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The IRT1 protein from Arabidopsis thaliana is a metal transporter with a broad substrate range 总被引:25,自引:0,他引:25
Korshunova Yulia O. Eide David Gregg Clark W. Lou Guerinot Mary Pakrasi Himadri B. 《Plant molecular biology》1999,40(1):37-44
The molecular basis for the transport of manganese across membranes in plant cells is poorly understood. We have found that IRT1, an Arabidopsis thaliana metal ion transporter, can complement a mutant Saccharomyces cerevisiae strain defective in high-affinity manganese uptake (smf1). The IRT1 protein has previously been identified as an iron transporter. The current studies demonstrated that IRT1, when expressed in yeast, can transport manganese as well. This manganese uptake activity was inhibited by cadmium, iron(II) and zinc, suggesting that IRT1 can transport these metals. The IRT1 cDNA also complements a zinc uptake-deficient yeast mutant strain (zrt1zrt2), and IRT1-dependent zinc transport in yeast cells is inhibited by cadmium, copper, cobalt and iron(III). However, IRT1 did not complement a copper uptake-deficient yeast mutant (ctr1), implying that this transporter is not involved in the uptake of copper in plant cells. The expression of IRT1 is enhanced in A. thaliana plants grown under iron deficiency. Under these conditions, there were increased levels of root-associated manganese, zinc and cobalt, suggesting that, in addition to iron, IRT1 mediates uptake of these metals into plant cells. Taken together, these data indicate that the IRT1 protein is a broad-range metal ion transporter in plants. 相似文献
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Tosoni K Costa A Sarno S D'Alessandro S Sparla F Pinna LA Zottini M Ruzzene M 《Molecular and cellular biochemistry》2011,356(1-2):245-254
The ubiquitous Ser/Thr protein kinase CK2, which phosphorylates hundreds of substrates and is essential for cell life, plays important roles also in plants; however, only few plant substrates have been identified so far. During a study aimed at identifying proteins targeted by CK2 in plant response to salicylic acid (SA), we found that the Arabidopsis co-chaperone protein p23 is a CK2 target, readily phosphorylated in vitro by human and maize CK2, being also a substrate for an endogenous casein kinase activity present in Arabidopsis extracts, which displays distinctive characteristics of protein kinase CK2. We also demonstrated that p23 and the catalytic subunit of CK2 interact in vitro and possibly in Arabidopsis mesophyll protoplasts, where they colocalize in the cytosol and in the nucleus. Although its exact function is presently unknown, p23 is considered a co-chaperone because of its ability to associate to the chaperone protein Hsp90; therefore, an involvement of p23 in plant signal transduction pathways, such as SA signaling, is highly conceivable, and its phosphorylation may represent a fine mechanism for the regulation of cellular responses. 相似文献
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While considerable progress has been achieved in plant CDPK studies in the past decade, there is relatively no information about the potential substrates of CRKs. In this report, a yeast two-hybrid screen was performed with truncated form of AtCRK3 as bait to identify its interacting proteins in an effort to dissect its physiological roles. One gene encoding cytosolic glutamine synthetase AtGLN1;1 was isolated. Further analyses indicated that AtGLN1;1 could interact specifically with AtCRK3 and the kinase domain of AtCRK3 and the catalytic domain of AtGLN1;1 were responsible for such interaction, respectively. Furthermore, in vitro and in vivo co-immunoprecipitation results strongly supported that they could physically interact with each other. Phosphorylation assays revealed that AtGLN1;1 could be specifically phosphorylated by AtCRK3 in vitro. All the results demonstrate that AtGLN1;1 may be a substrate of AtCRK3. In addition, both AtGLN1;1 and AtCRK3 could be induced by natural or artificially induced leaf senescence, implying that such interaction may be involved in the regulation of nitrogen remobilization during leaf senescence. 相似文献
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BTF3 is a potential new substrate of protein kinase CK2 总被引:2,自引:0,他引:2
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Roberto Costa Giorgio Arrigoni Giorgio Cozza Graziano Lolli Roberto Battistutta Juan Carlos Izpisua Belmonte Lorenzo A. Pinna Stefania Sarno 《Biochimica et Biophysica Acta - Proteins and Proteomics》2014,1844(4):722-729
Protein kinase CK2 is a pleiotropic serine/threonine kinase responsible for the generation of a substantial proportion of the human phosphoproteome. CK2 is generally found as a tetramer with two catalytic, α and α′ and two non catalytic β subunits. CK2α C-terminal tail phosphorylation is regulated during the mitotic events and the absence of these phosphosites in α′ suggests an isoform specialization. We used a proteomic approach to identify proteins specifically phosphorylated by a CK2α phosphomimetic mutant, CK2αT344ET360ES362ES370E (CK2α4E), in human neuroblastoma SKNBE cellular extract. One of these proteins is lysine-specific demethylase 1 (LSD1 or KDM1A), an important player of the epigenetic machinery. LSD1 is a FAD-dependent amine oxidase and promotes demethylation of lysine 4 and lysine 9 of mono- and di-methylated histone H3. We found that LSD1 is a new substrate and an interacting partner of protein kinase CK2. Three CK2 phosphosites, (Ser131, Ser137 and Ser166) in the N-terminal region of LSD1 have been identified. This domain is found in all chordates but not in more ancient organisms and it is not essential for LSD1 catalytic event while it could modulate the interaction with CK2 and with other partners in gene repressing and activating complexes. Our data support the view that the phosphorylation of the N-terminal domain by CK2 may represent a mechanism for regulating histone methylation, disclosing a new role for protein kinase CK2 in epigenetics. 相似文献
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Following prenylation, the proteins are subject to two prenyl-dependent modifications at their C-terminal end, which are required for their subcellular targeting. First, the three C-terminal residues of the CAAX box prenylation signaling motif are removed, which is followed by methylation of the free carboxyl group of the prenyl cysteine moiety. An Arabidopsis homologue of the yeast CAAX protease STE24 (AFC1) was cloned and expressed in rce1 Delta ste24 Delta mutant yeast to demonstrate functional complementation. The petunia calmodulin CaM53 is a prenylated protein terminating in a CTIL CAAX box. Coupled methylation proteolysis assays demonstrated the processing of CaM53 by AtSTE24. In addition, AtSTE24 promoted plasma membrane association of the GFP-Rac fusion protein, which terminates with a CLLM CAAX box. Interestingly, a plant homologue of the second and major CAAX protease in yeast and animal cells, RCE1, was not identified despite the availability of vast amounts of sequence data. Taken together, these data suggest that AtSTE24 may process several prenylated proteins in plant cells, unlike its yeast homologue, which processes only a-mating factor, and its mammalian homologue, for which prenyl-CAAX substrates have not been established. Transient expression of GFPAtSTE24 in leaf epidermal cells of Nicotiana benthamiana showed that AtSTE24 is exclusively localized in the endoplasmic reticulum, suggesting that prenylated proteins in plants are first targeted to the endoplasmic reticulum following their prenylation. 相似文献
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Identification and functional analysis of in vivo phosphorylation sites of the Arabidopsis BRASSINOSTEROID-INSENSITIVE1 receptor kinase 总被引:7,自引:0,他引:7
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Wang X Goshe MB Soderblom EJ Phinney BS Kuchar JA Li J Asami T Yoshida S Huber SC Clouse SD 《The Plant cell》2005,17(6):1685-1703
Brassinosteroids (BRs) regulate multiple aspects of plant growth and development and require an active BRASSINOSTEROID-INSENSITIVE1 (BRI1) and BRI1-ASSOCIATED RECEPTOR KINASE1 (BAK1) for hormone perception and signal transduction. Many animal receptor kinases exhibit ligand-dependent oligomerization followed by autophosphorylation and activation of the intracellular kinase domain. To determine if early events in BR signaling share this mechanism, we used coimmunoprecipitation of epitope-tagged proteins to show that in vivo association of BRI1 and BAK1 was affected by endogenous and exogenous BR levels and that phosphorylation of both BRI1 and BAK1 on Thr residues was BR dependent. Immunoprecipitation of epitope-tagged BRI1 from Arabidopsis thaliana followed by liquid chromatography-tandem mass spectrometry (LC/MS/MS) identified S-838, S-858, T-872, and T-880 in the juxtamembrane region, T-982 in the kinase domain, and S-1168 in C-terminal region as in vivo phosphorylation sites of BRI1. MS analysis also strongly suggested that an additional two residues in the juxtamembrane region and three sites in the activation loop of kinase subdomain VII/VIII were phosphorylated in vivo. We also identified four specific BAK1 autophosphorylation sites in vitro using LC/MS/MS. Site-directed mutagenesis of identified and predicted BRI1 phosphorylation sites revealed that the highly conserved activation loop residue T-1049 and either S-1044 or T-1045 were essential for kinase function in vitro and normal BRI1 signaling in planta. Mutations in the juxtamembrane or C-terminal regions had only small observable effects on autophosphorylation and in planta signaling but dramatically affected phosphorylation of a peptide substrate in vitro. These findings are consistent with many aspects of the animal receptor kinase model in which ligand-dependent autophosphorylation of the activation loop generates a functional kinase, whereas phosphorylation of noncatalytic intracellular domains is required for recognition and/or phosphorylation of downstream substrates. 相似文献
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J H Lin A Makris C McMahon S E Bear C Patriotis V R Prasad R Brent E A Golemis P N Tsichlis 《The Journal of biological chemistry》1999,274(21):14706-14715
Tvl-1 is a 269-amino acid ankyrin repeat protein expressed primarily in thymus, lung, and testes that was identified by screening a murine T-cell two-hybrid cDNA library for proteins that associate with the serine-threonine kinase Raf-1. The interaction of Tvl-1 with Raf-1 was confirmed by co-immunoprecipitation of the two proteins from COS-1 cells transiently transfected with Tvl-1 and Raf-1 expression constructs as well as by co-immunoprecipitation of the endogenous proteins from CV-1 and NB2 cells. Tvl-1 interacts with Raf-1 via its carboxyl-terminal ankyrin repeat domain. The same domain also mediates Tvl-1 homodimerization. Tvl-1 was detected by immunofluorescence in both the cytoplasm and the nucleus suggesting that in addition to Raf-1 it may also interact with nuclear proteins. Activated Raf-1 phosphorylates Tvl-1 both in vitro and in vivo. In baculovirus-infected Sf9 insect cells, Tvl-1 potentiates the activation of Raf-1 by Src and Ras while in COS-1 cells it potentiates the activation of Raf-1 by EGF. These data suggest that Tvl-1 is both a target as well as a regulator of Raf-1. The human homologue of Tvl-1 maps to chromosome 19p12, upstream of MEF2B with the two genes in a head to head arrangement. 相似文献
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Apoprotein A-1 (apo A-1), the predominant protein constituent of high density lipoproteins (HDL), was phosphorylated by protein kinase C (PKC). Optimal phosphorylation of lipid-free apo A-1 occurs in the absence of calcium, phosphatidyl serine (PS), and diolein (DO). However, HDL-bound apo A-1 was not phosphorylated by PKC. Furthermore, addition of either native or reconstituted HDL particles to lipid-free apo A-1 resulted in a concentration-dependent inhibition of phosphorylation. It appears that the phosphorylatable sites on apo A-1 are involved in hydrophobic interaction with the lipids of HDL. Apo A-1 is a novel substrate of PKC because it does not require calcium and lipid cofactors for optimal phosphorylation. 相似文献
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The Arabidopsis NPR1 protein is a receptor for the plant defense hormone salicylic acid 总被引:3,自引:0,他引:3
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Lichtenthaler SF Dominguez DI Westmeyer GG Reiss K Haass C Saftig P De Strooper B Seed B 《The Journal of biological chemistry》2003,278(49):48713-48719
The aspartyl protease BACE1 cleaves the amyloid precursor protein and the sialyltransferase ST6Gal I and is important in the pathogenesis of Alzheimer's disease. The normal function of BACE1 and additional physiological substrates have not been identified. Here we show that BACE1 acts on the P-selectin glycoprotein ligand 1 (PSGL-1), which mediates leukocyte adhesion in inflammatory reactions. In human monocytic U937 and human embryonic kidney 293 cells expressing endogenous or transfected BACE1, PSGL-1 was cleaved by BACE1 to generate a soluble ectodomain and a C-terminal transmembrane fragment. No evidence of the cleavage fragment was seen in primary cells derived from mice deficient in BACE1. By using deletion constructs and enzymatic deglycosylation of the C-terminal PSGL-1 fragments, the cleavage site in PSGL-1 was mapped to the juxtamembrane region within the ectodomain. In an in vitro assay BACE1 catalyzed the formation of the PSGL-1 products seen in vivo. The cleavage occurred at a Leu-Ser peptide bond as identified by mass spectrometry using a synthetic peptide. We conclude that PSGL-1 is an additional substrate for BACE1. 相似文献
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Forzani C Carreri A de la Fuente van Bentem S Lecourieux D Lecourieux F Hirt H 《The FEBS journal》2011,278(7):1126-1136
In Arabidopsis thaliana, the serine/threonine protein kinase oxidative signal-inducible 1 (OXI1), mediates oxidative stress signalling. Its activity is required for full activation of the mitogen-activated protein kinases (MAPKs), MPK3 and MPK6, in response to oxidative stress. In addition, the serine/threonine protein kinase Pto-interacting 1-2 (PTI1-2) has been positioned downstream from OXI1, but whether PTI1-2 signals through MAPK cascades is unclear. Using a yeast two-hybrid screen we show that OXI1 also interacts with PTI1-4. OXI1 and PTI1-4 are stress-responsive genes and are expressed in the same tissues. Therefore, studies were undertaken to determine whether PTI1-4 is positioned in the OXI1/MAPK signalling pathway. The interaction between OXI1 and PTI1-4 was confirmed by using in vivo co-immunoprecipitation experiments. OXI1 and PTI1-4 were substrates of MPK3 and MPK6 in vitro. Although no direct interaction was detected between OXI1 and MPK3 or MPK6, in vitro binding studies showed interactions between MPK3 or MPK6 with PTI1-4. In addition, PTI1-4 and MPK6 were found in vivo in the same protein complex. These results demonstrate that PTI1-4 signals via OXI1 and MPK6 signalling cascades. 相似文献
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Arabidopsis cell growth defect factor-1 (Cdf1 in yeast, At5g23040) was originally isolated as a cell growth suppressor of yeast from genetic screening. To investigate the in vivo role of Cdf1 in plants, a T-DNA insertion line was analyzed. A homozygous T-DNA insertion mutant (cdf1/cdf1) was embryo lethal and showed arrested embryogenesis at the globular stage. The Cdf1 protein, when fused with green fluorescent protein, was localized to the plastid in stomatal guard cells and mesophyll cells. A promoter-β-glucuronidase assay found expression of Cdf1 in the early heart stage of embryogenesis, suggesting that Cdf1 was essential for Arabidopsis embryogenesis during the transition of the embryo from the globular to heart stage. 相似文献
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The thiol oxidase Erv1 and the redox-regulated receptor Mia40/Tim40 are components of a disulfide relay system which mediates import of proteins into the intermembrane space (IMS) of mitochondria. Here we report that Erv1 requires Mia40 for its import into mitochondria. After passage across the translocase of the mitochondrial outer membrane Erv1 interacts via disulfide bonds with Mia40. Erv1 does not contain twin “CX3C” or twin “CX9C” motifs which are crucial for import of typical substrates of this pathway and it does not need two “CX2C” motifs for import into mitochondria. Thus, Erv1 represents an unusual type of substrate of the Mia40-dependent import pathway. 相似文献