Unexpected Heterodivalent Recruitment of NOS1AP to nNOS Reveals Multiple Sites for Pharmacological Intervention in Neuronal Disease Models

Li, Li-Li; Mera, Raquel M Melero-Fernandez de; Chen, Jia; Ba, Wang; Kasri, Nael Nadif; Zhang, Mingjie; Courtney, Michael J.

doi:10.1523/jneurosci.0037-15.2015

Cited by 32 publications

(78 citation statements)

References 60 publications

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“…Moreover, an intracellular signaling array was applied to screen important signaling cascades in CAPON-S-overexpressing glioma cells. In comparison to p38MAPK inactivation in hippocampal neurons [24,25], overexpression of CAPON-S remarkably suppressed the phosphorylation of Akt (Thr308 and Ser473) and S6 (Ser235/236), in glioma cells, without affecting any signaling molecule in the MAPK pathway (Table S1). Interestingly, the decreased phosphorylation of mTOR (Ser2448) as found by antibody array was not confirmed by Western blot.…”

Section: Discussionmentioning

confidence: 99%

Low Expression of CAPON in Glioma Contributes to Cell Proliferation via the Akt Signaling Pathway

Gao

Wang

Zhang

et al. 2016

IJMS

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CAPON is an adapter protein for nitric oxide synthase 1 (NOS1). CAPON has two isoforms in the human brain: CAPON-L (long form of CAPON) and CAPON-S (short form of CAPON). Recent studies have indicated the involvement of CAPON in tumorigenesis beyond its classical role in NOS1 activity regulation. In this study, we found that the protein levels of CAPON-S, but not than CAPON-L, were significantly decreased in glioma tissues. Therefore, we established lentivirus-mediated stable cell lines with CAPON-S overexpression or down-regulation, and investigated the role of CAPON-S in the proliferation of glioma cells by using CCK8, EdU, and flow cytometry assays. Overexpression of CAPON-S reduced the cell variability and the percentage of EdU-positive cells, and arrested the cells in the G1 phase in glioma cells. Silencing of CAPON by short-hairpin RNA showed the opposite effects. Furthermore, an intracellular signaling array revealed that overexpression of CAPON-S resulted in a remarkable reduction in the phosphorylation of Akt and S6 ribosomal protein in glioma cells, which was further confirmed by Western blot. These findings suggest that CAPON may function as a tumor suppressor in human brain glioma and that the inactivation of the Akt signaling pathway caused by CAPON-S overexpression may provide insight into the underlying mechanism of CAPON in glioma cell proliferation.

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Section: Discussionmentioning

confidence: 99%

Low Expression of CAPON in Glioma Contributes to Cell Proliferation via the Akt Signaling Pathway

Gao

Wang

Zhang

et al. 2016

IJMS

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“…It is interesting, in this context, that several proteins described to associate to the nNOS PDZ domain such as C-terminal binding protein and phosphofructokinase-M failed to do so in a large screening of binding partners for PDZ domains present in the mouse proteome (9). Furthermore, it has been recently proposed that binding of the PDZ domain of nNOS to the C termini of protein targets does not proceed as a canonical peptide/PDZ domain interaction because the low affinity of this association might not be physiologically relevant (25). Thus, a heterodivalent interaction between NOS1AP/ CAPON and the PDZ domain of nNOS has been put forward in which the insertion of the C terminus within the PDZ binding groove becomes accompanied by a secondary, lateral interaction that increases the overall binding affinity.…”

Section: Neuronal Nos (Nnos)mentioning

confidence: 99%

Insights into the C-terminal Peptide Binding Specificity of the PDZ Domain of Neuronal Nitric-oxide Synthase

Merino-Gracia

Costas-Insua

Canales

et al. 2016

Journal of Biological Chemistry

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Neuronal nitric-oxide synthase, unlike its endothelial and inducible counterparts, displays a PDZ (PSD-95/Dlg/ZO-1) domain located at its N terminus involved in subcellular targeting. The C termini of various cellular proteins insert within the binding groove of this PDZ domain and determine the subcellular distribution of neuronal NOS (nNOS). The molecular mechanisms underlying these interactions are poorly understood because the PDZ domain of nNOS can apparently exhibit class I, class II, and class III binding specificity. In addition, it has been recently suggested that the PDZ domain of nNOS binds with very low affinity to the C termini of target proteins, and a necessary simultaneous lateral interaction must take place for binding to occur. We describe herein that the PDZ domain of nNOS can behave as a bona fide class III PDZ domain and bind to C-terminal sequences with acidic residues at the P ؊2 position with low micromolar binding constants. Binding to C-terminal sequences with a hydrophobic residue at the P ؊2 position plus an acidic residue at the P ؊3 position (class II) can also occur, although interactions involving residues extending up to the P ؊7 position mediate this type of binding. This promiscuous behavior also extends to its association to class I sequences, which must display a Glu residue at P ؊3 and a Thr residue at P ؊2 . By means of site-directed mutagenesis and NMR spectroscopy, we have been able to identify the residues involved in each specific type of binding and rationalize the mechanisms used to recognize binding partners. Finally, we have analyzed the high affinity association of the PDZ domain of nNOS to claudin-3 and claudin-14, two tight junction tetraspan membrane proteins that are essential components of the paracellular barrier. Neuronal NOS (nNOS)2 is expressed constitutively in specific neurons of the brain and in the spinal cord, peripheral nitrergic nerves, epithelial cells of various organs, pancreatic islet cells, and vascular smooth muscle (1). nNOS differs from the two other mammalian isoforms, endothelial NOS and inducible NOS by an additional ϳ300-residue N-terminal extension mostly involved in specific subcellular targeting. The N terminus of nNOS contains a PDZ domain (first found in the proteins PSD-95, Dlg, and ZO-1) (2), a ␤-hairpin module that associates to ␣1-syntrophin and PSD-95 (3), a DYNLL1 binding site (4), and a stretch known to bind to repeats R16/R17 of dystrophin (5). Brain nNOS is found in particulate and soluble forms in cells, and the differential subcellular localization of nNOS in various tissues may contribute to its diverse functions. Reinforcement of the idea that the subcellular targeting is exquisitely governed by this N-terminal extension came from the observation that an N-terminal deletion mutant of nNOS is an active, mislocalized enzyme (6). In the nervous system, the PDZ domain of PSD-95 is known to couple NMDA receptors to nNOS so that ⅐ NO release becomes reversibly regulated by Ca 2ϩ /calmodulin binding (1). PDZ domains are modula...

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“…His-nNOS(1–155) was generated as described Li et al (2015) as was GST-nNOS(1–155) and GST. All His-nNOS[1–155] used for fluorescence polarization (FP) assays was polished by Superdex200 size-exclusion chromatography as described Li et al (2015).…”

Section: Methodsmentioning

confidence: 99%

Efficient Binding of the NOS1AP C-Terminus to the nNOS PDZ Pocket Requires the Concerted Action of the PDZ Ligand Motif, the Internal ExF Site and Structural Integrity of an Independent Element

Cisek

Courtney

2017

Front. Mol. Neurosci.

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Neuronal nitric oxide synthase is widely regarded as an important contributor to a number of disorders of excitable tissues. Recently the adaptor protein NOS1AP has emerged as a contributor to several nNOS-linked conditions. As a consequence, the unexpectedly complex mechanisms of interaction between nNOS and its effector NOS1AP have become a particularly interesting topic from the point of view of both basic research and the potential for therapeutic applications. Here we demonstrate that the concerted action of two previously described motif regions contributing to the interaction of nNOS with NOS1AP, the ExF region and the PDZ ligand motif, efficiently excludes an alternate ligand from the nNOS-PDZ ligand-binding pocket. Moreover, we identify an additional element with a denaturable structure that contributes to interaction of NOS1AP with nNOS. Denaturation does not affect the functions of the individual motifs and results in a relatively mild drop, ∼3-fold, of overall binding affinity of the C-terminal region of NOS1AP for nNOS. However, denaturation selectively prevents the concerted action of the two motifs that normally results in efficient occlusion of the PDZ ligand-binding pocket, and results in 30-fold reduction of competition between NOS1AP and an alternate PDZ ligand.

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Unexpected Heterodivalent Recruitment of NOS1AP to nNOS Reveals Multiple Sites for Pharmacological Intervention in Neuronal Disease Models

Cited by 32 publications

References 60 publications

Low Expression of CAPON in Glioma Contributes to Cell Proliferation via the Akt Signaling Pathway

Low Expression of CAPON in Glioma Contributes to Cell Proliferation via the Akt Signaling Pathway

Insights into the C-terminal Peptide Binding Specificity of the PDZ Domain of Neuronal Nitric-oxide Synthase

Efficient Binding of the NOS1AP C-Terminus to the nNOS PDZ Pocket Requires the Concerted Action of the PDZ Ligand Motif, the Internal ExF Site and Structural Integrity of an Independent Element

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