Mutation of the Protein Kinase C Phosphorylation Site on Rat α1 Na+,K+-ATPase Alters Regulation of Intracellular Na+ and pH and Influences Cell Shape and Adhesiveness

Belusa, Roger; Wang, Zhengming; Matsubara, Takako; Sahlgren, B.; Dulubova, Irina; Nairn, Angus C.; Ruoslahti, Erkki; Greengard, Paul; Aperia, Anita

doi:10.1074/jbc.272.32.20179

Cited by 56 publications

(32 citation statements)

References 30 publications

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“…GFP-NKA␣1 was stably expressed in COS-7 cells (12). pGFP-NKA␣1, pGFP-NKA␣1.M32, pEF-GSTm49-IRES-GFP, or pEGFP-actin was transiently transfected into RPT cells on culture day 2 using CLONfectin (Clontech).…”

Section: Methodsmentioning

confidence: 99%

Cell Signaling Microdomain with Na,K-ATPase and Inositol 1,4,5-Trisphosphate Receptor Generates Calcium Oscillations

Miyakawa-Naito

Uhlén

Lal

et al. 2003

Journal of Biological Chemistry

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157

158

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Section: Methodsmentioning

confidence: 99%

Cell Signaling Microdomain with Na,K-ATPase and Inositol 1,4,5-Trisphosphate Receptor Generates Calcium Oscillations

Miyakawa-Naito

Uhlén

Lal

et al. 2003

Journal of Biological Chemistry

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157

158

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“…In this regard, it has been demonstrated that the ␣-subunit of Na ϩ ,K ϩ -ATPase can be phosphorylated by PKA and PKC in vitro (Bertorello et al, 1991;Chibalin et al, 1992;Feschenko and Sweadner, 1995) and in intact cells (Middleton et al, 1993;Béguin et al, 1994;Féraille et al, 1995;Fisone et al, 1995;Carranza et al, 1996b). Recent studies in transfected cells indicate that serine-threonine phosphorylation of the catalytic ␣-subunit by PKA or PKC is a key event in the short-term regulation of Na ϩ ,K ϩ -ATPase activity (Fisone et al, 1994;Belusa et al, 1997;Pedemonte et al, 1997;Chibalin et al, 1998). The physiological relevance of this process is supported by the correlation between the phosphorylation level of the ␣-subunit and the activity of Na ϩ ,K ϩ -ATPase in response to PKA (Carranza et al, 1996b) or PKC activation (Carranza et al, 1996a) in isolated rat PCT.…”

Section: Introductionmentioning

confidence: 99%

Insulin-induced Stimulation of Na⁺,K⁺-ATPase Activity in Kidney Proximal Tubule Cells Depends on Phosphorylation of the α-Subunit at Tyr-10

Féraille

Carranza

Gonin

et al. 1999

MBoC

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Phosphorylation of the ␣-subunit of Na ϩ ,K ϩ -ATPase plays an important role in the regulation of this pump. Recent studies suggest that insulin, known to increase solute and fluid reabsorption in mammalian proximal convoluted tubule (PCT), is stimulating Na ϩ ,K ϩ -ATPase activity through the tyrosine phosphorylation process. This study was therefore undertaken to evaluate the role of tyrosine phosphorylation of the Na ϩ ,K ϩ -ATPase ␣-subunit in the action of insulin. In rat PCT, insulin and orthovanadate (a tyrosine phosphatase inhibitor) increased tyrosine phosphorylation level of the ␣-subunit more than twofold. Their effects were not additive, suggesting a common mechanism of action. Insulin-induced tyrosine phosphorylation was prevented by genistein, a tyrosine kinase inhibitor. The site of tyrosine phosphorylation was identified on Tyr-10 by controlled trypsinolysis in rat PCTs and by site-directed mutagenesis in opossum kidney cells transfected with rat ␣-subunit. The functional relevance of Tyr-10 phosphorylation was assessed by 1) the abolition of insulin-induced stimulation of the ouabain-sensitive 86 Rb uptake in opossum kidney cells expressing mutant rat ␣1-subunits wherein tyrosine was replaced by alanine or glutamine; and 2) the similarity of the time course and dose dependency of the insulin-induced increase in ouabain-sensitive 86 Rb uptake and tyrosine phosphorylation. These findings indicate that phosphorylation of the Na ϩ ,K ϩ -ATPase ␣-subunit at Tyr-10 likely participates in the physiological control of sodium reabsorption in PCT.

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“…[17] Thus, the changes observed in this study should be regarded as representative only for the α1-subunit phosphorylation degree at Ser-23 and not as phosphorylation rates of other sites. [35,36] However, phosphorylation at the Ser-23 is responsible for most of the PKC dependent phosphorylation of the enzyme in the rat, since it has been demonstrated that PKC dependent Na + K + ATPase phosphorylation is almost suppressed when the Ser-23 site is mutated. [35,36] Though in the current experiments PKC activity was not measured, results in the CRF experimental model would suggest a constitutive activation of the enzyme in mTAL segments.…”

Section: Discussionmentioning

confidence: 99%

Mechanisms of PKC-Dependent Na⁺K⁺ATPase Phosphorylation in the Rat Kidney with Chronic Renal Failure

et al. 2007

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The present work was designed to study Na + K + ATPase α1-subunit phosphorylation in rats with chronic renal failure (CRF) in comparison with normal rats. Na + K + ATPase α1-subunit phosphorylation degree was measured by binding the McK-1 antibody to dephosphorylated Ser-23 in microdissected medullary thick ascending limb of Henle (mTAL) segments. In addition, the total Na + K + ATPase α1-subunit expression and activity were also measured in the outer renal medulla homogenates and membranes.CRF rats showed a higher Na + K + ATPase activity, as compared with control rats (18.95 ± 2.4 vs. 11.21 ± 1.5 μmol Pi/mg prot/h, p < 0.05), accompanied by a higher total Na + K + ATPase expression (0.54 ± 0.04 vs. 0.27 ± 0.02 normalized arbitrary units (NU), p < 0.05). When McK-1 antibody was used, a higher immunosignal in mTAL of CRF rats was observed, as compared with controls (6.3 ± 0.35 vs.4.1 ± 0.33 NU, p < 0.05). The ratio Na + K + ATPase α1-subunit phosphorylation / total Na + K + ATPase α1-subunit expression per μg protein showed a nonsignificant difference between CRF and control rats in microdissected mTAL segments (2.11 ± 0.12 vs.2.26 ± 0.18 NU, p = NS). The PKC inhibitor RO-318220 10 −6 M increased immunosignal (lower phosphorylation degree) in mTAL of CRF rats to 128.43 ± 7.08% (p < 0.05) but did not alter McK1 binding in control rats. Both phorbol 12-myristate 13-acetate (PMA) 10 −6 M and dopamine 10 −6 M decreased immunosignal in CRF rats, corresponding to a higher Na + K + ATPase α1-subunit phosphorylation degree at Ser-23 (55.26 ± 11.17% and 53.27 ± 7.12% compared with basal, p < 0.05). In mTAL of CRF rats, the calcineurin inhibitor FK-506 10 −6 M did not modify phosphorylation degree at Ser-23 of Na + K + ATPase α1-subunit (100.21 ± 3.00% compared with basal CRF). In control rats, FK 506 10M decreased the immunosignal, which corresponds to a higher Na + K + ATPase α1-subunit phosphorylation degree at Ser-23. The data suggest that the regulation of basal Na + K + ATPase α1-subunit phosphorylation degree at Ser-23 in mTAL segments of CRF rats was primarily dependent on PKC activation rather than calcineurin dependent mechanisms.

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Mutation of the Protein Kinase C Phosphorylation Site on Rat α1 Na+,K+-ATPase Alters Regulation of Intracellular Na+ and pH and Influences Cell Shape and Adhesiveness

Cited by 56 publications

References 30 publications

Cell Signaling Microdomain with Na,K-ATPase and Inositol 1,4,5-Trisphosphate Receptor Generates Calcium Oscillations

Cell Signaling Microdomain with Na,K-ATPase and Inositol 1,4,5-Trisphosphate Receptor Generates Calcium Oscillations

Insulin-induced Stimulation of Na⁺,K⁺-ATPase Activity in Kidney Proximal Tubule Cells Depends on Phosphorylation of the α-Subunit at Tyr-10

Mechanisms of PKC-Dependent Na⁺K⁺ATPase Phosphorylation in the Rat Kidney with Chronic Renal Failure

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Mutation of the Protein Kinase C Phosphorylation Site on Rat α1 Na+,K+-ATPase Alters Regulation of Intracellular Na+ and pH and Influences Cell Shape and Adhesiveness

Cited by 56 publications

References 30 publications

Cell Signaling Microdomain with Na,K-ATPase and Inositol 1,4,5-Trisphosphate Receptor Generates Calcium Oscillations

Cell Signaling Microdomain with Na,K-ATPase and Inositol 1,4,5-Trisphosphate Receptor Generates Calcium Oscillations

Insulin-induced Stimulation of Na+,K+-ATPase Activity in Kidney Proximal Tubule Cells Depends on Phosphorylation of the α-Subunit at Tyr-10

Mechanisms of PKC-Dependent Na+K+ATPase Phosphorylation in the Rat Kidney with Chronic Renal Failure

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Insulin-induced Stimulation of Na⁺,K⁺-ATPase Activity in Kidney Proximal Tubule Cells Depends on Phosphorylation of the α-Subunit at Tyr-10

Mechanisms of PKC-Dependent Na⁺K⁺ATPase Phosphorylation in the Rat Kidney with Chronic Renal Failure