The expression of 80K/MARCKS, a major substrate of protein kinase C (PKC), is down-regulated through both PKC-dependent and -independent pathways. Effects of bombesin, platelet-derived growth factor, and cAMP.
We have examined the regulation of expression of 80K/MARCKS, a major and specific protein kinase C (PKC) substrate of Swiss 3T3 fibroblasts. Addition of bombesin (10 nM) to confluent quiescent cultures of these cells induced a dramatic and sustained down-regulation of 80-kDa mRNA and protein levels to a minimum of 5% of control within 8 and 48 h, respectively, without depletion of PKC activity. In contrast, the effect of phorbol 12,13-dibutyrate on 80K/MARCKS mRNA levels was transient, and recovery of these transcripts correlated with the loss of PKC activity. The ability of bombesin to down-regulate 80K/MARCKS mRNA levels was dose-dependent (ED50 0.5 nM) and was abolished by both the specific bombesin antagonist [Leu13 psi (CH2NH),Leu14]bombesin and by prior depletion of PKC. Of a range of agents tested, platelet-derived growth factor (PDGF), but not insulin or Ca2+ ionophore, also down-regulated 80K/MARCKS mRNA to 24% of control within 5 h. Prior down-regulation of PKC abolished the effect of PDGF at a concentration of 7 ng/ml. Surprisingly, at higher doses (25 ng/ml), PDGF induced the down-regulation of 80K/MARCKS mRNA in a PKC-independent manner. Furthermore, elevation of cAMP, either through receptor-mediated mechanisms (e.g. prostaglandin E1) or by direct stimulation of adenylate cyclase (e.g. forskolin), also caused a marked dose-dependent depletion of 80K/MARCKS mRNA levels, which were further reduced by co-administration with cAMP-phosphodiesterase inhibitors. The rate of transcription of the 80K/MARCKS gene was unaltered by treatment of cells with either bombesin, PDGF, or forskolin/1-methyl-3-isobutylxanthine. These results indicate a role for both PKC-dependent and -independent pathways in growth factor-induced down-regulation of 80K/MARCKS expression, through a post-transcriptional mechanism.
Brooks
S F
SF
Imperial Cancer Research Fund, Lincoln's Inn Fields, London, United Kingdom.
Herget
T
T
Broad
S
S
Rozengurt
E
E
D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D016475:3T3 Cells; D000818:Animals; D015152:Blotting, Northern; D001839:Bombesin / Q000494:pharmacology*; D002467:Cell Nucleus / Q000187:drug effects / Q000502:physiology*; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism*; D004789:Enzyme Activation; D006706:Homeostasis; D007328:Insulin / Q000494:pharmacology; D047908:Intracellular Signaling Peptides and Proteins; D007700:Kinetics; D008565:Membrane Proteins; D051379:Mice; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology*; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology*; D011493:Protein Kinase C / Q000378:metabolism*; D011506:Proteins / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D013997:Time Factors; D014158:Transcription, Genetic / Q000187:drug effects
D007328:Insulin; D047908:Intracellular Signaling Peptides and Proteins; C000619711:Marcks protein, mouse; D008565:Membrane Proteins; D010982:Platelet-Derived Growth Factor; D011506:Proteins; D012333:RNA, Messenger; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D005576:Colforsin; D015240:Phorbol 12,13-Dibutyrate; D000242:Cyclic AMP; D011493:Protein Kinase C; D001839:Bombesin; D015056:1-Methyl-3-isobutylxanthine