The protein kinase complex CBL10–CIPK8–SOS1 functions in Arabidopsis to regulate salt tolerance

Abstract Salt tolerance in plants is mediated by Na+ extrusion from the cytosol by the plasma membrane Na+/H+ antiporter SOS1. This is activated in Arabidopsis root by the protein kinase complex SOS2–SOS3 and in Arabidopsis shoot by the protein kinase complex CBL10–SOS2, with SOS2 as a key node in the two pathways. The sos1 mutant is more sensitive than the sos2 mutant, suggesting that other partners may positively regulate SOS1 activity. Arabidopsis has 26 CIPK family proteins of which CIPK8 is the closest homolog to SOS2. It is hypothesized that CIPK8 can activate Na+ extrusion by SOS1 similarly to SOS2. The plasma membrane Na+/H+ exchange activity of transgenic yeast co-expressing CBL10, CIPK8, and SOS1 was higher than that of untransformed and SOS1 transgenic yeast, resulting in a lower Na+ accumulation and a better growth phenotype under salinity. However, CIPK8 could not interact with SOS3, and the co-expression of SOS3, CIPK8, and SOS1 in yeast did not confer a significant salt tolerance phenotype relative to SOS1 transgenic yeast. Interestingly, cipk8 displayed a slower Na+ efflux, a higher Na+ level, and a more sensitive phenotype than wild-type Arabidopsis, but grew better than sos2 under salinity stress. As expected, sos2cipk8 exhibited a more severe salt damage phenotype relative to cipk8 or sos2. Overexpression of CIPK8 in both cipk8 and sos2cipk8 attenuated the salt sensitivity phenotype. These results suggest that CIPK8-mediated activation of SOS1 is CBL10-dependent and SOS3-independent, indicating that CIPK8 and SOS2 activity in shoots is sufficient for regulating Arabidopsis salt tolerance.

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Protein kinase D inhibits plasma membrane Na+/H+exchanger activity

AJP Cell Physiology ◽

10.1152/ajpcell.1999.277.6.c1202 ◽

1999 ◽

Vol 277 (6) ◽

pp. C1202-C1209 ◽

Cited By ~ 33

Author(s):

Robert S. Haworth ◽

James Sinnett-Smith ◽

Enrique Rozengurt ◽

Metin Avkiran

Keyword(s):

Plasma Membrane ◽

Protein Kinase ◽

Phorbol Ester ◽

Fluorescent Protein ◽

Dominant Negative ◽

Protein Kinase D ◽

Wild Type ◽

Ph Indicator

The regulation of plasma membrane Na+/H+exchanger (NHE) activity by protein kinase D (PKD), a novel protein kinase C- and phorbol ester-regulated kinase, was investigated. To determine the effect of PKD on NHE activity in vivo, intracellular pH (pHi) measurements were made in COS-7 cells by microepifluorescence using the pH indicator cSNARF-1. Cells were transfected with empty vector (control), wild-type PKD, or its kinase-deficient mutant PKD-K618M, together with green fluorescent protein (GFP). NHE activity, as reflected by the rate of acid efflux ( J H), was determined in single GFP-positive cells following intracellular acidification. Overexpression of wild-type PKD had no significant effect on J H(3.48 ± 0.25 vs. 3.78 ± 0.24 mM/min in control at pHi 7.0). In contrast, overexpression of PKD-K618M increased J H (5.31 ± 0.57 mM/min at pHi 7.0; P < 0.05 vs. control). Transfection with these constructs produced similar effects also in A-10 cells, indicating that native PKD may have an inhibitory effect on NHE in both cell types, which is relieved by a dominant-negative action of PKD-K618M. Exposure of COS-7 cells to phorbol ester significantly increased J H in control cells but failed to do so in cells overexpressing either wild-type PKD (due to inhibition by the overexpressed PKD) or PKD-K618M (because basal J Hwas already near maximal). A fusion protein containing the cytosolic regulatory domain (amino acids 637–815) of NHE1 (the ubiquitous NHE isoform) was phosphorylated in vitro by wild-type PKD, but with low stoichiometry. These data suggest that PKD inhibits NHE activity, probably through an indirect mechanism, and represents a novel pathway in the regulation of the exchanger.

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Akt2 phosphorylates Synip to regulate docking and fusion of GLUT4-containing vesicles

The Journal of Cell Biology ◽

10.1083/jcb.200408182 ◽

2005 ◽

Vol 168 (6) ◽

pp. 921-928 ◽

Cited By ~ 63

Author(s):

Eijiro Yamada ◽

Shuichi Okada ◽

Tsugumichi Saito ◽

Kihachi Ohshima ◽

Minoru Sato ◽

...

Keyword(s):

Plasma Membrane ◽

Protein Kinase ◽

Membrane Fusion ◽

Glucose Transporter ◽

Protein Kinase B ◽

Specific Substrate ◽

Glucose Transporter 4 ◽

Wild Type ◽

Phosphorylation Motif

We have identified an unusual potential dual Akt/protein kinase B consensus phosphorylation motif in the protein Synip (RxKxRS97xS99). Surprisingly, serine 97 is not appreciably phosphorylated, whereas serine 99 is only a specific substrate for Akt2 but not Akt1 or Akt3. Although wild-type Synip (WT-Synip) undergoes an insulin-stimulated dissociation from Syntaxin4, the Synip serine 99 to phenylalanine mutant (S99F-Synip) is resistant to Akt2 phosphorylation and fails to display insulin-stimulated Syntaxin4 dissociation. Furthermore, overexpression of WT-Synip in 3T3L1 adipocytes had no effect on insulin-stimulated recruitment of glucose transporter 4 (GLUT4) to the plasma membrane, whereas overexpression of S99F-Synip functioned in a dominant-interfering manner by preventing insulin-stimulated GLUT4 recruitment and plasma membrane fusion. These data demonstrate that insulin activation of Akt2 specifically regulates the docking/fusion step of GLUT4-containing vesicles at the plasma membrane through the regulation of Synip phosphorylation and Synip–Syntaxin4 interaction.

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Glucose-independent inhibition of yeast plasma-membrane H+-ATPase by calmodulin antagonists

Biochemical Journal ◽

10.1042/bj3220823 ◽

1997 ◽

Vol 322 (3) ◽

pp. 823-828 ◽

Cited By ~ 12

Author(s):

Irma ROMERO ◽

Ana M. MALDONADO ◽

Pilar ERASO

Keyword(s):

Plasma Membrane ◽

Protein Kinase ◽

Atpase Activity ◽

Plasma Membranes ◽

Glucose Regulation ◽

Wild Type ◽

Calmodulin Antagonists ◽

Dependent Protein Kinase ◽

Yeast Plasma Membrane ◽

Dependent Protein

Glucose metabolism causes activation of the yeast plasma-membrane H+-ATPase. The molecular mechanism of this regulation is not known, but it is probably mediated by phosphorylation of the enzyme. The involvement in this process of several kinases has been suggested but their actual role has not been proved. The physiological role of a calmodulin-dependent protein kinase in glucose-induced activation was investigated by studying the effect of specific calmodulin antagonists on the glucose-induced ATPase kinetic changes in wild-type and two mutant strains affected in the glucose regulation of the enzyme. Preincubation of the cells with calmidazolium or compound 48/80 impeded the increase in ATPase activity by reducing the Vmax of the enzyme without modifying the apparent affinity for ATP in the three strains. In one mutant, pma1-T912A, the putative calmodulin-dependent protein kinase-phosphorylatable Thr-912 was eliminated, and in the other, pma1-P536L, H+-ATPase was constitutively activated, suggesting that the antagonistic effect was not mediated by a calmodulin-dependent protein kinase and not related to glucose regulation. This was corroborated when the in vitroeffect of the calmodulin antagonists on H+-ATPase activity was tested. Purified plasma membranes from glucose-starved or glucose-fermenting cells from both pma1-P890X, another constitutively activated ATPase mutant, and wild-type strains were preincubated with calmidazolium or melittin. In all cases, ATP hydrolysis was inhibited with an IC50 of ≈1 μM. This inhibition was reversed by calmodulin. Analysis of the calmodulin-binding protein pattern in the plasma-membrane fraction eliminates ATPase as the calmodulin target protein. We conclude that H+-ATPase inhibition by calmodulin antagonists is mediated by an as yet unidentified calmodulin-dependent membrane protein.

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Overexpression of plasma membrane Na+/H+ antiporter OsSOS1 gene improves salt tolerance in transgenic rice plants

ORYZA- An International Journal on Rice ◽

10.35709/ory.2020.57.4.3 ◽

2020 ◽

Vol 57 (4) ◽

pp. 277-287

Author(s):

Sushma M Awaji ◽

Prashantkumar S Hanjagi ◽

Pushpa BN ◽

Sashidhar VR

Keyword(s):

Plasma Membrane ◽

Salt Tolerance ◽

Cell Viability ◽

Chlorophyll Content ◽

Transgenic Rice ◽

Screening Test ◽

Wild Type ◽

Rice Plants ◽

Transgenic Rice Plants ◽

Salt Screening

Crop productivity is greatly affected by soil salinity; therefore, improvement in salinity tolerance of crops is a major goal in salt-tolerant breeding. The Salt Overly Sensitive (SOS) signal-transduction pathway plays a key role in ion homeostasis and salt tolerance in plants. In plants pumping of Na+ from the root cells is mediated by the plasma membrane Na+/H+ antiporter (SOS1) which plays important role in preventing the accumulation of toxic levels of Na+ in cytosol. In the present study, OsSOS1 (NHX7), gene was overexpressed in rice (var-Vikas) by Agrobacterium mediated In Planta transformation technique. To screen putative T1 plants for salt tolerance, stringent salt screening test was followed and root and shoot growth of transformants were used as selection criterion. Some of the putative transgenics showed significantly higher root growth compared to wild type. To confirm the presence of transgene in putative T1 transgenic plants, PCR based approach was followed using genomic DNA. The result showed that 16 % of the selected seedlings from the stringent salt screening test were PCR positives. Five selected lines were positive for RT-PCR analysis. Physiological studies such as chlorophyll content, membrane permeability, cell viability and sodium /potassium content analysis were also conducted to assess their levels of tolerance. Some of the T1 transformants showed lower percent reduction in chlorophyll content and less membrane leakage, higher cell viability and maintained higher K/Na ratio after NaCl treatment compared to wild type. These results clearly demonstrate that transgenic rice plants overexpressing OsSOS1 have better salt-tolerance. This could be attributed to extrusion of excess Na+ from cytosol into the apoplast and thereby reducing the toxic effects of Na+in the cell.

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Selective loss of substrate recognition induced by the tumour-associated D294G point mutation in protein kinase Cα

Biochemical Journal ◽

10.1042/bj3340393 ◽

1998 ◽

Vol 334 (2) ◽

pp. 393-397 ◽

Cited By ~ 11

Author(s):

Corinne PRÉVOSTEL ◽

Véronique ALVARO ◽

Alice VALLENTIN ◽

Annick MARTIN ◽

Susan JAKEN ◽

...

Keyword(s):

Plasma Membrane ◽

Protein Kinase ◽

Protein Interactions ◽

Kinase Activity ◽

Substrate Recognition ◽

Mutant Enzyme ◽

Wild Type ◽

Wild Type Enzyme ◽

Selective Loss ◽

Protein Kinase Cα

The tumour-associated D294G mutant of protein kinase Cα (PKCα) was recently shown not to be translocated to the plasma membrane on stimulation with PMA, in contrast with the wild-type enzyme. Using recombinant wild-type and mutant PKCα, we establish here that, although the PKCα intrinsic lipid-dependent catalytic activity remains unaltered by the D294G mutation, the mutant enzyme exhibits a selective loss of substrate recognition. Indeed, whereas the mutant enzyme is still able to phosphorylate histone IIIS with comparable efficiency to that of the wild-type enzyme, it exhibits a lack of kinase activity towards the previously cloned 35F and 35H substrates for PKC. Overlay experiments demonstrate that this selective loss of kinase activity is correlated with a decrease in binding of D294G PKCα to the 35F and 35H proteins compared with that of the wild-type enzyme. Because the 35H and 35F proteins are predicted to be PKCα-anchoring proteins, these findings suggest a selective loss of PKCα–protein interactions that might fail to stabilize the location of the PKCα mutant at the plasma membrane.

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A Single Point Mutation in the V3 Region Affects Protein Kinase Cα Targeting and Accumulation at Cell-Cell Contacts

Molecular and Cellular Biology ◽

10.1128/mcb.21.10.3351-3363.2001 ◽

2001 ◽

Vol 21 (10) ◽

pp. 3351-3363 ◽

Cited By ~ 24

Author(s):

Alice Vallentin ◽

Thi-Chang Lo ◽

Dominique Joubert

Keyword(s):

Plasma Membrane ◽

Protein Kinase ◽

Single Point ◽

Single Point Mutation ◽

Actin Network ◽

Wild Type ◽

Cell Contacts ◽

Regulatory Processes ◽

Protein Kinase Cα ◽

Cell Cell

ABSTRACT Given the importance of intercellular adhesion for many regulatory processes, we have investigated the control of protein kinase Cα (PKCα) targeting to the cell-cell contacts. We have previously shown that, upon treatment of the pituitary cell line GH3B6 with thyrotropin-releasing hormone (TRH) or phorbol 12-myristate 13-acetate (PMA), human PKCα (hPKCα) is selectively targeted to the cell-cell contacts (42). Here we show that the D294G mutation of hPKCα, previously identified in a subpopulation of human tumors, induces the loss of this selective targeting. The D294G mutant is instead targeted to the entire plasma membrane, including the cell-cell contacts, and the duration of the first rapid and transient translocation induced by TRH (42) is longer than that of the wild-type enzyme (93.3 versus 22.5 s), coinciding with the duration of the [Ca2+]i increase. We found that in the presence or absence of PMA, RACK1 is never localized at the cell-cell contacts nor was it coimmunoprecipitated with hPKCα wild type or the D294G mutant. In contrast, PMA treatment or long-term TRH stimulation resulted in the presence of F-actin and β-catenin at the cell-cell contacts and their exclusion from the rest of the plasma membrane. Upon disruption of the F-actin network with phalloidin or cytochalasin D, wild-type hPKCα translocates but did not accumulate at the plasma membrane and β-catenin did not accumulate at the cell-cell contacts. In contrast, the disruption of the F-actin network affected neither translocation nor accumulation of the D294G mutant. These results show that the presence of PKCα at the cell-cell contacts is a regulated process which depends upon the integrity of both PKCα and the actin microfilament network.

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Adenoviral expression of NHERF-1 in NHERF-1 null mouse renal proximal tubule cells restores Npt2a regulation by low phosphate media and parathyroid hormone

AJP Renal Physiology ◽

10.1152/ajprenal.00036.2006 ◽

2006 ◽

Vol 291 (4) ◽

pp. F896-F901 ◽

Cited By ~ 24

Author(s):

Rochelle Cunningham ◽

Deborah Steplock ◽

Xiaofei E ◽

Rajat S. Biswas ◽

Fengying Wang ◽

...

Keyword(s):

Plasma Membrane ◽

Parathyroid Hormone ◽

Protein Kinase ◽

Proximal Tubule ◽

Phosphate Transport ◽

Null Mouse ◽

Wild Type ◽

Proximal Tubule Cells ◽

Sodium Dependent ◽

Tubule Cells

Sodium-dependent phosphate transport in NHERF-1−/− proximal tubule cells does not increase when grown in a low phosphate media and is resistant to the normal inhibitory effects of parathyroid hormone (PTH). The current experiments employ adenovirus-mediated gene transfer in primary cultures of mouse proximal tubule cells from NHERF-1 null mice to explore the specific role of NHERF-1 on regulated Npt2a trafficking and sodium-dependent phosphate transport. NHERF-1 null cells have decreased sodium-dependent phosphate transport compared with wild-type cells. Infection of NHERF-1 null cells with adenovirus-GFP-NHERF-1 increased phosphate transport and plasma membrane abundance of Npt2a. Adenovirus-GFP-NHERF-1 infected NHERF-1 null proximal tubule cells but not cells infected with adenovirus-GFP demonstrated increased phosphate transport and Npt2a abundance in the plasma membrane when grown in low phosphate (0.1 mM) compared with high phosphate media (1.9 mM). PTH inhibited phosphate transport and decreased Npt2a abundance in the plasma membrane of adenovirus-GFP-NHERF-1-infected NHERF-1 null proximal tubule cells but not cells infected with adenovirus-GFP. Interestingly, phosphate transport is inhibited by activation of protein kinase A and protein kinase C in wild-type proximal tubule cells but not in NHERF-1−/− cells. Together, these results highlight the requirement for NHERF-1 for physiological control of Npt2a trafficking and suggest that the Npt2a/NHERF-1 complex represents a unique PTH-responsive pool of Npt2a in renal microvilli.

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