Protein kinase C activation potently down-regulates the expression of its major substrate, 80K, in Swiss 3T3 cells.

Abstract

The amino acid sequence of 80K, the major acidic protein kinase C (PKC) substrate of Swiss 3T3 fibroblasts, was deduced from a cDNA nucleotide sequence. Overall, 25% of the predicted amino acid sequence is supported by direct protein sequence data. Southern blot analysis suggests that the mouse genome contains a single copy of this gene. Two 80K mRNA species, a major band of 2.25 kb and a minor band of 3.9 kb, were detected by Northern blot analysis. Stimulation of PKC by biologically active phorbol esters, including phorbol‐12, 13‐dibutyrate (PDB), reduced the steady state level of 80K mRNA to 8.8% of control within 5–7 h. This effect was dose‐dependent, and was abolished by prior depletion of PKC. The PDB‐induced down‐regulation of 80K mRNA levels was transient, and recovery coincided with the disappearance of PKC activity. A similar transient decrease in 80K mRNA levels was also demonstrated in tertiary cultures of mouse embryo fibroblasts. The down‐regulation of 80K mRNA levels was completely abolished by actinomycin D, cycloheximide or anisomycin if added up to 30 min after PDB addition. Since the rate of transcription of the 80K gene was unaltered by PDB treatment, we concluded that the PKC‐induced down‐regulation of 80K mRNA is mediated by a post‐transcriptional mechanism. In addition, PDB transiently decreased the level of 80K protein within 14–18 h, thus reflecting the effects of this phorbol ester on mRNA expression.

The expression of PDGF‐α but not PDGF‐β receptors is suppressed in Swiss/3T3 fibroblasts over‐expressing protein kinase C‐α

“…We have shown that the 100 kDa 2-5A synthetase, the enzymatic products of which have the capacity to inhibit protein synthesis and the stability of specific mRNAs, is up-regulated by PMCd at a post~transcriptional level 1471. In another study, Brooks et al [48] reported that activation of PKC in Swiss/3T3 cells by phorbol esters reduced the expression of its major phosphorylation substrate, ~80, apparently also by a post-transcriptional mechanism affecting the stability of its mRNA. It is therefore possible that similar molecular m~hanisms might be active in regulating the expression of EGF, PDGF-ol and other receptor molecules.…”

Overexpression of the myristoylated alanine‐rich C‐kinase substrate in Rat1 cells increases sensitivity to calmodulin antagonists

“…Various cDNA sequences inserted into the EcoRI site of pMV7 were stably expressed and yielded high-level production of the corresponding protein [ll]. For the present study, the 80-kDa MARCKS cDNA p803.3 isolated from a Swiss 3T3 cDNA library [7] was digested with EcoRI and the l-kb insert ligated into the EcoRI site down-stream of the 5' LTR of the pMV7 plasmid after gel purification. The resulting construct was called pMV7-80K.…”

“…10 pg DNA was digested with the restriction enzymes EcoRI or XbaI, separated in a 1% agarose gel, then transferred onto a Hybond-N' membrane (Amersham). The filters were hybridised with a gel-purified 843-bp Ball-EcoRI fragment of the 80-kDa MARCKS-coding region of clone p809.1 isolated from a Swiss 3T3 cDNA library [7]. The specific activity of the random-primed probe was 5 X 10' c p d p g .…”

“…Western-blot analyses were performed with two different antisera. One antiserum was raised against a synthetic peptide comprising the 11 C-terminal amino acids of the Swiss 3T3 80-kDa MARCKS protein and used at 1 : 500 dilution [7]. A second antiserum was raised against a recombinant 80-kDa MARCKS-glutathionine-S-transferase fusion protein and used at 1:lOOO dilution [6].…”

Multisite phosphorylation of the 80 kDa (MARCKS) protein kinase C substrate in C3H/10T1/2 fibroblasts Quantitative analysis of individual sites by solid‐phase microsequencing

“…Phosphorylation occurs on serine at similar sites (up to 2.8 moles of s2P/mole of protein) in vitro and in vivo [5,8,9]. The 87 kDa protein has been cloned and sequenced from the brain and nonneuronal tissue of many species [10][11][12][13]. The gene codes for proteins of approximately 330 amino acids that are 60-70% identical in sequence although the phospho- rylated region is completely conserved.…”