Bradykinin and bombesin rapidly stimulate tyrosine phosphorylation of a 120-kDa group of proteins in Swiss 3T3 cells

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Bradykinin and bombesin rapidly stimulate tyrosine phosphorylation of a 120-kDa group of proteins in Swiss 3T3 cells. / Leeb-Lundberg, L. M Fredrik; Song, Xin Hua.

In: Journal of Biological Chemistry, Vol. 266, No. 12, 25.04.1991, p. 7746-7749.

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T1 - Bradykinin and bombesin rapidly stimulate tyrosine phosphorylation of a 120-kDa group of proteins in Swiss 3T3 cells

AU - Leeb-Lundberg, L. M Fredrik

AU - Song, Xin Hua

PY - 1991/4/25

Y1 - 1991/4/25

N2 - We have examined the effect of bradykinin (BK) and other peptide mediators with related cellular actions on tyrosine phosphorylation in confluent Swiss 3T3 fibroblast cells using an anti-phosphotyrosine antibody. Immunoblots of extracts from cells stimulated with BK showed a major heterogeneous band centered at Mr 120,000. Three phosphorylated protein species were present within this band. The lower of these three phosphoproteins was occasionally present under basal conditions. The detection of this group of phosphoproteins by the antibody was prevented by coincubation with an excess of phosphotyrosine but not with an excess of phosphoserine or phosphothreonine. The BK-promoted increase in phosphorylation was rapid and transient with the peak response apparent following BK exposure for 1 min. The response was dose-dependent with half-maximal effect occurring at 10-30 nM BK. The antagonist Arg0, Hyp3, Thi5,8, D-Phe7-BK completely inhibited the response indicating that BK was acting via a B2 kinin receptor. Bombesin, at 0.1 μM, stimulated an increase in phosphorylation of the 120-kDa group of proteins with the same efficacy as 0.1 μM BK. On the other hand, 1 μM vasopressin was considerably less efficaceous than either of the former agonists. Short-term preexposure to 0.1 μM 12-O-tetradecanoyl-phorbol-13-acetate (1 min), a protein kinase C stimulator, or 30 μM H7 (15 min), a protein kinase C inhibitor, had no significant effect either on the basal or BK-promoted increase in tyrosine phosphorylation of these proteins. BK also stimulated inositol phosphate formation in these cells. Genistein, a tyrosine kinase inhibitor, inhibited BK stimulation of tyrosine phosphorylation. In addition, genistein partially inhibited BK stimulation of inositol phosphate formation. These results show that an increase in tyrosine phosphorylation of a 120-kDa group of proteins is an early protein kinase C-independent cellular signal elicited by both bradykinin and bombesin.

AB - We have examined the effect of bradykinin (BK) and other peptide mediators with related cellular actions on tyrosine phosphorylation in confluent Swiss 3T3 fibroblast cells using an anti-phosphotyrosine antibody. Immunoblots of extracts from cells stimulated with BK showed a major heterogeneous band centered at Mr 120,000. Three phosphorylated protein species were present within this band. The lower of these three phosphoproteins was occasionally present under basal conditions. The detection of this group of phosphoproteins by the antibody was prevented by coincubation with an excess of phosphotyrosine but not with an excess of phosphoserine or phosphothreonine. The BK-promoted increase in phosphorylation was rapid and transient with the peak response apparent following BK exposure for 1 min. The response was dose-dependent with half-maximal effect occurring at 10-30 nM BK. The antagonist Arg0, Hyp3, Thi5,8, D-Phe7-BK completely inhibited the response indicating that BK was acting via a B2 kinin receptor. Bombesin, at 0.1 μM, stimulated an increase in phosphorylation of the 120-kDa group of proteins with the same efficacy as 0.1 μM BK. On the other hand, 1 μM vasopressin was considerably less efficaceous than either of the former agonists. Short-term preexposure to 0.1 μM 12-O-tetradecanoyl-phorbol-13-acetate (1 min), a protein kinase C stimulator, or 30 μM H7 (15 min), a protein kinase C inhibitor, had no significant effect either on the basal or BK-promoted increase in tyrosine phosphorylation of these proteins. BK also stimulated inositol phosphate formation in these cells. Genistein, a tyrosine kinase inhibitor, inhibited BK stimulation of tyrosine phosphorylation. In addition, genistein partially inhibited BK stimulation of inositol phosphate formation. These results show that an increase in tyrosine phosphorylation of a 120-kDa group of proteins is an early protein kinase C-independent cellular signal elicited by both bradykinin and bombesin.

M3 - Article

C2 - 2019598

AN - SCOPUS:0025744009

VL - 266

SP - 7746

EP - 7749

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 1083-351X

IS - 12

ER -