Control of phosphatidylserine synthase II activity in Chinese hamster ovary cells

Osamu Kuge, Kyoko Saito, Masahiro Nishijima

Research output: Contribution to journalArticle

29 Citations (Scopus)

Abstract

Phosphatidylserine (PtdSer) in Chinese hamster ovary (CHO) cells is synthesized through the action of PtdSer synthase (PSS) I and II, which catalyzes the exchange of L-serine with the base moiety of phosphatidylcholine and phosphatidylethanolamine, respectively. The PtdSer synthesis in a CHO cell mutant, PSA-3, which lacks PSS I but has normal PSS II activity, was almost completely inhibited by the addition of PtdSer to the culture medium, like that in the wild-type CHO-K1 cells. In contrast, the PtdSer synthesis in a PSS II-over-producing stable transformant of CHO-K1, K1/wt-pssB, was reduced by only 35% upon addition of PtdSer. The serine exchange activity in a membrane fraction of K1/wt-pssB cells was not inhibited by PtdSer at all, whereas those of PSA-3 and CHO-K1 cells were inhibited by >95%. These results indicated that PSS II activity in PSA-3 and CHO-K1 cells is inhibited by exogenous PtdSer and that overproduction of PSS II leads to the loss of normal control of PSS II activity by exogenous PtdSer. Although overproduced PSS II in K1/wt-pssB cells was not normally controlled by exogenous PtdSer, K1/wt-pssB cells cultivated without exogenous PtdSer exhibited a normal PtdSer biosynthetic rate similar to that in CHO-K1 cells. In contrast to K1/wt-pssB cells, another stable transformant of CHO- K1, K1/R97K-pssB, which overproduces R97K mutant PSS II, exhibited a ~4-fold higher PtdSer biosynthetic rate compared with that in CHO-K1 cells. These results suggested that for maintenance of a normal PtdSer biosynthetic rate, the activity of overproduced wild-type PSS II in K1/wt-pssB cells is depressed by an as yet unknown post-translational mechanisms other than those for the exogenous PtdSer-mediated inhibition and that Arg-97 of PSS II is critical for this depression of overproduced PSS II activity. When the cDNA- directed wild-type and R97K mutant PSS II activities were expressed at nonoverproduction levels in a PSS I- and PSS II-defective mutant of CHO-K1 cells, expression of the mutant PSS II activity but not that of the wild-type PSS II activity induced the PtdSer-resistant PtdSer biosynthesis. This suggested that Arg-97 of PSS II is critical also for the exogenous PtdSer- mediated inhibition of PSS II.

Original languageEnglish
Pages (from-to)23844-23849
Number of pages6
JournalJournal of Biological Chemistry
Volume274
Issue number34
DOIs
Publication statusPublished - Aug 20 1999
Externally publishedYes

Fingerprint

Phosphatidylserines
Cricetulus
Ovary
Cells
Glycogen Synthase
Mouse Ptdss2 protein
Serine
Biosynthesis
Phosphatidylcholines

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

Control of phosphatidylserine synthase II activity in Chinese hamster ovary cells. / Kuge, Osamu; Saito, Kyoko; Nishijima, Masahiro.

In: Journal of Biological Chemistry, Vol. 274, No. 34, 20.08.1999, p. 23844-23849.

Research output: Contribution to journalArticle

Kuge, Osamu ; Saito, Kyoko ; Nishijima, Masahiro. / Control of phosphatidylserine synthase II activity in Chinese hamster ovary cells. In: Journal of Biological Chemistry. 1999 ; Vol. 274, No. 34. pp. 23844-23849.
@article{83b60786fdb2463ba04e05ff1f4e6b22,
title = "Control of phosphatidylserine synthase II activity in Chinese hamster ovary cells",
abstract = "Phosphatidylserine (PtdSer) in Chinese hamster ovary (CHO) cells is synthesized through the action of PtdSer synthase (PSS) I and II, which catalyzes the exchange of L-serine with the base moiety of phosphatidylcholine and phosphatidylethanolamine, respectively. The PtdSer synthesis in a CHO cell mutant, PSA-3, which lacks PSS I but has normal PSS II activity, was almost completely inhibited by the addition of PtdSer to the culture medium, like that in the wild-type CHO-K1 cells. In contrast, the PtdSer synthesis in a PSS II-over-producing stable transformant of CHO-K1, K1/wt-pssB, was reduced by only 35{\%} upon addition of PtdSer. The serine exchange activity in a membrane fraction of K1/wt-pssB cells was not inhibited by PtdSer at all, whereas those of PSA-3 and CHO-K1 cells were inhibited by >95{\%}. These results indicated that PSS II activity in PSA-3 and CHO-K1 cells is inhibited by exogenous PtdSer and that overproduction of PSS II leads to the loss of normal control of PSS II activity by exogenous PtdSer. Although overproduced PSS II in K1/wt-pssB cells was not normally controlled by exogenous PtdSer, K1/wt-pssB cells cultivated without exogenous PtdSer exhibited a normal PtdSer biosynthetic rate similar to that in CHO-K1 cells. In contrast to K1/wt-pssB cells, another stable transformant of CHO- K1, K1/R97K-pssB, which overproduces R97K mutant PSS II, exhibited a ~4-fold higher PtdSer biosynthetic rate compared with that in CHO-K1 cells. These results suggested that for maintenance of a normal PtdSer biosynthetic rate, the activity of overproduced wild-type PSS II in K1/wt-pssB cells is depressed by an as yet unknown post-translational mechanisms other than those for the exogenous PtdSer-mediated inhibition and that Arg-97 of PSS II is critical for this depression of overproduced PSS II activity. When the cDNA- directed wild-type and R97K mutant PSS II activities were expressed at nonoverproduction levels in a PSS I- and PSS II-defective mutant of CHO-K1 cells, expression of the mutant PSS II activity but not that of the wild-type PSS II activity induced the PtdSer-resistant PtdSer biosynthesis. This suggested that Arg-97 of PSS II is critical also for the exogenous PtdSer- mediated inhibition of PSS II.",
author = "Osamu Kuge and Kyoko Saito and Masahiro Nishijima",
year = "1999",
month = "8",
day = "20",
doi = "10.1074/jbc.274.34.23844",
language = "English",
volume = "274",
pages = "23844--23849",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "34",

}

TY - JOUR

T1 - Control of phosphatidylserine synthase II activity in Chinese hamster ovary cells

AU - Kuge, Osamu

AU - Saito, Kyoko

AU - Nishijima, Masahiro

PY - 1999/8/20

Y1 - 1999/8/20

N2 - Phosphatidylserine (PtdSer) in Chinese hamster ovary (CHO) cells is synthesized through the action of PtdSer synthase (PSS) I and II, which catalyzes the exchange of L-serine with the base moiety of phosphatidylcholine and phosphatidylethanolamine, respectively. The PtdSer synthesis in a CHO cell mutant, PSA-3, which lacks PSS I but has normal PSS II activity, was almost completely inhibited by the addition of PtdSer to the culture medium, like that in the wild-type CHO-K1 cells. In contrast, the PtdSer synthesis in a PSS II-over-producing stable transformant of CHO-K1, K1/wt-pssB, was reduced by only 35% upon addition of PtdSer. The serine exchange activity in a membrane fraction of K1/wt-pssB cells was not inhibited by PtdSer at all, whereas those of PSA-3 and CHO-K1 cells were inhibited by >95%. These results indicated that PSS II activity in PSA-3 and CHO-K1 cells is inhibited by exogenous PtdSer and that overproduction of PSS II leads to the loss of normal control of PSS II activity by exogenous PtdSer. Although overproduced PSS II in K1/wt-pssB cells was not normally controlled by exogenous PtdSer, K1/wt-pssB cells cultivated without exogenous PtdSer exhibited a normal PtdSer biosynthetic rate similar to that in CHO-K1 cells. In contrast to K1/wt-pssB cells, another stable transformant of CHO- K1, K1/R97K-pssB, which overproduces R97K mutant PSS II, exhibited a ~4-fold higher PtdSer biosynthetic rate compared with that in CHO-K1 cells. These results suggested that for maintenance of a normal PtdSer biosynthetic rate, the activity of overproduced wild-type PSS II in K1/wt-pssB cells is depressed by an as yet unknown post-translational mechanisms other than those for the exogenous PtdSer-mediated inhibition and that Arg-97 of PSS II is critical for this depression of overproduced PSS II activity. When the cDNA- directed wild-type and R97K mutant PSS II activities were expressed at nonoverproduction levels in a PSS I- and PSS II-defective mutant of CHO-K1 cells, expression of the mutant PSS II activity but not that of the wild-type PSS II activity induced the PtdSer-resistant PtdSer biosynthesis. This suggested that Arg-97 of PSS II is critical also for the exogenous PtdSer- mediated inhibition of PSS II.

AB - Phosphatidylserine (PtdSer) in Chinese hamster ovary (CHO) cells is synthesized through the action of PtdSer synthase (PSS) I and II, which catalyzes the exchange of L-serine with the base moiety of phosphatidylcholine and phosphatidylethanolamine, respectively. The PtdSer synthesis in a CHO cell mutant, PSA-3, which lacks PSS I but has normal PSS II activity, was almost completely inhibited by the addition of PtdSer to the culture medium, like that in the wild-type CHO-K1 cells. In contrast, the PtdSer synthesis in a PSS II-over-producing stable transformant of CHO-K1, K1/wt-pssB, was reduced by only 35% upon addition of PtdSer. The serine exchange activity in a membrane fraction of K1/wt-pssB cells was not inhibited by PtdSer at all, whereas those of PSA-3 and CHO-K1 cells were inhibited by >95%. These results indicated that PSS II activity in PSA-3 and CHO-K1 cells is inhibited by exogenous PtdSer and that overproduction of PSS II leads to the loss of normal control of PSS II activity by exogenous PtdSer. Although overproduced PSS II in K1/wt-pssB cells was not normally controlled by exogenous PtdSer, K1/wt-pssB cells cultivated without exogenous PtdSer exhibited a normal PtdSer biosynthetic rate similar to that in CHO-K1 cells. In contrast to K1/wt-pssB cells, another stable transformant of CHO- K1, K1/R97K-pssB, which overproduces R97K mutant PSS II, exhibited a ~4-fold higher PtdSer biosynthetic rate compared with that in CHO-K1 cells. These results suggested that for maintenance of a normal PtdSer biosynthetic rate, the activity of overproduced wild-type PSS II in K1/wt-pssB cells is depressed by an as yet unknown post-translational mechanisms other than those for the exogenous PtdSer-mediated inhibition and that Arg-97 of PSS II is critical for this depression of overproduced PSS II activity. When the cDNA- directed wild-type and R97K mutant PSS II activities were expressed at nonoverproduction levels in a PSS I- and PSS II-defective mutant of CHO-K1 cells, expression of the mutant PSS II activity but not that of the wild-type PSS II activity induced the PtdSer-resistant PtdSer biosynthesis. This suggested that Arg-97 of PSS II is critical also for the exogenous PtdSer- mediated inhibition of PSS II.

UR - http://www.scopus.com/inward/record.url?scp=0033588336&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0033588336&partnerID=8YFLogxK

U2 - 10.1074/jbc.274.34.23844

DO - 10.1074/jbc.274.34.23844

M3 - Article

VL - 274

SP - 23844

EP - 23849

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 34

ER -