C, suggesting that Cts1 and calcineurin function coordinately during thermal stress. GFP-Cts1 exhibits slower electrophoretic mobility for cells grown at 37°C than for cells grown at 24°C, and the shift to a higher molecular weight is more pronounced in the presence of the calcineurin inhibitor FK506. In vitro treatment with calf intestinal alkaline phosphatase (CIP) restores faster electrophoretic mobility to GFP-Cts1, suggesting that Cts1 is phosphorylated at 37°C and may be dephosphorylated in a calcineurindependent manner. mCherry-Cts1 also coimmunoprecipitates with GFP-Cna1, with greater complex formation at 37°C than at 24°C. Taken together, these findings support potential roles for Cts1 in endocytic trafficking, mRNA processing, and cytokinesis and suggest that Cts1 is a substrate of calcineurin during high-temperature stress responses.Cryptococcus neoformans is a human fungal pathogen that causes life-threatening meningitis, primarily in immunocompromised patients (5, 17). Cryptococcal meningitis is one of the most important HIV-related opportunistic infections, with ϳ1 million cases occurring each year (36). Identification of novel therapeutic targets for cryptococcosis is essential given the toxic effects of existing therapies and the rising prevalence of drug-resistant strains (38).The ability to survive, grow, and divide at human physiological temperature is an important C. neoformans virulence attribute and is orchestrated by elaborate signaling pathways that are not yet fully elucidated (3, 19). Calcineurin, a calcium/ calmodulin-dependent serine/threonine-specific protein phosphatase, is essential for C. neoformans growth at 37°C and for its virulence (1, 21, 35). Calcineurin consists of two subunits, the catalytic A (Cna1) and the regulatory B (Cnb1) subunits, both of which are necessary for enzymatic function and survival of C. neoformans at 37°C (1,12,35). Studies of both pathogenic and nonpathogenic fungi have revealed important roles for calcineurin in myriad physiological processes, including morphogenesis, cell cycle progress...

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Blockade of NFAT Activation by the Second Calcineurin Binding Site

Abstract: Activation of NFAT transcription factors requires their dephosphorylation by the phosphatase calcineurin (CN).

Cited by 65 publications

References 44 publications

C/EBPβ and Nuclear Factor of Activated T Cells Differentially Regulate Adamts-1 Induction by Stimuli Associated with Vascular Remodeling

C/EBPβ and Nuclear Factor of Activated T Cells Differentially Regulate Adamts-1 Induction by Stimuli Associated with Vascular Remodeling

FHL2 Binds Calcineurin and Represses Pathological Cardiac Growth

The C2 Domain Protein Cts1 Functions in the Calcineurin Signaling Circuit during High-Temperature Stress Responses in Cryptococcus neoformans

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