Induction of system A amino acid transport through long‐term treatment with ouabain: Correlation with increased (Na<sup>+</sup>/K<sup>+</sup>)‐ATpase activity

Schenerman, Mark A.; Leister, Kirk J.; Trachtenberg, David K.; Racker, Efraim

doi:10.1002/jcp.1041350202

Cited by 7 publications

(7 citation statements)

References 21 publications

(16 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The cells were grown and harvested as described previously (9,10). Plasma membranes from Rat-1 cells were prepared according to Thom et al (11) and stored at -70'C in 10%o (wt/vol) sucrose/20 mM Hepes (pH 7.4)/1 mM dithiothreitol.…”

Section: Methodsmentioning

confidence: 99%

Decreased susceptibility of a 70-kDa protein to cathepsin L after phosphorylation by protein kinase C.

Laumas

Abdel-Ghany²,

Leister³

et al. 1989

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

A 70-kDa protein is phosphorylated in cellfree preparations from rat or mouse fibroblasts by an endogenous protein kinase. This protein is immunologically related to a group of 68-kDa to 87-kDa proteins described in the literature as substrates for protein kinase C (PK-C). Although the phosphorylation of the 70-kDa protein by isolated plasma membranes takes place in the presence of EGTA, we conclude that the reaction is catalyzed by PK-C based on its inhibition by staurosporin. As shown previously, pure PK-C phosphorylates a synthetic random polymer of arginine and serine in the absence of Ca2+ and lipids, a reaction markedly stimulated by an endogenous unidentified activator of PK-C. When the '70-kDa protein from normal fibroblasts was exposed to the cytosol of chemically or ras-transformed fibroblasts, it disappeared as measured by phosphorylation by added PK-C. Cytosol ofnormal fibroblasts was much less effective (ca. 20%). Cathepsin L purified from rat kidney or from the medium of transformed cells had an effect similar to that of the cytosol of transformed cells. When the 70-kDa protein was phosphorylated by PK-C prior to exposure to cathepsin L or to the cytosol of transformed cells, there was a marked protection of the 70-kDa protein. We conclude that the 70-kDa protein is degraded by cathepsin L as ascertained by both immunological and biochemical assays and that it is protected by prior phosphorylation with PK-C. The possible role of this effect in signal transduction is discussed.

show abstract

Section: Methodsmentioning

confidence: 99%

Decreased susceptibility of a 70-kDa protein to cathepsin L after phosphorylation by protein kinase C.

Laumas

Abdel-Ghany²,

Leister³

et al. 1989

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

show abstract

“…We have described in the preceding paper (Schenerman et al, 1988) an increase in System A amino acid transport and Na+/K+ pump activity following the treatment of cells with ouabain. Under these con-ditions, enhanced Na + /K + pump activity, following removal of ouabain, correlated with high [Nafli.…”

mentioning

confidence: 94%

Energetic mechanism of system a amino acid transport in normal and transformed mouse fibroblasts

Leister

Schenerman

Racker

1988

Journal Cellular Physiology

Self Cite

View full text Add to dashboard Cite

Ouabain treatment (0.4 mM) of normal and transformed C3H-10T1/2 cells caused a progressive increase in 2-aminoisobutyrate (AIB) transport reaching a maximum after 16 to 18 h exposure. There was a virtually complete blockage of this stimulated rate when 3 microM cycloheximide (CHX) was added together with ouabain at T = 0. In the transformed cell, addition of CHX after 14 h had no effect; in the normal cell, it inhibited (ca. 50%) the final AIB transport rate achieved after 24 h. The t1/2 for reaching maximal activity (insensitive to CHX exposure) was thus shifted from 8 h in the transformed cell to 15 h in the normal cell. Since the rate of achieving maximal activity in the absence of CHX was about the same in the two cells, the shift in t1/2 in the presence of CHX suggests that the rate of degradation is more rapid in the normal cell. Following ouabain treatment, the apparent Km for Na+ was decreased in both cells. The Km returned to the basal level 1 h after ouabain removal in the normal cell, but remained low in the transformed cell during this time period. The stimulation of AIB transport following ouabain removal was largely abolished by a proton ionophore (1799), a lipophilic cation (tetraphenyl-phosphonium), or ouabain. These results suggest that, under the conditions of ouabain stress, there is a switch in the bioenergetic mechanism. The Na+/K+ pump and System A transporter appear to be linked and the membrane potential generated by the Na+/K+ pump activity becomes a major driving force for AIB uptake.

show abstract

“…Both carriers, the A system and the Na',K+-ATPase, are thus related mechanistically by sharing Na' as a substrate. It is therefore not surprising that the activities of both these systems are closely interrelated (16)(17)(18)(19)(20)(21)(22). Besides the concentration of Na' affecting the activity of the Na',K+-ATPase, under certain conditions that lead to increases in Na+ content there is an associated increase in the number of Na+,K+-ATPase molecules on the plasma membrane (16,(23)(24)(25)(26)(27)(28)(29)(30) and an increase in the abundance of their corresponding mRNA (29,30).…”

mentioning

confidence: 99%

Evidence for coordinate regulation of the A system for amino acid transport and the mRNA for the alpha 1 subunit of the Na+,K(+)-ATPase gene in Chinese hamster ovary cells.

Qian

Pastor‐Anglada

Englesberg

1991

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

Previous work suggested that the structural gene for the A system transporter and the mRNA for the a subunit of the Na+,K+-ATPase in Chinese hamster ovary cells CHO-K1 [wild type (WT)] are coordinately controlled by regulatory gene R1. This conclusion was based on analysis of a mutant for the A system, alar4. This mutant had a constitutive level of A system transport activity equal to the level found in derepressed WT cells and a 4 times increase in abundance ofthe a, subunit of Na',K'-ATPase mRNA over that found in repressed WT. The level of Na+ per cell in alar4 was not significantly greater than that found in the WT. To further characterize the likely coregulation of both genes, we have studied the A system activity and Na+,K+-ATPase mRNA a,-subunit levels in cells grown under various conditions that result in repression or derepression of the A system in the WT. System A activity increased up to 2-3 times the basal transport rate (repressed state) and Na+,K+-ATPase mRNA a,-subunit levels showed a 3-fold increase after amino acid starvation (derepressed state). These changes occurred along with a decrease in intracellular Na+ levels. N-Methyl-a-aminoisobutyric acid and fi-alanine, previously shown to be corepressors for the A system, prevented to a similar extent A system derepression and Na+,K+-ATPase mRNA a,-subunit accumulation. On the other hand, phenylalanine and lysine, amino acids that are not corepressors of the A system, failed to significantly prevent derepression of both genes. Hybrids between the WT and ala'4 have the phenotype of the WT when grown under repressed conditions. These results give further support to the proposition that both the A system transporter and mRNA for the aI subunit of the Na+,K+-ATPase are coordinately controlled by regulatory gene R1 and elevated Na+ concentrations are not involved. No Na+,K+-ATPase activity was detected in derepressed cells. Activity was restored by the addition of monensin. However, this activity was no greater than that obtained in repressed cells. Indications are that the reduced Na+ content in derepressed cells inhibits Na+,K+-ATPase activity and that conditions that favored derepression do not allow for de novo synthesis of the Na+,K+-ATPase.Evidence has been presented suggesting that the activity of the A system of amino acid transport and the Na+,K+-ATPase is coordinately regulated at the transcriptional level by means of repression and negative control by the A system regulatory gene R1 (1).The A system and the Na+ ,K+-ATPase are ubiquitous integral membrane proteins in mammalian cells that function as secondary and primary active transporters, respectively. The A system accepts as substrates most neutral amino acids but shows a strong preference for proline and small straight-chain amino acids such as alanine (2). It is Na' dependent and transports one Na' into the cells with each amino acid (3). This system is inducible by hormones (4-6), repressible by amino acids, which it generally transports (7-11), and present with increased activ...

show abstract

Induction of system A amino acid transport through long‐term treatment with ouabain: Correlation with increased (Na⁺/K⁺)‐ATpase activity

Cited by 7 publications

References 21 publications

Decreased susceptibility of a 70-kDa protein to cathepsin L after phosphorylation by protein kinase C.

Decreased susceptibility of a 70-kDa protein to cathepsin L after phosphorylation by protein kinase C.

Energetic mechanism of system a amino acid transport in normal and transformed mouse fibroblasts

Evidence for coordinate regulation of the A system for amino acid transport and the mRNA for the alpha 1 subunit of the Na+,K(+)-ATPase gene in Chinese hamster ovary cells.

Contact Info

Product

Resources

About

Induction of system A amino acid transport through long‐term treatment with ouabain: Correlation with increased (Na+/K+)‐ATpase activity

Cited by 7 publications

References 21 publications

Decreased susceptibility of a 70-kDa protein to cathepsin L after phosphorylation by protein kinase C.

Decreased susceptibility of a 70-kDa protein to cathepsin L after phosphorylation by protein kinase C.

Energetic mechanism of system a amino acid transport in normal and transformed mouse fibroblasts

Evidence for coordinate regulation of the A system for amino acid transport and the mRNA for the alpha 1 subunit of the Na+,K(+)-ATPase gene in Chinese hamster ovary cells.

Contact Info

Product

Resources

About

Induction of system A amino acid transport through long‐term treatment with ouabain: Correlation with increased (Na⁺/K⁺)‐ATpase activity