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Identification of p125, a component of a group of 120-kDa proteins that are phosphorylated on tyrosine residues in response to bradykinin and bombesin stimulation, in anti-ras-GTPase-activating protein immunoprecipitates of swiss 3T3 Cells

Leeb-Lundberg, L. M Fredrik LU and Song, Xin Hua (1993) In Journal of Biological Chemistry 268(11). p.8151-8157
Abstract

Bradykinin (BK) and bombesin (BN) stimulate an increase in the tyrosine phosphorylation of a 120-kDa group of proteins (pps120) in Swiss 3T3 cells (Leeb-Lundberg, L. M. F., and Song X.-H. (1991) J. Biol. Chem. 266, 7746-7749). Here, we show that a component of pps120, p125, was specifically immunoprecipitated with antibodies against the p21ras GTPase-activating protein (GAP). The major portion of GAP in nonstimulated cells (96%) was located in the cytosol, and this distribution was not affected by exposure of cells to 1 μM BK for 1 min. A significant amount of GAP in nonstimulated cells was recovered in anti-phosphotyrosine (anti-Tyr(P)) immunoprecipitates, and the cellular distribution of this GAP essentially paralleled that... (More)

Bradykinin (BK) and bombesin (BN) stimulate an increase in the tyrosine phosphorylation of a 120-kDa group of proteins (pps120) in Swiss 3T3 cells (Leeb-Lundberg, L. M. F., and Song X.-H. (1991) J. Biol. Chem. 266, 7746-7749). Here, we show that a component of pps120, p125, was specifically immunoprecipitated with antibodies against the p21ras GTPase-activating protein (GAP). The major portion of GAP in nonstimulated cells (96%) was located in the cytosol, and this distribution was not affected by exposure of cells to 1 μM BK for 1 min. A significant amount of GAP in nonstimulated cells was recovered in anti-phosphotyrosine (anti-Tyr(P)) immunoprecipitates, and the cellular distribution of this GAP essentially paralleled that of total GAP. Recovery of GAP in anti-Tyr(P) immunoprecipitates of nonstimulated cells appeared to be caused at least in part by the presence of GAP complexed to a 190-kDa tyrosine-phosphorylated protein (p190). Exposure of cells to 1 μM BK for 1 min resulted in an increase in the recovery of a portion of the cellular GAP in anti-Tyr(P) inmmunoprecipitates. This increase was paralleled by the appearance of a tyrosine-phosphorylated protein species of 125 kDa (p125) in anti-GAP immunoprecipitates. Tyrosine-phosphorylated p125 was present also in anti-GAP immunoprecipitates after exposure of cells to 1 μM BN. High performance gel exclusion liquid chromatography of the anti-GAP-immunoprecipitated proteins on a Protein-Pak 300SW column revealed that p125 is not GAP. Anti-GAP immunoprecipitation of p125 was prevented by prior denaturation of cell lysates in sodium dodecyl sulfate suggesting that p 125 is physically associated with GAP. Chromatography of cell lysates revealed that the pps120 group of tyrosine phosphoproteins includes a 125- and a 120-kDa protein. The anti-GAP-immunoprecipitable p125 migrated identically to the 125-kDa phosphoprotein component of pps120. These observations show that the pps120 group of tyrosine phosphoproteins is composed of at least two physically distinct protein components, p125 and p120. p125 is associated in some manner with a portion of the cellular GAP after exposure of cells to BK and BN.

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author
publishing date
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Contribution to journal
publication status
published
subject
in
Journal of Biological Chemistry
volume
268
issue
11
pages
8151 - 8157
publisher
ASBMB
external identifiers
  • pmid:7681835
  • scopus:0027418334
ISSN
0021-9258
language
English
LU publication?
no
id
97f87976-863b-432a-9cea-a0bd53904849
date added to LUP
2019-06-12 11:43:51
date last changed
2019-11-25 09:31:23
@article{97f87976-863b-432a-9cea-a0bd53904849,
  abstract     = {<p>Bradykinin (BK) and bombesin (BN) stimulate an increase in the tyrosine phosphorylation of a 120-kDa group of proteins (pps120) in Swiss 3T3 cells (Leeb-Lundberg, L. M. F., and Song X.-H. (1991) J. Biol. Chem. 266, 7746-7749). Here, we show that a component of pps120, p125, was specifically immunoprecipitated with antibodies against the p21<sub>ras</sub> GTPase-activating protein (GAP). The major portion of GAP in nonstimulated cells (96%) was located in the cytosol, and this distribution was not affected by exposure of cells to 1 μM BK for 1 min. A significant amount of GAP in nonstimulated cells was recovered in anti-phosphotyrosine (anti-Tyr(P)) immunoprecipitates, and the cellular distribution of this GAP essentially paralleled that of total GAP. Recovery of GAP in anti-Tyr(P) immunoprecipitates of nonstimulated cells appeared to be caused at least in part by the presence of GAP complexed to a 190-kDa tyrosine-phosphorylated protein (p190). Exposure of cells to 1 μM BK for 1 min resulted in an increase in the recovery of a portion of the cellular GAP in anti-Tyr(P) inmmunoprecipitates. This increase was paralleled by the appearance of a tyrosine-phosphorylated protein species of 125 kDa (p125) in anti-GAP immunoprecipitates. Tyrosine-phosphorylated p125 was present also in anti-GAP immunoprecipitates after exposure of cells to 1 μM BN. High performance gel exclusion liquid chromatography of the anti-GAP-immunoprecipitated proteins on a Protein-Pak 300SW column revealed that p125 is not GAP. Anti-GAP immunoprecipitation of p125 was prevented by prior denaturation of cell lysates in sodium dodecyl sulfate suggesting that p 125 is physically associated with GAP. Chromatography of cell lysates revealed that the pps120 group of tyrosine phosphoproteins includes a 125- and a 120-kDa protein. The anti-GAP-immunoprecipitable p125 migrated identically to the 125-kDa phosphoprotein component of pps120. These observations show that the pps120 group of tyrosine phosphoproteins is composed of at least two physically distinct protein components, p125 and p120. p125 is associated in some manner with a portion of the cellular GAP after exposure of cells to BK and BN.</p>},
  author       = {Leeb-Lundberg, L. M Fredrik and Song, Xin Hua},
  issn         = {0021-9258},
  language     = {eng},
  month        = {04},
  number       = {11},
  pages        = {8151--8157},
  publisher    = {ASBMB},
  series       = {Journal of Biological Chemistry},
  title        = {Identification of p125, a component of a group of 120-kDa proteins that are phosphorylated on tyrosine residues in response to bradykinin and bombesin stimulation, in anti-ras-GTPase-activating protein immunoprecipitates of swiss 3T3 Cells},
  volume       = {268},
  year         = {1993},
}