S-Nitrosylation Induces Both Autonomous Activation and Inhibition of Calcium/Calmodulin-dependent Protein Kinase II δ

NO is known to modulate calcium handling and cellular signaling in the myocardium, but key targets for NO in the heart remain unidentified. Recent reports have implied that NO can activate calcium/calmodulin (Ca²⁺/CaM)-dependent protein kinase II (CaMKII) in neurons and the heart. Here we use our novel sensor of CaMKII activation, Camui, to monitor changes in the conformation and activation of cardiac CaMKII (CaMKIIδ) activity after treatment with the NO donor S-nitrosoglutathione (GSNO). We demonstrate that exposure to NO after Ca²⁺/CaM binding to CaMKIIδ results in autonomous kinase activation, which is abolished by mutation of the Cys-290 site. However, exposure of CaMKIIδ to GSNO prior to Ca²⁺/CaM exposure strongly suppresses kinase activation and conformational change by Ca²⁺/CaM. This NO-induced inhibition was ablated by mutation of the Cys-273 site. We found parallel effects of GSNO on CaM/CaMKIIδ binding and CaMKIIδ-dependent ryanodine receptor activation in adult cardiac myocytes. We conclude that NO can play a dual role in regulating cardiac CaMKIIδ activity.     

Nitric Oxide Induces Ca2+-independent Activity of the Ca2+/Calmodulin-dependent Protein Kinase II (CaMKII)

Both signaling by nitric oxide (NO) and by the Ca²⁺/calmodulin (CaM)-dependent protein kinase II α isoform (CaMKIIα) are implicated in two opposing forms of synaptic plasticity underlying learning and memory, as well as in excitotoxic/ischemic neuronal cell death. For CaMKIIα, these functions specifically involve also Ca²⁺-independent autonomous activity, traditionally generated by Thr-286 autophosphorylation. Here, we demonstrate that NO-induced S-nitrosylation of CaMKIIα also directly generated autonomous activity, and that CaMKII inhibition protected from NO-induced neuronal cell death. NO induced S-nitrosylation at Cys-280/289, and mutation of either site abolished autonomy, indicating that simultaneous nitrosylation at both sites was required. Additionally, autonomy was generated only when Ca²⁺/CaM was present during NO exposure. Thus, generation of this form of CaMKIIα autonomy requires simultaneous signaling by NO and Ca²⁺. Nitrosylation also significantly reduced subsequent CaMKIIα autophosphorylation specifically at Thr-286, but not at Thr-305. A previously described reduction of CaMKII activity by S-nitrosylation at Cys-6 was also observed here, but only after prolonged (>5 min) exposure to NO donors. These results demonstrate a novel regulation of CaMKII by another second messenger system and indicate its involvement in excitotoxic neuronal cell death.     

Partial Purification and Characterization of a Ca2+-Dependent Protein Kinase from the Green Alga, Dunaliella salina

A calcium-dependent protein kinase was partially purified and characterized from the green alga Dunaliella salina. The enzyme was activated at free Ca²⁺ concentrations above 10⁻⁷ molar. and half-maximal activation was at about 3 × 10⁻⁷ molar. The optimum pH for its Ca²⁺-dependent activity was 7.5. The addition of various phospholipids and diolein had no effects on enzyme activity and did not alter the sensitivity of the enzyme toward Ca²⁺. The enzyme was inhibited by calmodulin antagonists, N-(6-aminohexyl)-1-naphthalene sulfonamide and N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide in a dose-dependent manner while the protein kinase C inhibitor, sphingosine, had little effect on enzyme activity up to 800 micromolar. Immunoassay showed some calmodulin was present in the kinase preparations. However, it is unlikely the kinase was calmodulin regulated, since it still showed stimulation by Ca²⁺ in gel assays after being electrophoretically separted from calmodulin by two different methods. This gel method of detection of the enzyme indicated that a protein band with an apparent molecular weight of 40,000 showed protein kinase activity at each one of the several steps in the purification procedure. Gel assay analysis also showed that after native gel isoelectric focusing the partially purified kinase preparations had two bands with calcium-dependent activity, at isoelectric points 6.7 and 7.1. By molecular weight, by isoelectric point, and by a comparative immunoassay, the Dunaliella kinase appears to differ from at least some of the calcium-dependent, but calmodulin and phospholipid independent kinases described from higher plants.     

Ca-dependent binding of calcium-binding protein 1 to presynaptic group III metabotropic glutamate receptors and blockage by phosphorylation of the receptors

Presynaptic group III metabotropic glutamate receptors (mGluRs) and Ca²⁺ channels are the main neuronal activity-dependent regulators of synaptic vesicle release, and they use common molecules in their signaling cascades. Among these, calmodulin (CaM) and the related EF-hand Ca²⁺-binding proteins are of particular importance as sensors of presynaptic Ca²⁺, and a multiple of them are indeed utilized in the signaling of Ca²⁺ channels. However, despite its conserved structure, CaM is the only known EF-hand Ca²⁺-binding protein for signaling by presynaptic group III mGluRs. Because the mGluRs and Ca²⁺ channels reciprocally regulate each other and functionally converge on the regulation of synaptic vesicle release, the mGluRs would be expected to utilize more EF-hand Ca²⁺-binding proteins in their signaling. Here I show that calcium-binding protein 1 (CaBP1) bound to presynaptic group III mGluRs competitively with CaM in a Ca²⁺-dependent manner and that this binding was blocked by protein kinase C (PKC)-mediated phosphorylation of these receptors. As previously shown for CaM, these results indicate the importance of CaBP1 in signal cross talk at presynaptic group III mGluRs, which includes many molecules such as cAMP, Ca²⁺, PKC, G protein, and Munc18-1. However, because the functional diversity of EF-hand calcium-binding proteins is extraordinary, as exemplified by the regulation of Ca²⁺ channels, CaBP1 would provide a distinct way by which presynaptic group III mGluRs fine-tune synaptic transmission.     

Guard Cells Possess a Calcium-Dependent Protein Kinase That Phosphorylates the KAT1 Potassium Channel

Increasing evidence suggests that changes in cytosolic Ca²⁺ levels and phosphorylation play important roles in the regulation of stomatal aperture and as ion transporters of guard cells. However, protein kinases responsible for Ca²⁺ signaling in guard cells remain to be identified. Using biochemical approaches, we have identified a Ca²⁺-dependent protein kinase with a calmodulin-like domain (CDPK) in guard cell protoplasts of Vicia faba. Both autophosphorylation and catalytic activity of CDPK are Ca²⁺ dependent. CDPK exhibits a Ca²⁺-induced electrophoretic mobility shift and its Ca²⁺-dependent catalytic activity can be inhibited by the calmodulin antagonists trifluoperazine and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide. Antibodies to soybean CDPKα cross-react with CDPK. Micromolar Ca²⁺ concentrations stimulate phosphorylation of several proteins from guard cells; cyclosporin A, a specific inhibitor of the Ca²⁺-dependent protein phosphatase calcineurin enhances the Ca²⁺-dependent phosphorylation of several soluble proteins. CDPK from guard cells phosphorylates the K⁺ channel KAT1 protein in a Ca²⁺-dependent manner. These results suggest that CDPK may be an important component of Ca²⁺ signaling in guard cells.     

Hippo Signaling Mediates Proliferation,Invasiveness, and Metastatic Potential of Clear Cell Renal Cell Carcinoma

Recent work has identified dysfunctional Hippo signaling to be involved in maintenance and progression of various human cancers, although data on clear cell renal cell carcinoma (ccRCC) have been limited. Here, we provide evidence implicating aberrant Hippo signaling in ccRCC proliferation, invasiveness, and metastatic potential. Nuclear overexpression of the Hippo target Yes-associated protein (YAP) was found in a subset of patients with ccRCC. Immunostaining was particularly prominent at the tumor margins and highlighted neoplastic cells invading the tumor-adjacent stroma. Short hairpin RNA-mediated knockdown of YAP significantly inhibited proliferation, migration, and anchorage-independent growth of ccRCC cells in soft agar and led to significantly reduced murine xenograft growth. Microarray analysis of YAP knockdown versus mock-transduced ccRCC cells revealed down-regulation of endothelin 1, endothelin 2, cysteine-rich, angiogenic inducer, 61 (CYR61), and c-Myc in ccRCC cells as well as up-regulation of the cell adhesion molecule cadherin 6. Signaling pathway impact analysis revealed activation of the p53 signaling and cell cycle pathways as well as inhibition of mitogen-activated protein kinase signaling on YAP down-regulation. Our data suggest CYR61 and c-Myc as well as signaling through the endothelin axis as bona fide downstream effectors of YAP and establish aberrant Hippo signaling as a potential therapeutic target in ccRCC.     

cAMP/PKA signalling reinforces the LATS–YAP pathway to fully suppress YAP in response to actin cytoskeletal changes

Actin cytoskeletal damage induces inactivation of the oncoprotein YAP (Yes‐associated protein). It is known that the serine/threonine kinase LATS (large tumour suppressor) inactivates YAP by phosphorylating its Ser127 and Ser381 residues. However, the events downstream of actin cytoskeletal changes that are involved in the regulation of the LATS–YAP pathway and the mechanism by which LATS differentially phosphorylates YAP on Ser127 and Ser381 in vivo have remained elusive. Here, we show that cyclic AMP (cAMP)‐dependent protein kinase (PKA) phosphorylates LATS and thereby enhances its activity sufficiently to phosphorylate YAP on Ser381. We also found that PKA activity is involved in all contexts previously reported to trigger the LATS–YAP pathway, including actin cytoskeletal damage, G‐protein‐coupled receptor activation, and engagement of the Hippo pathway. Inhibition of PKA and overexpression of YAP cooperate to transform normal cells and amplify neural progenitor pools in developing chick embryos. We also implicate neurofibromin 2 as an AKAP (A‐kinase‐anchoring protein) scaffold protein that facilitates the function of the cAMP/PKA–LATS–YAP pathway. Our study thus incorporates PKA as novel component of the Hippo pathway.     

Oxidative stress-CBP axis modulates MOB1 acetylation and activates the Hippo signaling pathway

Reactive oxygen species (ROS) are constantly produced in cells, an excess of which causes oxidative stress. ROS has been linked to regulation of the Hippo pathway; however, the underlying detailed mechanisms remain unclear. Here, we report that MOB1, a substrate of MST1/2 and co-activator of LATS1/2 in the canonical Hippo pathway, interacts with and is acetylated at lysine 11 by acetyltransferase CBP and deacetylated by HDAC6. MOB1-K11 acetylation stabilizes itself by reducing its binding capacity with E3 ligase Praja2 and subsequent ubiquitination. MOB1-K11 acetylation increases its phosphorylation and activates LATS1. Importantly, upstream oxidative stress signals promote MOB1 acetylation by suppressing CBP degradation, independent of MST1/2 kinase activity and HDAC6 deacetylation effect, thereby linking oxidative stress to activation of the Hippo pathway. Functionally, the acetylation-deficient mutant MOB1-K11R promotes lung cancer cell proliferation, migration and invasion in vitro and accelerates tumor growth in vivo, compared to the wild-type MOB1. Clinically, acetylated MOB1 corresponds to better prediction of overall survival in patients with non-small cell lung cancer. Therefore, as demonstrated, an oxidative stress-CBP regulatory axis controls MOB1-K11 acetylation and activates LATS1, thereby activating the Hippo pathway and suppressing YAP/TAZ nuclear translocation and tumor progression.     

Spectral studies of the Ca2+-dependent interaction of trifluoperazine with S100b

S100b is a calcium-binding protein that will bind to many calmodulin target molecules in a Ca²⁺-dependent manner. In order to study the Ca²⁺-dependent binding properties of S100b, its interaction with a calmodulin antagonist, trifluoperazine (TFP), was investigated using [¹⁹F]- and [¹H]-NMR and UV-difference spectroscopy. It was estimated from [¹⁹F]-NMR that in the absence of Ca²⁺, thek _1/2 value of TFP was 130 µM, while itsk _1/2 value decreased to 28 µM in the presence of Ca²⁺. The addition of KCl was not antagonistic to the Ca²⁺-dependent interaction of TFP to S100b. The chemical exchange rate of TFP with Ca²⁺-bound S100b was estimated to be 9×10² sec^–1. By comparison with TFP-calmodulin exchange rates, it is suggested that the TFP-binding site on S100b is structurally different from its binding sites on calmodulin. Proton NMR resonance broadening in the range 6.8–7.2 ppm, corresponding to phenylalanine nuclei of S100b, indicates that these residues may be involved in TFP binding. Addition of Ca²⁺ to a 1:1 mixture of S100b and TFP resulted in a red-shifted UV-difference spectrum, while no significant difference spectrum was detected when Mg²⁺ was added to a S100b-TFP solution. Thus, we suggest that Ca²⁺ induces the exposure of a hydrophobic domain on S100b containing one or more phenylalanine residues that will bind TFP but that this domain is different from the hydrophobic domain on calmodulin.     

Studies on the kinetics of cation-associated fluorescence changes in chloroplast membranes

A soluble Ca²⁺- and Ca²⁺—calmodulin-activated protein kinase was partially purified from wheat germ. The phosphorylation of histones and casein catalyzed by this enzyme is largely Ca²⁺-dependent. After repeated gel filtration of the protein kinase in the presence of 1 mM EGTA, the phosphorylation of casein and histones by the enzyme is activated 3-fold and up to 16-fold, respectively, by added calmodulin (12.5 μM). Such activation of the protein kinase by calmodulin is Ca²⁺-dependent. The protein kinase binds to calmodulin—Sepharose 4B in a Ca²⁺-dependent fashion. This type of Ca²⁺-activated protein kinase may be involved in stimulus—response coupling in plants.