The EMBO Journal vol.10 no.9 pp.2497-2505, 1991

Protein kinase C activation potently down-regulates the expression of its major substrate, 80K, in Swiss 3T3 cells Susan F.Brooks, Thomas Herget, Jorge D.Erusalimsky and Enrique Rozengurt Imperial Cancer Research Fund, PO Box 123, Lincoln's Inn Fields, London WC2A 3PX, UK Communicated by E.Rozengurt

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, cyclohexnmide 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 posttranscriptional 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 ex-

pression. Key words: cellular signalling/molecular cloning/mRNA expression/phorbol ester/protein kinase C substrate

Introduction Protein kinase C (PKC) comprises a family of closely related that are activated by the second messenger diacylglycerol and serve as the target for tumour-promoting phorbol esters (Nishizuka, 1986, 1988). These enzymes are involved in the signal transduction of many short-term responses including secretion of hormones and enzymes, neurotransmitter release, alterations in membrane ionic conductance and muscle contraction (Kikkawa and Nishizuka, 1986). Furthermore, PKC activation has been implicated in the regulation of long-term responses such as gene expression and cell proliferation (Rozengurt and Sinnett-Smith, 1988). Since the mechanisms through which PKC mediates these cellular responses remain largely enzymes

(C) Oxford University Press

unknown, it is essential to characterize the physiological substrates of this kinase family. An acidic protein that migrates with an apparent molecular mass of 80 kDa in SDS -polyacrylamide gels, termed 80K, has been identified as a major and specific substrate for PKC in quiescent mouse 3T3 fibroblasts (Rozengurt et al., 1983, 1984; Rodriguez-Pena and Rozengurt, 1985, 1986a; Tsuda et al., 1985). This protein is reversibly phosphorylated by a variety of growth factors and pharmacological agents, including bombesin, vasopressin, bradykinin and phorbol esters (Rozengurt et al., 1983; Rodriguez-Pena and Rozengurt, 1985, 1986b; Isacke et al., 1986; RodriguezPena et al., 1986; Zachary et al., 1986; Erusalimsky et al., 1988; Erusalimsky and Rozengurt, 1989; Issandou and Rozengurt, 1990) all of which are known to stimulate PKC. A reduction in the level and phosphorylation of 80K has been shown to accompany both a loss of mitogenic responsiveness to phorbol esters in 3T3 cell variants (Beimann and Erickson, 1990), and conversion to the transformed phenotype in ras, src orfins transformed NIH3T3 fibroblasts (Wolfman et al., 1987). These findings raise the possibility that 80K is involved in mitogenic signal transduction and transformation by certain oncogenes, and suggest that this protein may be regulated through alterations in both phosphorylation and expression. We have recently reported the purification and partial amino acid sequence of 80K from Swiss 3T3 fibroblasts (Brooks et al., 1990). Acidic cellular 65-78 kDa PKC substrates that appear to be related to the protein found in Swiss 3T3 fibroblasts have also been identified in a wide variety of mammalian cultured cells (Rozengurt et al., 1983; Coughlin et al., 1985; Rodriguez-Pena and Rozengurt, 1985; Blackshear et al., 1985, 1986; Hornbeck and Paul, 1986; Aderem et al., 1988; Hirai and Shimizu, 1989; Hornbeck et al., 1989) as well as in brain (Blackshear et al., 1986; Albert et al., 1986; Morris and Rozengurt, 1988; Graff et al., 1989a; Erusalimsky et al., 1991) and other tissues (Albert et al., 1986; Blackshear et al., 1986). Comparison of the amino acid sequences deduced from the cDNA clones encoding both rat brain 80K (Erusalimsky et al., 1991) and the bovine brain 87 kDa PKC substrate, termed MARCKS (Stumpo et al., 1989), has revealed that they are related but not identical. Furthermore, a cDNA encoding an acidic 80 kDa PKC substrate from epidermal carcinoma cells predicts an entirely different amino acid sequence (Sakai et al., 1989). These findings indicate that the acidic 6587 kDa proteins may be more diverse than previously thought. Thus, in order to assess the functional implications of this diversity and to analyse the long-term regulation of this protein, it is necessary to define the structure of each individual substrate in detail. The non-tumorigenic murine Swiss 3T3 fibroblast cell line has proved to be a useful model system for identifying both extracellular factors that modulate cell growth and for elucidating the early signals that lead to mitogenesis 2497

S.F.Brooks et al.

(Rozengurt, 1986). In the present study, we have deduced the complete amino acid sequence of the Swiss 3T3 fibroblast 80K protein from its cDNA sequence and have investigated the role of PKC in the regulation of 80K mRNA and protein expression. Surprisingly, we find that activation of PKC leads to a striking down-regulation of 80K mRNA levels in Swiss 3T3 fibroblasts through a post-transcriptional mechanism. Results cDNA sequence of Swiss 3T3 fibroblast 80K A 7lmer oligonucleotide probe, spanning a region highly conserved between the mouse fibroblast (Brooks et al. , 1990) and rat brain 80K proteins (Erusalimsky et al., 199 1), was synthesized and used to screen a Swiss 3T3 fibroblast XZAPII cDNA library. A total of 19 positive clones were isolated, 14 of which were shown to contain homologous sequences by restriction mapping and partial DNA sequencing. The complete DNA sequence encoding the 80K protein was determined by sequencing three overlapping clones and TTT TAA AAA AAC CTC OTT OCT GAG TAT TTA GGT GTG TGT

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is 1900 bp in length, including a putative poly(A) tail of 15 bases (Figure 1). At the 5' end, 282 nucleotides precede an ATG codon, which is present in the consensus sequence for translation initiation (Kozak, 1987). After the translation start site, there is a single open reading frame, which predicts a protein of 309 amino acid residues. Overall, 25 % of the deduced amino acid sequence is supported by direct protein sequence data (Brooks et al., 1990), allowing the unequivocal identification of the cDNA clones. At the 3' end of the coding region, an in-frame stop codon at position 1210 is followed by 688 bp of untranslated sequence. A consensus polyadenylation signal (AATAAA) is present 25 bp upstream from the putative poly(A) tail.

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TOO GCA GCC CCC GOC GOG TOG COG GAG COG GAG CCC GAG TOO AGT COG GAG GCG K C SL..P..L....A S A A P A A S P F P C P

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Fig. 1. Nucleotide sequence of the 80K cDNA clone and the deduced primary structure of the protein. The complete cDNA sequence was obtained by sequencing three overlapping clones [p803.3 (97-1572 bp), p809.1 (1-1233 bp) and p8017.1 (391-1900 bp)]. Numbers on the left indicate the position of the amino acids, and on the right, positions of the nucleotides. Peptide sequences confirmed by Edman degradation are underlined. In the 3' untranslated region, the polyadenylation signal is underlined.

2498

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TTT CCC CCC AGT TTG TTT GTT GGA GTG GTG CCA GOT ACT GOT TOT GGA GMA OTT GTO TAO MGC CAG GGA TTG ATT TTA MAG ATT TTT TTT TMA ATT TGA GAT TTT TTT

AMA AMA AMA

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Fig. 2. (A) Primer extension analysis. A 5' end-labelled oligonucleotide probe complementary to the region between bases 20-50 of the 80K cDNA clone was hybridized to either 3T3 or tobacco plant RNA, and the extension products were analysed on a 6% sequencing gel. The positions of fragments of pBluescript SK( -) digested with HpaII and labelled by fill-in reaction with [a32p]dCTP is shown on the left. (B) Genomic Southern blot analysis. Genomic DNA from Swiss 3T3 fibroblasts was digested with EcoRI, HindIll, PstI or TaqI and analysed by Southern blotting using a 843 bp BalI-EcoRI cDNA fragment of p809.1 as a probe. The positions of the XDNA/HindIII fragments are shown on the right. (C) Northern blot analysis. Total cellular RNA (10 /og) isolated from Swiss 3T3 fibroblasts was analysed by Northern blotting using the 843 bp BalI-EcoRI probe. The positions of the 28S and 18S ribosomal RNAs are shown on the right.

Down-regulation of 80K mRNA

The extent of the 5' untranslated region of the 80K mRNA was determined by primer extension analysis of total RNA from Swiss 3T3 fibroblasts. The most prominent extension products were a doublet 157 - 159 bases upstream of the 5' end of the primer, which corresponds to a position 107-109 bp from the 5' end of the cDNA clone (Figure 2A). Therefore, the complete 5' untranslated region is 389-391 bp in length. No extension products were detected

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