Role of Ca2+/Calmodulin-dependent Protein Kinase II in Drosophila Photoreceptors

Ca²⁺ modulates the visual response in both vertebrates and invertebrates. In Drosophila photoreceptors, an increase of cytoplasmic Ca²⁺ mimics light adaptation. Little is known regarding the mechanism, however. We explored the role of the sole Drosophila Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) to mediate light adaptation. CaMKII has been implicated in the phosphorylation of arrestin 2 (Arr2). However, the functional significance of Arr2 phosphorylation remains debatable. We identified retinal CaMKII by anti-CaMKII antibodies and by its Ca²⁺-dependent autophosphorylation. Moreover, we show that phosphorylation of CaMKII is greatly enhanced by okadaic acid, and indeed, purified PP2A catalyzes the dephosphorylation of CaMKII. Significantly, we demonstrate that anti-CaMKII antibodies co-immunoprecipitate, and CaMKII fusion proteins pull down the catalytic subunit of PP2A from fly extracts, indicating that PP2A interacts with CaMKII to form a protein complex. To investigate the function of CaMKII in photoreceptors, we show that suppression of CaMKII in transgenic flies affects light adaptation and increases prolonged depolarizing afterpotential amplitude, whereas a reduced PP2A activity brings about reduced prolonged depolarizing afterpotential amplitude. Taken together, we conclude that CaMKII is involved in the negative regulation of the visual response affecting light adaptation, possibly by catalyzing phosphorylation of Arr2. Moreover, the CaMKII activity appears tightly regulated by the co-localized PP2A.Visual transduction is the process that converts the signal of light (photons) into a change of membrane potential in photoreceptors (see Ref. 1 for review). Visual signaling is initiated upon the activation of rhodopsins by light: light switches on rhodopsin to generate metarhodopsin, which activates the heterotrimeric G_q in Drosophila (2). Subsequently, the GTP-bound Gα_q subunit activates phospholipase Cβ4 encoded by the norpA (no receptor potential A) gene (3). Phospholipase Cβ4 catalyzes the breakdown of phosphoinositol 4,5-bisphosphate to generate diacylglycerol, which or its metabolite has been implicated in gating the transient receptor potential (TRP)² and TRP-like channels (4, 5). TRP is the major Ca²⁺ channel that mediates the light-dependent depolarization response leading to an increase of cytosolic Ca²⁺ in photoreceptors. The rise of intracellular Ca²⁺ modulates several aspects of the visual response including activation, deactivation, and light adaptation (6). For example, Ca²⁺ together with diacylglycerol activates a classical protein kinase C, eye-PKC, which is critical for the negative regulation of visual signaling by modulating deactivation and light adaptation (7–11).Light adaptation is the process by which photoreceptors adjust the visual sensitivity in response to ambient background light by down-regulating rhodopsin-mediated signaling. Light adaptation can be arbitrarily subdivided into long term and short term adaptation and may involve multiple regulations to reduce the efficiency of rhodopsin, G protein, or cation channels. For example, translocation of both G_q (12, 13) and TRP-like channels (14, 15) out of the visual organelle may contribute to long term adaptation in Drosophila. In contrast, short term adaptation may be orchestrated by modulating the activity of signaling proteins by protein kinases. Hardie and co-workers (16) demonstrated that an increase of cytoplasmic [Ca²⁺] mimicked light adaptation, leading to inhibition of the light-induced current. These authors also showed that light adaptation is independent of eye-PKC. Thus the effect of cytoplasmic Ca²⁺ to control light adaptation is likely mediated via calmodulin and CaMKII. The contribution of CaMKII to light adaptation has not been explored.CaMKII is a multimeric Ca²⁺/calmodulin-dependent protein kinase that modulates diverse signaling processes (17). Drosophila contains one CaMKII gene (18) that gives rise to at least four protein isoforms (19). These CaMKII isoforms share over 85% sequence identities with the α isoform of vertebrate CaMKII. For insights into the in vivo physiological role of CaMKII, Griffith et al. (20) generated transgenic flies (ala) expressing an inhibitory domain of the rat CaMKII under the control of a heat shock promoter, hsp70. They demonstrated that, upon heat shock treatment, the overexpression of the inhibitory peptide resulted in a suppression of the endogenous CaMKII activity in the transgenic flies (20). It has been shown that inhibition of CaMKII affects learning and memory (20) and neuronal functions (21–24). In photoreceptors, CaMKII has been implicated in the phosphorylation of the major visual arrestin, Arr2 (25, 26). However, how phosphorylation of Arr2 by CaMKII modifies the visual signaling remains to be elucidated.Here we report the biochemical and electrophysiological analyses of CaMKII in Drosophila retina. We demonstrate that suppression of CaMKII in ala¹ transgenic flies leads to a phenotype indicative of defective light adaptation. The ala¹ flies also display greater visual response, suggesting a defect in Arr2. These results support the notion that CaMKII plays a role in the negative regulation of the visual response. Our biochemical analyses demonstrate that dephosphorylation of CaMKII is mediated by protein phosphatase 2A (PP2A). Importantly, we show that PP2A interacts with CaMKII, indicating that CaMKII forms a stable protein complex with PP2A to ensure a tight regulation of the kinase activity. Thus a partial loss of function in PP2A would elevate the CaMKII activity. Indeed, we show that mts heterozygotes display reduced prolonged depolarizing potential (PDA) amplitude. This PDA phenotype strongly suggests that Arr2 becomes more effective to terminate the visual signaling in mts flies. Together, our findings indicate that the ability of Arr2 to terminate metarhodopsin is increased upon phosphorylation by CaMKII, and the retinal CaMKII activity is regulated by PP2A.     

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.     

Sorbitol Uptake in Plasma Membrane Vesicles Isolated from Immortalized Rabbit TALH Cells: Activation by a Ca2+/Calmodulin-dependent Protein Kinase

Apical plasma membrane vesicles were isolated from cultures of immortalized thick ascending limb of Henle's loop (TALH) cells and sorbitol uptake was investigated using a rapid filtration technique. In the presence of Mg²⁺, Ca²⁺, ATP, and GTP sorbitol equilibrated within three minutes with the intravesicular space; this uptake was reduced by 75% when the incubation temperature was decreased from 37°C to 4°C. A lower level of uptake was also observed in the presence of 100 μm quinidine and when Ca²⁺ or ATP were omitted from the medium. Membranes preincubated with Mg²⁺, Ca²⁺, ATP, and GTP showed, however, a high sorbitol uptake in ATP-free medium. Staurosporine, but only at high concentrations of 200 nm, inhibited sorbitol uptake when present during the transport experiments or during the preincubation with ATP. Similar results were obtained with 1 μm trifluoperazine. Protein kinase C inhibitory peptide was ineffective whereas 20 nm KT 5926, at low concentrations a specific inhibitor of Ca²⁺/calmodulin-dependent kinase, attenuated the activation. On the basis of these data we suggest that a Ca²⁺/calmodulin-dependent kinase is a mediator of regulation of sorbitol plasma membrane permeability in renal medullary cells. Received: 31 March 1997/Revised: 11 June 1997     

LKB1 Mediates the Development of Conventional and Innate T Cells via AMP-Dependent Kinase Autonomous Pathways

The present study has examined the role of the serine/threonine kinase LKB1 in the survival and differentiation of CD4/8 double positive thymocytes. LKB1-null DPs can respond to signals from the mature α/β T-cell-antigen receptor and initiate positive selection. However, in the absence of LKB1, thymocytes fail to mature to conventional single positive cells causing severe lymphopenia in the peripheral lymphoid tissues. LKB1 thus appears to be dispensable for positive selection but important for the maturation of positively selected thymocytes. LKB1 also strikingly prevented the development of invariant Vα14 NKT cells and innate TCR αβ gut lymphocytes. Previous studies with gain of function mutants have suggested that the role of LKB1 in T cell development is mediated by its substrate the AMP-activated protein kinase (AMPK). The present study now analyses the impact of AMPK deletion in DP thymocytes and shows that the role of LKB1 during the development of both conventional and innate T cells is mediated by AMPK-independent pathways.     

Adiponectin blocks interleukin-18-mediated endothelial cell death via APPL1-dependent AMP-activated protein kinase (AMPK) activation and IKK/NF-kappaB/PTEN suppression

The adipocyte-derived cytokine adiponectin is known to exert anti-inflammatory and anti-apoptotic effects. In patients with atherosclerotic cardiovascular disease, circulating levels of adiponectin correlate inversely with those of the proinflammatory, proapoptotic cytokine interleukin (IL)-18. The opposing actions of IL-18 and adiponectin on both cell survival and inflammation led us to investigate whether adiponectin signaling antagonizes IL-18-mediated endothelial cell death and to identify the underlying molecular mechanisms. Treatment with IL-18 suppressed Akt phosphorylation and its associated kinase activity, induced IkappaB kinase (IKK)-NF-kappaB-dependent PTEN activation, and promoted endothelial cell death. Pretreatment with adiponectin stimulated APPL1-dependent AMPK activation, reversed Akt inhibition in a phosphatidylinositol 3-kinase-dependent manner, blocked IKK-NF-kappaB-PTEN signaling, reduced caspase-3 activity, blocked Bax translocation, and inhibited endothelial cell death. The cytoprotective effect of adiponectin signaling was recapitulated by treatment with the pharmacological AMPK activator 5-aminoimidazole-4-carboxamide-1-beta-riboside. Collectively, these results demonstrated that adiponectin reverses IL-18-mediated endothelial cell death through an AMPK-associated mechanism, which may thus have therapeutic potential for diminishing IL-18-dependent vascular injury and inflammation.     

Dentin Phosphophoryn Activates Smad Protein Signaling through Ca2+-Calmodulin-dependent Protein Kinase II in Undifferentiated Mesenchymal Cells

Dentin phosphophoryn (DPP) is a major noncollagenous protein in the dentin matrix. In this study, we demonstrate that pluripotent stem cells such as C3H10T1/2 and human bone marrow cells can be committed to the osteogenic lineage by DPP. Treatment with DPP can stimulate the release of intracellular Ca²⁺. This calcium flux triggered the activation of Ca²⁺-calmodulin-dependent protein kinase II (CaMKII). Activated CaMKII induced the phosphorylation of Smad1 and promoted nuclear translocation of p-Smad1. Inhibition of store Ca²⁺ depletion by 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis(acetoxymethyl ester) or down-regulation of CaMKII by KN-62, a selective cell-permeable pharmacological inhibitor or a dominant negative plasmid of CaMKII, blocked DPP-mediated Smad1 phosphorylation. Activation of Smad1 resulted in the expression of osteogenic markers such as Runx2, Osterix, DMP1, Bone sialoprotein, Osteocalcin, NFATc1, and Schnurri-2, which have been implicated in osteoblast differentiation. These findings suggest that DPP is capable of triggering commitment of pluripotent stem cells to the osteogenic lineage.     

Calcium/Calmodulin-dependent Protein Kinase Kinase 2: Roles in Signaling and Pathophysiology

Many cellular Ca(2+)-dependent signaling cascades utilize calmodulin (CaM) as the intracellular Ca(2+) receptor. Ca(2+)/CaM binds and activates a plethora of enzymes, including CaM kinases (CaMKs). CaMKK2 is one of the most versatile of the CaMKs and will phosphorylate and activate CaMKI, CaMKIV, and AMP-activated protein kinase. Cell expression of CaMKK2 is limited, yet CaMKK2 is involved in regulating many important physiological and pathophysiological processes, including energy balance, adiposity, glucose homeostasis, hematopoiesis, inflammation, and cancer. Here, we explore known functions of CaMKK2 and discuss its potential as a target for therapeutic intervention.     

Ca2+-independent Activation of Ca2+/Calmodulin-dependent Protein Kinase II Bound to the C-terminal Domain of CaV2.1 Calcium Channels

Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) forms a major component of the postsynaptic density where its functions in synaptic plasticity are well established, but its presynaptic actions are poorly defined. Here we show that CaMKII binds directly to the C-terminal domain of Ca_V2.1 channels. Binding is enhanced by autophosphorylation, and the kinase-channel signaling complex persists after dephosphorylation and removal of the Ca²⁺/CaM stimulus. Autophosphorylated CaMKII can bind the Ca_V2.1 channel and synapsin-1 simultaneously. CaMKII binding to Ca_V2.1 channels induces Ca²⁺-independent activity of the kinase, which phosphorylates the enzyme itself as well as the neuronal substrate synapsin-1. Facilitation and inactivation of Ca_V2.1 channels by binding of Ca²⁺/CaM mediates short term synaptic plasticity in transfected superior cervical ganglion neurons, and these regulatory effects are prevented by a competing peptide and the endogenous brain inhibitor CaMKIIN, which blocks binding of CaMKII to Ca_V2.1 channels. These results define the functional properties of a signaling complex of CaMKII and Ca_V2.1 channels in which both binding partners are persistently activated by their association, and they further suggest that this complex is important in presynaptic terminals in regulating protein phosphorylation and short term synaptic plasticity.     

AMP-activated Protein Kinase Stimulates Warburg-like Glycolysis and Activation of Satellite Cells during Muscle Regeneration

Satellite cells are the major myogenic stem cells residing inside skeletal muscle and are indispensable for muscle regeneration. Satellite cells remain largely quiescent but are rapidly activated in response to muscle injury, and the derived myogenic cells then fuse to repair damaged muscle fibers or form new muscle fibers. However, mechanisms eliciting metabolic activation, an inseparable step for satellite cell activation following muscle injury, have not been defined. We found that a noncanonical Sonic Hedgehog (Shh) pathway is rapidly activated in response to muscle injury, which activates AMPK and induces a Warburg-like glycolysis in satellite cells. AMPKα1 is the dominant AMPKα isoform expressed in satellite cells, and AMPKα1 deficiency in satellite cells impairs their activation and myogenic differentiation during muscle regeneration. Drugs activating noncanonical Shh promote proliferation of satellite cells, which is abolished because of satellite cell-specific AMPKα1 knock-out. Taken together, AMPKα1 is a critical mediator linking noncanonical Shh pathway to Warburg-like glycolysis in satellite cells, which is required for satellite activation and muscle regeneration.     

Modulation of Inositol 1,4,5-Trisphosphate Receptor Type 2 Channel Activity by Ca2+/Calmodulin-dependent Protein Kinase II (CaMKII)-mediated Phosphorylation

InsP₃-mediated calcium release through the type 2 inositol 1,4,5-trisphosphate receptor (InsP₃R2) in cardiac myocytes results in the activation of associated CaMKII, thus enabling the kinase to act on downstream targets, such as histone deacetylases 4 and 5 (HDAC4 and HDAC5). The CaMKII activity also feedback modulates InsP₃R2 function by direct phosphorylation and results in a dramatic decrease in the receptor-channel open probability (P_o). We have identified S150 in the InsP₃R2 core suppressor domain (amino acids 1–225) as the specific residue that is phosphorylated by CaMKII. Site-directed mutagenesis reveals that S150 is the CaMKII phosphorylation site responsible for modulation of channel activity. Nonphosphorylatable (S150A) and phosphomimetic (S150E) mutations were studied in planar lipid bilayers. The InsP₃R2 S150A channel showed no decrease in activity when treated with CaMKII. Conversely, the phosphomimetic (S150E) channel displayed a very low P_o under normal recording conditions in the absence of CaMKII (2 μm InsP₃ and 250 nm [Ca²⁺]_FREE) and mimicked a WT channel that has been phosphorylated by CaMKII. Phopho-specific antibodies demonstrate that InsP₃R2 Ser-150 is phosphorylated in vivo by CaMKIIδ. The results of this study show that serine 150 of the InsP₃R2 is phosphorylated by CaMKII and results in a decrease in the channel open probability.     

Trimethyltin Stimulates Protein Kinase C Translocation Through Receptor-Mediated Phospholipase C Activation in PC12 Cells

Abstract: Trimethyltin (TMT) is a potent neurotoxic compound that initiates a delayed neuronal cell death. Previously we have shown that TMT-induced cytotoxicity is associated with protein kinase C (PKC) translocation and activation. The present study investigates the mechanism underlying TMT-stimulated PKC translocation in PC12 cells. TMT exposure led to a rapid increase in intracellular levels of inositol 1,4,5-trisphosphate (IP₃), a product of phospholipase C (PLC). This was significantly decreased by pretreating cells with antagonists to either the cholinergic muscarinic receptor (atropine) or the glutamatergic metabotropic receptor [(+)-α-methyl-4-carboxyphenylglycine; (+)-MCPG]. Furthermore, the rise in IP₃ level was blocked by pretreating cells with a PLC inhibitor (U-73122) or by a combination of atropine and (+)-MCPG. This pretreatment also significantly decreased TMT-stimulated PKC translocation, indicating that TMT-mediated PKC translocation was related to PLC activation, presumably through formation of diacylglycerol, an endogenous activator of PKC and product of PLC. It is interesting that atropine and (+)-MCPG did not provide protection against TMT-induced cytotoxicity in these cells. However, these data suggest that TMT causes the release of cellular constituents that activate G protein-coupled receptors, ultimately leading to PKC translocation.     

Thromboxane A2 Receptor Activates a Rho-associated Kinase/LKB1/PTEN Pathway to Attenuate Endothelium Insulin Signaling

This study was conducted to elucidate the molecular mechanisms of thromboxane A2 receptor (TP)-induced insulin resistance in endothelial cells. Exposure of human umbilical vein endothelial cells (HUVECs) or mouse aortic endothelial cells to either IBOP or U46619, two structurally related thromboxane A₂ mimetics, significantly reduced insulin-stimulated phosphorylation of endothelial nitric-oxide synthase (eNOS) at Ser¹¹⁷⁷ and Akt at Ser⁴⁷³. These effects were abolished by pharmacological or genetic inhibitors of TP. TP-induced suppression of both eNOS and Akt phosphorylation was accompanied by up-regulation of PTEN (phosphatase and tension homolog deleted on chromosome 10), Ser³⁸⁰/Thr^382/383 PTEN phosphorylation, and PTEN lipid phosphatase activity. PTEN-specific small interference RNA restored insulin signaling in the face of TP activation. The small GTPase, Rho, was also activated by TP stimulation, and pretreatment of HUVECs with Y27632, a Rho-associated kinase inhibitor, rescued TP-impaired insulin signaling. Consistent with this result, pertussis toxin abrogated IBOP-induced dephosphorylation of both Akt and eNOS, implicating the G_i family of G proteins in the suppressive effects of TP. In mice, high fat diet-induced diabetes was associated with aortic PTEN up-regulation, PTEN-Ser³⁸⁰/Thr^382/383 phosphorylation, and dephosphorylation of both Akt (at Ser⁴⁷³) and eNOS (at Ser¹¹⁷⁷). Importantly, administration of TP antagonist blocked these changes. We conclude that TP stimulation impairs insulin signaling in vascular endothelial cells by selectively activating the Rho/Rho-associated kinase/LKB1/PTEN pathway.Insulin exerts multiple biological actions relating to not only metabolism but also to endothelial functions (1, 2). Insulin has beneficial effects on the vasculature, primarily because of its ability to enhance endothelial nitric-oxide synthase (eNOS)² activation and expression. These effects, in turn, enhance the bioavailability of nitric oxide (3), which engenders a wide array of antiatherogenic effects. Global insulin resistance is a key feature of the metabolic syndrome leading to cardiovascular disease. In an insulin-resistant state, a systemic deregulation of the insulin signal leads to a combined deregulation of insulin-regulated metabolism and endothelial functions (4), resulting in glucose intolerance and cardiovascular disease. Insulin resistance is associated with endothelial dysfunction (5), a hallmark of atherosclerosis, and predicts adverse cardiovascular events (6). Therefore, endothelium-specific insulin resistance (impaired insulin-stimulated phosphorylation of Akt and eNOS) may play an important role in the development of cardiovascular diseases.Prostanoids have critical roles in the development of endothelial dysfunction (7). Thromboxane A₂ (TXA₂) is believed to be a prime mediator of a variety of cardiovascular and pulmonary diseases such as atherosclerosis, myocardial infarction, and primary pulmonary hypertension. TXA₂ perturbs the normal quiescent phenotype of endothelial cells (ECs). This results in leukocyte adhesion to the vessel wall as well as increased vascular permeability and expression of adhesion molecules on ECs, all important components of the inflammatory response. In smooth muscle cells, TXA₂ promotes proliferation (8) and migration, contributing to neointima formation (9). TXA₂ binds to the thromboxane A₂ receptor (TP), which has two isoforms TPα and TPβ in human (10–12), activation of which is implicated in atherosclerosis and inflammation (13–16). Atherosclerosis is accelerated by diabetes and is associated with increased levels of TXA₂ and other eicosanoids that stimulate TP (14). TP expression and plasma levels of TP ligands are elevated, both locally and systemically, in several vascular and thrombotic diseases (17). Importantly, TP activation induces EC apoptosis (15, 18) and prevents tube formation (19) by inhibiting Akt phosphorylation (18). TP activation also inhibits vascular endothelial growth factor-induced EC migration and angiogenesis by decreasing Akt and eNOS phosphorylation (20). However, the regulatory mechanisms underlying Akt inhibition by TP stimulation remain largely undefined. Moreover, whether TP activation impairs endothelial insulin signaling is also unclear.Here, we investigated whether TP ligands interfere with insulin signaling. Our results reveal that activation of TP using a potent and stable ligand (IBOP) abrogates insulin signaling in ECs. We also show that Rho/ROCK/LKB1-mediated PTEN (phosphatase and tensin homolog deleted on chromosome ten) up-regulation is required for TP-induced inhibition of insulin signaling in ECs.     

Ca2+ Regulates Hormone Secretion and Proopiomelanocortin Gene Expression in Melanotrope Cells via the Calmodulin and the Protein Kinase C Pathways

The mechanism by which Ca2+ regulates proopiomelanocortin (POMC)-derived peptide secretion and POMC mRNA levels was investigated in primary cultures of porcine intermediate lobe (IL) cells maintained in serum-free medium. POMC gene expression was evaluated by the dot blot hybridization assay with a 32P-labeled DNA probe complementary to the full-length sequence of porcine POMC mRNA. Treatment of IL cells for 24 h with the calmodulin (CAM) antagonists W7 and W13 reduced POMC mRNA levels by a maximum of 50% in a dose-dependent manner (ED50 approximately 10(-8) M). Accumulation of alpha-melanocyte-stimulating hormone (alpha-MSH) in the medium was also depressed by 50% after 8 h of treatment. The role of protein kinase C (PKC) was investigated by depleting the IL cell PKC content with phorbol ester treatment. Phorbol 12-myristate 13-acetate (PMA) at 5 X 10(-8) M induced a rapid translocation of cytoplasmic PKC activity toward the membrane. After 12 h of PMA treatment, PKC activity was undetectable in either the cytoplasmic or the particulate fractions. The same dose of PMA induced a time-dependent decrease in POMC mRNA levels (50% inhibition after 24 h). The same effect was seen with the phorbol ester phorbol 12,13-dibutyrate at 5 X 10(-8) M, whereas the inactive phorbol ester 4 alpha-phorbol at 5 X 10(-8) M was without effect after 24 h of treatment. PMA treatment had a biphasic effect on alpha-MSH secretion.(ABSTRACT TRUNCATED AT 250 WORDS)