Phosphorylation of proteins is an important post-translational modification. Methods to determine the phosphorylation state of proteins are very important to evaluate diverse biological processes. CRK5 is the CDPK-rel...Phosphorylation of proteins is an important post-translational modification. Methods to determine the phosphorylation state of proteins are very important to evaluate diverse biological processes. CRK5 is the CDPK-related protein kinase in Arabidopsis, WD-repeat protein (WDRP) might be CRK5-interact-protein based on Y2H results. Here, we used bimolecular fluorescence complementation (BiFC) further to study and visualize the interaction between CRK5 and WDRP in living cells. Then, we combined Phos-tagTM SDS-PAGE with western blot (WB) analysis, using WDRP antibody and the anti-6×His antibody, to detect phosphorylated WDRP. This approach confirmed that WDRP might be phosphorylated by CRK5 in vitro. Site mutation analysis suggested that serine-70 might be the amino acid phosphorylated by CRK5 in WDRP. Cell extracts isolated from WT, OERK5, and crk5 used to analyze the kinase reaction using recombinant WDRP as substrate. These results demonstrated that WDRP was phosphorylated by cell extracts and that there may be additional kinases that phosphorylate WDRP in Arabidopsis. Phos-tagTM SDS-PAGE thus provides a suitable and convenient method for analysis of phosphorylation in plants.展开更多
Patch clamp techniques were employed to investigate if calcium dependent protein kinases (CDPKs) be involved in the signal transduction pathways of stomatal movement regulation by the phytohormone abscisic acid (ABA...Patch clamp techniques were employed to investigate if calcium dependent protein kinases (CDPKs) be involved in the signal transduction pathways of stomatal movement regulation by the phytohormone abscisic acid (ABA) in Vicia faba. Stomatal opening was completely inhibited by external application of 1 μmol/L ABA, and such ABA inhibition was significantly reversed by the addition of CDPK inhibitor trifluoperazine (TFP). The inward whole cell K + currents were inhibited by 60% in the presence of 1 μmol/L intracellular ABA, and this inhibition was completely abolished by the addition of CDPK competitive substrate histone Ⅲ S. The results suggest that CDPKs may be involved in the signal transduction cascades of ABA regulated stomatal movements.展开更多
By screening tobacco cDNA library with MCK1 as a probe, we isolated a cDNA clone NtCPK5 （accession number AY971376）, which encodes a typical calcium-dependent protein kinase. Sequence analyses indicated that NtCPK5 ...By screening tobacco cDNA library with MCK1 as a probe, we isolated a cDNA clone NtCPK5 （accession number AY971376）, which encodes a typical calcium-dependent protein kinase. Sequence analyses indicated that NtCPK5 is related to both CPKs and CRKs superfamilies and has all of the three conserved domains of CPKs. The biochemical activity of NtCPK5 was calcium-dependent. NtCPK5 had Vmax and Km of 526nmol/min/mg and 210μg/ml respectively with calf thymus histone （fraction Ⅲ, abbreviated to histone Ⅲs） as substrate. For substrate syntide-2, NtCPK5 showed a higher Vmax of 2008 nmol/min/mg and a lower Km of 30μM. The K0.5 of calcium activation was 0.04μM or 0.06μM for histone Ⅲs or syntide-2 respectively. The putative myristoylation and palmitoylation consensus sequence of NtCPK5 suggests that it could be a membrane-anchoring protein. Indeed, our transient expression experiments with wild type and mutant forms of NtCPK5/GFP fusion proteins showed that NtCPK5 was localized to the plasma membrane of onion epidermal cells and that the localization required the N-terminal acylation sites of NtCPK5/GFP. Taking together, our data have demonstrated the biochemical characteristics of a novel protein NtCPK5 and its subcellular localization as a membrane-anchoring protein.展开更多
Iron(Fe)is an essential micronutrient for all organisms.Fe availability in the soil is usually much lower than that required for plant growth,and Fe deficiencies seriously restrict crop growth and yield.Calcium(Ca2+)i...Iron(Fe)is an essential micronutrient for all organisms.Fe availability in the soil is usually much lower than that required for plant growth,and Fe deficiencies seriously restrict crop growth and yield.Calcium(Ca2+)is a second messenger in all eukaryotes;however,it remains largely unknown how Ca2+regulates Fe deficiency.In this study,mutations in CPK21 and CPK23,which are two highly homologous calcium-dependent protein kinases,conferredimpaired growth and rootdevelopment under Fe-deficient conditions,whereas constitutively active CPK21 and CPK23 enhanced plant tolerance to Fe-deficient conditions.Furthermore,we found that CPK21 and CPK23 interacted with and phosphorylated the Fe transporter IRONREGULATED TRANSPORTER1(IRT1)at the Ser149 residue.Biochemical analyses and complementation of Fe transport in yeast and plants indicated that IRT1 Ser149 is critical for IRT1 transport activity.Taken together,these findings suggest that the CPK21/23-IRT1 signaling pathway is critical for Fe homeostasis in plants and provides targets for improving Fe-deficient environments and breeding crops resistant to Fe-deficient conditions.展开更多
14-3-3 proteins play an important role in the regulation of many cellular processes. The Arabidopsis vacuolar two-pore K＋ channel 1 （TPK1） interacts with the 14-3-3 protein GRF6 （GF14-λ）. Upon phosphorylation of...14-3-3 proteins play an important role in the regulation of many cellular processes. The Arabidopsis vacuolar two-pore K＋ channel 1 （TPK1） interacts with the 14-3-3 protein GRF6 （GF14-λ）. Upon phosphorylation of the putative binding motif in the N-terminus of TPK1, GRF6 binds to TPK1 and activates the potassium channel. In order to gain a deeper understanding of this 14-3-3-mediated signal transduction, we set out to identify the respective kinases, which regulate the phosphorylation status of the 14-3-3 binding motif in TPK1. Here, we report that the calcium-dependent protein kinases （CDPKs） can phosphorylate and thereby activate the 14-3-3 binding motif in TPK1. Focusing on the stress-activated kinase CPK3, we visualized direct and specific interaction of TPK1 with the kinase at the tonoplast in vivo. In line with its proposed role in K＋ homeostasis, TPK1 phosphorylation was found to be induced by salt stress in planta, and both cpk3 and tpkl mutants displayed salt-sensitive phenotypes. Molecular modeling of the TPK1-CPK3 interaction domain provided mechanistic insights into TPK1 stress-regulated phosphorylation responses and pinpointed two arginine residues in the N-terminal 14-3-3 binding motif in TPK1 critical for kinase interaction. Taken together, our studies provide evidence for an essential role of the vacuolar potassium channel TPK1 in salt-stress adaptation as a target of calcium-regulated stress signaling pathways involving Ca2＋, Ca2＋-dependent kinases, and 14-3-3 proteins.展开更多
ABSTRACT Calcium, as a ubiquitous second messenger, plays essential roles in tip-growing cells, such as animal neu-rons, plant pollen tubes, and root hairs. However, little is known concerning the regulatory mechanism...ABSTRACT Calcium, as a ubiquitous second messenger, plays essential roles in tip-growing cells, such as animal neu-rons, plant pollen tubes, and root hairs. However, little is known concerning the regulatory mechanisms that code anddecode Ca2＋ signals in plants. The evidence presented here indicates that a calcium-dependent protein kinase, CPK32,controls polar growth of pollen tubes. Overexpression of CPK32 disrupted the polar growth along with excessive Ca2＋accumulation in the tip. A search of downstream effector molecules for CPK32 led to identification of a cyclic nucleotide-gated channel, CNGC18, as an interacting partner for CPK32. Co-expression of CPK32 and CNGC18 resulted in activationof CNGC18 in Xenopus oocytes where expression of CNGC18 alone did not exhibit significant calcium channel activity.Overexpression of CNGC18 produced a growth arrest phenotype coupled with accumulation of calcium in the tip, simi-lar to that induced by CPK32 overexpression. Co-expression of CPK32 and CNGC18 had a synergistic effect leading tomore severe depolarization of pollen tube growth. These results provide a potential feed-forward mechanism in whichcalcium-activated CPK32 activates CNGC18, further promoting calcium entry during the elevation phase of Ca2＋ oscilla-tions in the polar growth of pollen tubes.展开更多
文摘Phosphorylation of proteins is an important post-translational modification. Methods to determine the phosphorylation state of proteins are very important to evaluate diverse biological processes. CRK5 is the CDPK-related protein kinase in Arabidopsis, WD-repeat protein (WDRP) might be CRK5-interact-protein based on Y2H results. Here, we used bimolecular fluorescence complementation (BiFC) further to study and visualize the interaction between CRK5 and WDRP in living cells. Then, we combined Phos-tagTM SDS-PAGE with western blot (WB) analysis, using WDRP antibody and the anti-6×His antibody, to detect phosphorylated WDRP. This approach confirmed that WDRP might be phosphorylated by CRK5 in vitro. Site mutation analysis suggested that serine-70 might be the amino acid phosphorylated by CRK5 in WDRP. Cell extracts isolated from WT, OERK5, and crk5 used to analyze the kinase reaction using recombinant WDRP as substrate. These results demonstrated that WDRP was phosphorylated by cell extracts and that there may be additional kinases that phosphorylate WDRP in Arabidopsis. Phos-tagTM SDS-PAGE thus provides a suitable and convenient method for analysis of phosphorylation in plants.
文摘Patch clamp techniques were employed to investigate if calcium dependent protein kinases (CDPKs) be involved in the signal transduction pathways of stomatal movement regulation by the phytohormone abscisic acid (ABA) in Vicia faba. Stomatal opening was completely inhibited by external application of 1 μmol/L ABA, and such ABA inhibition was significantly reversed by the addition of CDPK inhibitor trifluoperazine (TFP). The inward whole cell K + currents were inhibited by 60% in the presence of 1 μmol/L intracellular ABA, and this inhibition was completely abolished by the addition of CDPK competitive substrate histone Ⅲ S. The results suggest that CDPKs may be involved in the signal transduction cascades of ABA regulated stomatal movements.
基金supported in part by the National Natural Science Foundation of China(Grant No.30230050)the Program for Changjiang Scholars and Innovative Research Team in University to Ying Tang LU.
文摘By screening tobacco cDNA library with MCK1 as a probe, we isolated a cDNA clone NtCPK5 （accession number AY971376）, which encodes a typical calcium-dependent protein kinase. Sequence analyses indicated that NtCPK5 is related to both CPKs and CRKs superfamilies and has all of the three conserved domains of CPKs. The biochemical activity of NtCPK5 was calcium-dependent. NtCPK5 had Vmax and Km of 526nmol/min/mg and 210μg/ml respectively with calf thymus histone （fraction Ⅲ, abbreviated to histone Ⅲs） as substrate. For substrate syntide-2, NtCPK5 showed a higher Vmax of 2008 nmol/min/mg and a lower Km of 30μM. The K0.5 of calcium activation was 0.04μM or 0.06μM for histone Ⅲs or syntide-2 respectively. The putative myristoylation and palmitoylation consensus sequence of NtCPK5 suggests that it could be a membrane-anchoring protein. Indeed, our transient expression experiments with wild type and mutant forms of NtCPK5/GFP fusion proteins showed that NtCPK5 was localized to the plasma membrane of onion epidermal cells and that the localization required the N-terminal acylation sites of NtCPK5/GFP. Taking together, our data have demonstrated the biochemical characteristics of a novel protein NtCPK5 and its subcellular localization as a membrane-anchoring protein.
基金supported by the National Natural Science Foundation of China(32222008,32100215,31900236)Northwest A&F University(Z111021604)+1 种基金the open funds of China Postdoctoral Science Foundation(2018M643740)Natural Science Basic Research Plan in Shaanxi Province of China(2019JQ-150).
文摘Iron(Fe)is an essential micronutrient for all organisms.Fe availability in the soil is usually much lower than that required for plant growth,and Fe deficiencies seriously restrict crop growth and yield.Calcium(Ca2+)is a second messenger in all eukaryotes;however,it remains largely unknown how Ca2+regulates Fe deficiency.In this study,mutations in CPK21 and CPK23,which are two highly homologous calcium-dependent protein kinases,conferredimpaired growth and rootdevelopment under Fe-deficient conditions,whereas constitutively active CPK21 and CPK23 enhanced plant tolerance to Fe-deficient conditions.Furthermore,we found that CPK21 and CPK23 interacted with and phosphorylated the Fe transporter IRONREGULATED TRANSPORTER1(IRT1)at the Ser149 residue.Biochemical analyses and complementation of Fe transport in yeast and plants indicated that IRT1 Ser149 is critical for IRT1 transport activity.Taken together,these findings suggest that the CPK21/23-IRT1 signaling pathway is critical for Fe homeostasis in plants and provides targets for improving Fe-deficient environments and breeding crops resistant to Fe-deficient conditions.
基金the Austrian Science Foundation (FWF) to M.T.，grants of the DFG to R.H.，T.D.M.，D.B.
文摘14-3-3 proteins play an important role in the regulation of many cellular processes. The Arabidopsis vacuolar two-pore K＋ channel 1 （TPK1） interacts with the 14-3-3 protein GRF6 （GF14-λ）. Upon phosphorylation of the putative binding motif in the N-terminus of TPK1, GRF6 binds to TPK1 and activates the potassium channel. In order to gain a deeper understanding of this 14-3-3-mediated signal transduction, we set out to identify the respective kinases, which regulate the phosphorylation status of the 14-3-3 binding motif in TPK1. Here, we report that the calcium-dependent protein kinases （CDPKs） can phosphorylate and thereby activate the 14-3-3 binding motif in TPK1. Focusing on the stress-activated kinase CPK3, we visualized direct and specific interaction of TPK1 with the kinase at the tonoplast in vivo. In line with its proposed role in K＋ homeostasis, TPK1 phosphorylation was found to be induced by salt stress in planta, and both cpk3 and tpkl mutants displayed salt-sensitive phenotypes. Molecular modeling of the TPK1-CPK3 interaction domain provided mechanistic insights into TPK1 stress-regulated phosphorylation responses and pinpointed two arginine residues in the N-terminal 14-3-3 binding motif in TPK1 critical for kinase interaction. Taken together, our studies provide evidence for an essential role of the vacuolar potassium channel TPK1 in salt-stress adaptation as a target of calcium-regulated stress signaling pathways involving Ca2＋, Ca2＋-dependent kinases, and 14-3-3 proteins.
文摘ABSTRACT Calcium, as a ubiquitous second messenger, plays essential roles in tip-growing cells, such as animal neu-rons, plant pollen tubes, and root hairs. However, little is known concerning the regulatory mechanisms that code anddecode Ca2＋ signals in plants. The evidence presented here indicates that a calcium-dependent protein kinase, CPK32,controls polar growth of pollen tubes. Overexpression of CPK32 disrupted the polar growth along with excessive Ca2＋accumulation in the tip. A search of downstream effector molecules for CPK32 led to identification of a cyclic nucleotide-gated channel, CNGC18, as an interacting partner for CPK32. Co-expression of CPK32 and CNGC18 resulted in activationof CNGC18 in Xenopus oocytes where expression of CNGC18 alone did not exhibit significant calcium channel activity.Overexpression of CNGC18 produced a growth arrest phenotype coupled with accumulation of calcium in the tip, simi-lar to that induced by CPK32 overexpression. Co-expression of CPK32 and CNGC18 had a synergistic effect leading tomore severe depolarization of pollen tube growth. These results provide a potential feed-forward mechanism in whichcalcium-activated CPK32 activates CNGC18, further promoting calcium entry during the elevation phase of Ca2＋ oscilla-tions in the polar growth of pollen tubes.
