Early phosphorylation events following the treatment of Swiss 3T3 cells with bombesin and the mammalian bombesin-related peptide, gastrin-releasing peptide.

Abstract

Bombesin and the related mammalian peptides, such as gastrin‐releasing peptide (GRP), are potent mitogens for some fibroblast cell lines. Here we have examined the bombesin‐ and GRP‐mediated changes in the phosphorylation of proteins in Swiss 3T3 cells and compared these to the events observed after platelet‐derived growth factor (PDGF), epidermal growth factor (EGF) and tumor promoter treatment. In agreement with previous reports, bombesin, GRP and PDGF, but not EGF, increased the activity of protein kinase C. This was assayed by an inhibition of [125I]EGF binding, stimulation in phosphorylation of pp60c‐src on serine 12 and stimulation in phosphorylation of a group of 80 kd proteins. The different phosphorylated forms of the 80 kd proteins were examined by tryptic peptide mapping and shown to contain multiple phosphorylation sites. An investigation of the tyrosine phosphorylation events following mitogen treatment revealed a significant difference between PDGF and the bombesin peptides. PDGF treatment caused a marked increase in total cellular phosphotyrosine levels, and tyrosine phosphorylation both of known substrates and its own receptor. In contrast, bombesin and GRP treatments resulted in only a weak or undetectable increase in tyrosine phosphorylation of total cellular protein or known substrates. In this respect bombesin and GRP were more similar to EGF. The fact that the bombesin peptides do not induce a phosphorylation response identical with either PDGF or EGF suggests that there is not a single common signal pathway which is activated by all these mitogens.

The protein kinase C family

“…The acidic protein, myristoylated, alanine-rich, C-kinase substrate (MARCKS) has an apparent molecular mass of 80-87 kDa and is phosphorylated in cells rapidly after phorbol ester stimulation (Isacke et al, 1986;Rodriguez-Pena and Rozengurt, 1986;Wu et al, 1982). The protein (as derived from a cDNA clone) has 335 amino acids with a predicted molecular mass of 32 kDa (Stumpo et al, 1989) and thus migrates in polyacrylamide gels anomalously.…”

Protein kinase C-mediated negative-feedback inhibition of unstimulated and bombesin-stimulated polyphosphoinositide hydrolysis in Swiss-mouse 3T3 cells

“…Pretreatment of these cells for 48 h with high concentrations of phorbol esters causes a pronounced down-modulation of cellular protein kinase C, measured either as enzyme activity in cell extracts or as phorbol ester binding to intact cells (Rodriguez-Pena & Rozengurt, 1984;Collins & Rozengurt, 1984;Blackshear et al, 1985Blackshear et al, , 1986. Furthermore, the phosphorylation of a protein of Mr 80000, which is a well-characterized protein kinase C substrate (Blackshear et al, 1986;Isacke et al, 1986), is stimulated by TPA in control cultures, but not in the down-modulated cells (Blackshear et al, 1985). We have found (results not shown) that the down-modulated cells fail to exhibit TPA-mediated inhibition of 125I-EGF binding, an effect strongly correlated with activation of protein kinase C (Brown et al, 1979Friedman et al, 1984;Hunter et al, 1984;Davis & Czech, 1985Downward et al, 1985;Lin et al, 1986).…”

Differential protein phosphorylation in induction of thyroid cell proliferation by thyrotropin, epidermal growth factor, or phorbol ester.

“…The receptor for platelet-derived growth factor seems to activate both signal pathways. This could also be the case for the bombesin receptor, although its tyrosine kinase activity is still debated (7,19).…”