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.
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 microM 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 microM BK for 1 min resulted in an increase in the recovery of a portion of the cellular GAP in anti-Tyr(P) immunoprecipitates. 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 microM 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 p125 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.
Leeb-Lundberg
L M
LM
Department of Biochemistry, University of Texas Health Science Center, San Antonio 78284-7760.
Song
X H
XH
D016475:3T3 Cells; D000818:Animals; D000906:Antibodies; D001839:Bombesin / Q000494:pharmacology*; D001920:Bradykinin / Q000494:pharmacology*; D002455:Cell Division; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D020690:GTPase-Activating Proteins; D006801:Humans; D015151:Immunoblotting; D051379:Mice; D008970:Molecular Weight; D010750:Phosphoproteins / Q000302:isolation & purification / Q000378:metabolism*; D010766:Phosphorylation; D019000:Phosphotyrosine; D011506:Proteins / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000032:analysis; D020703:ras GTPase-Activating Proteins
D000906:Antibodies; D020690:GTPase-Activating Proteins; D010750:Phosphoproteins; D011506:Proteins; D020703:ras GTPase-Activating Proteins; D019000:Phosphotyrosine; D014443:Tyrosine; D001839:Bombesin; D001920:Bradykinin