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Guard Cells Possess a Calcium-Dependent Protein Kinase That
Phosphorylates the KAT1 Potassium Channel 总被引:25,自引:2,他引:23
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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. 相似文献
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Calcium-Dependent Protein Phosphorylation May Mediate the
Gibberellic Acid Response in Barley Aleurone 总被引:6,自引:0,他引:6
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Peptide substrates of well-defined protein kinases were microinjected into aleurone protoplasts of barley (Hordeum vulgare L. cv Himalaya) to inhibit, and therefore identify, protein kinase-regulated events in the transduction of the gibberellin (GA) and abscisic acid signals. Syntide-2, a substrate designed for Ca2+- and calmodulin (CaM)-dependent kinases, selectively inhibited the GA response, leaving constitutive and abscisic acid-regulated events unaffected. Microinjection of syntide did not affect the GA-induced increase in cytosolic [Ca2+], suggesting that it inhibited GA action downstream of the Ca2+ signal. When photoaffinity-labeled syntide-2 was electroporated into protoplasts and cross-linked to interacting proteins in situ, it selectively labeled proteins of approximately 30 and 55 kD. A 54-kD, soluble syntide-2 phosphorylating protein kinase was detected in aleurone cells. This kinase was activated by Ca2+ and was CaM independent, but was inhibited by the CaM antagonist N-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamide (250 μm), suggesting that it was a CaM-domain protein kinase-like activity. These results suggest that syntide-2 inhibits the GA response of the aleurone via an interaction with this kinase, implicating the 54-kD kinase as a Ca2+-dependent regulator of the GA response in these cells. 相似文献
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烟草GA合成调控转录因子RSG应答甲醇和乙醇刺激的分子机理初探 总被引:1,自引:0,他引:1
为了考察甲醇或乙醇促进植物生长与赤霉素(GA)的合成关系,该研究在MS固体培养基中培养并添加外源GA和GA合成抑制剂多效唑(PAC),分析其对2mmol/L甲醇或乙醇促进烟草生长的影响及GA合成调控转录因子RSG(for repression of shoot growth)应答甲醇或乙醇刺激的分子机理。结果显示:(1)外源添加GA可增强甲醇或乙醇对烟草生长的刺激作用,而添加PAC却抑制甲醇和乙醇对烟草生长的刺激作用。(2)14-3-3蛋白与RSG结合抑制RSG进入细胞核及其转录调控活性;甲醇和乙醇诱导烟草14-3-3基因的转录和表达,对RSG蛋白表达也有诱导作用。(3)甲醇和乙醇可降低14-3-3蛋白与RSG的相互作用,同时增强RSG与GA20ox1启动子的结合。研究表明,甲醇和乙醇刺激烟草的生长可能通过增加RSG表达,且减弱RSG与14-3-3蛋白的结合来增加RSG细胞核定位作用,从而增强RSG与GA20ox1启动子的结合,最终增加GA的合成,从而促进烟草的生长,这可能是甲醇和乙醇促进烟草生长的一种重要的分子机制。 相似文献
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A Bononi M Bonora S Marchi S Missiroli F Poletti C Giorgi P P Pandolfi P Pinton 《Cell death and differentiation》2013,20(12):1631-1643
The tumor suppressor activity of PTEN (phosphatase and tensin homolog deleted on chromosome 10) is thought to be largely attributable to its lipid phosphatase activity. PTEN dephosphorylates the lipid second messenger phosphatidylinositol 3,4,5-trisphosphate to directly antagonize the phosphoinositide 3-kinase-Akt pathway and prevent the activating phosphorylation of Akt. PTEN has also other proposed mechanisms of action, including a poorly characterized protein phosphatase activity, protein–protein interactions, as well as emerging functions in different compartment of the cells such as nucleus and mitochondria. We show here that a fraction of PTEN protein localizes to the endoplasmic reticulum (ER) and mitochondria-associated membranes (MAMs), signaling domains involved in calcium (2+) transfer from the ER to mitochondria and apoptosis induction. We demonstrate that PTEN silencing impairs ER Ca2+ release, lowers cytosolic and mitochondrial Ca2+ transients and decreases cellular sensitivity to Ca2+-mediated apoptotic stimulation. Specific targeting of PTEN to the ER is sufficient to enhance ER-to-mitochondria Ca2+ transfer and sensitivity to apoptosis. PTEN localization at the ER is further increased during Ca2+-dependent apoptosis induction. Importantly, PTEN interacts with the inositol 1,4,5-trisphosphate receptors (IP3Rs) and this correlates with the reduction in their phosphorylation and increased Ca2+ release. We propose that ER-localized PTEN regulates Ca2+ release from the ER in a protein phosphatase-dependent manner that counteracts Akt-mediated reduction in Ca2+ release via IP3Rs. These findings provide new insights into the mechanisms and the extent of PTEN tumor-suppressive functions, highlighting new potential strategies for therapeutic intervention. 相似文献
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Bin Song Bingquan Lai Zhihao Zheng Yuying Zhang Jingyan Luo Chong Wang Yuan Chen James R. Woodgett Mingtao Li 《The Journal of biological chemistry》2010,285(52):41122-41134
Glycogen synthase kinase-3 (GSK-3) plays a critical role in neuronal apoptosis. The two mammalian isoforms of the kinase, GSK-3α and GSK-3β, are inhibited by phosphorylation at Ser-21 and Ser-9, respectively. Depolarization, which is vital for neuronal survival, causes both an increase in Ser-21/9 phosphorylation and an inhibition of GSK-3α/β. However, the role of GSK-3 phosphorylation in depolarization-dependent neuron survival and the signaling pathway contributing to GSK-3 phosphorylation during depolarization remain largely unknown. Using several approaches, we showed that both isoforms of GSK-3 are important for mediating neuronal apoptosis. Nonphosphorylatable GSK-3α/β mutants (S21A/S9A) promoted apoptosis, whereas a peptide encompassing Ser-9 of GSK-3β protected neurons in a phosphorylation-dependent manner; these results indicate a critical role for Ser-21/9 phosphorylation on depolarization-dependent neuron survival. We found that Ser-21/9 phosphorylation of GSK-3 was mediated by Ca2+/calmodulin-dependent protein kinase II (CaMKII) but not by Akt/PKB, PKA, or p90RSK. CaMKII associated with and phosphorylated GSK-3α/β. Furthermore, the pro-survival effect of CaMKII was mediated by GSK-3 phosphorylation and inactivation. These findings identify a novel Ca2+/calmodulin/CaMKII/GSK-3 pathway that couples depolarization to neuronal survival. 相似文献
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A function for tyrosine phosphorylation of type 1 inositol 1,4,5-trisphosphate receptor in lymphocyte activation 总被引:1,自引:0,他引:1
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Sustained elevation of intracellular calcium by Ca2+ release–activated Ca2+ channels is required for lymphocyte activation. Sustained Ca2+ entry requires endoplasmic reticulum (ER) Ca2+ depletion and prolonged activation of inositol 1,4,5-trisphosphate receptor (IP3R)/Ca2+ release channels. However, a major isoform in lymphocyte ER, IP3R1, is inhibited by elevated levels of cytosolic Ca2+, and the mechanism that enables the prolonged activation of IP3R1 required for lymphocyte activation is unclear. We show that IP3R1 binds to the scaffolding protein linker of activated T cells and colocalizes with the T cell receptor during activation, resulting in persistent phosphorylation of IP3R1 at Tyr353. This phosphorylation increases the sensitivity of the channel to activation by IP3 and renders the channel less sensitive to Ca2+-induced inactivation. Expression of a mutant IP3R1-Y353F channel in lymphocytes causes defective Ca2+ signaling and decreased nuclear factor of activated T cells activation. Thus, tyrosine phosphorylation of IP3R1-Y353 may have an important function in maintaining elevated cytosolic Ca2+ levels during lymphocyte activation. 相似文献
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Summary Ca2+-dependent protein kinase (CDPK) has been proposed to mediate inhibition by Ca2+ of cytoplasmic streaming in the green algaChara. We have identified the in vivo substrate(s) of CDPK inChara by using vacuolar perfusion of individual internodal cells with [-32P]ATP. Phosphorylation of several polypeptides is enhanced when perfusions are performed at 10–4M free Ca2+ compared to <10–9M free Ca2+. The Ca2+-stimulated phosphorylation of these proteins is inhibited by the presence of a monoclonal antibody to soybean CDPK. One of these proteins is 16 to 18kDa and is recognized by an antibody against gizzard myosin light chains. These results demonstrate that inChara, several polypeptides are phophorylated by CDPK and one of these proteins has been tentatively identified as a myosin light chain. These observations support the hypothesis that Ca2+-regulated phosphorylation of myosin is involved in the regulation of cytoplasmic streaming.Abbreviations CDPK
calcium-dependent protein kinase
- mAb
monoclonal antibody 相似文献
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Imtiaz MS Zhao J Hosaka K von der Weid PY Crowe M van Helden DF 《Biophysical journal》2007,92(11):3843-3861
This study presents an investigation of pacemaker mechanisms underlying lymphatic vasomotion. We tested the hypothesis that active inositol 1,4,5-trisphosphate receptor (IP3R)-operated Ca2+ stores interact as coupled oscillators to produce near-synchronous Ca2+ release events and associated pacemaker potentials, this driving action potentials and constrictions of lymphatic smooth muscle. Application of endothelin 1 (ET-1), an agonist known to enhance synthesis of IP3, to quiescent lymphatic smooth muscle syncytia first enhanced spontaneous Ca2+ transients and/or intracellular Ca2+ waves. Larger near-synchronous Ca2+ transients then occurred leading to global synchronous Ca2+ transients associated with action potentials and resultant vasomotion. In contrast, blockade of L-type Ca2+ channels with nifedipine prevented ET-1 from inducing near-synchronous Ca2+ transients and resultant action potentials, leaving only asynchronous Ca2+ transients and local Ca2+ waves. These data were well simulated by a model of lymphatic smooth muscle with: 1), oscillatory Ca2+ release from IP3R-operated Ca2+ stores, which causes depolarization; 2), L-type Ca2+ channels; and 3), gap junctions between cells. Stimulation of the stores caused global pacemaker activity through coupled oscillator-based entrainment of the stores. Membrane potential changes and positive feedback by L-type Ca2+ channels to produce more store activity were fundamental to this process providing long-range electrochemical coupling between the Ca2+ store oscillators. We conclude that lymphatic pacemaking is mediated by coupled oscillator-based interactions between active Ca2+ stores. These are weakly coupled by inter- and intracellular diffusion of store activators and strongly coupled by membrane potential. Ca2+ store-based pacemaking is predicted for cellular systems where: 1), oscillatory Ca2+ release induces depolarization; 2), membrane depolarization provides positive feedback to induce further store Ca2+ release; and 3), cells are interconnected. These conditions are met in a surprisingly large number of cellular systems including gastrointestinal, lymphatic, urethral, and vascular tissues, and in heart pacemaker cells. 相似文献
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CDPKs CPK6 and CPK3 Function in ABA Regulation of Guard Cell S-Type Anion- and Ca2+- Permeable Channels and Stomatal Closure
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Izumi C Mori Yoshiyuki Murata Yingzhen Yang Shintaro Munemasa Yong-Fei Wang Shannon Andreoli Herv Tiriac Jose M Alonso Jeffery F Harper Joseph R Ecker June M Kwak Julian I Schroeder 《PLoS biology》2006,4(10)
Abscisic acid (ABA) signal transduction has been proposed to utilize cytosolic Ca2+ in guard cell ion channel regulation. However, genetic mutants in Ca2+ sensors that impair guard cell or plant ion channel signaling responses have not been identified, and whether Ca2+-independent ABA signaling mechanisms suffice for a full response remains unclear. Calcium-dependent protein kinases (CDPKs) have been proposed to contribute to central signal transduction responses in plants. However, no Arabidopsis CDPK gene disruption mutant phenotype has been reported to date, likely due to overlapping redundancies in CDPKs. Two Arabidopsis guard cell–expressed CDPK genes, CPK3 and CPK6, showed gene disruption phenotypes. ABA and Ca2+ activation of slow-type anion channels and, interestingly, ABA activation of plasma membrane Ca2+-permeable channels were impaired in independent alleles of single and double cpk3cpk6 mutant guard cells. Furthermore, ABA- and Ca2+-induced stomatal closing were partially impaired in these cpk3cpk6 mutant alleles. However, rapid-type anion channel current activity was not affected, consistent with the partial stomatal closing response in double mutants via a proposed branched signaling network. Imposed Ca2+ oscillation experiments revealed that Ca2+-reactive stomatal closure was reduced in CDPK double mutant plants. However, long-lasting Ca2+-programmed stomatal closure was not impaired, providing genetic evidence for a functional separation of these two modes of Ca2+-induced stomatal closing. Our findings show important functions of the CPK6 and CPK3 CDPKs in guard cell ion channel regulation and provide genetic evidence for calcium sensors that transduce stomatal ABA signaling. 相似文献