Isolation of cDNAs encoding a substrate for protein kinase C: nucleotide sequence and chromosomal mapping of the gene...

An acidic phosphoprotein of Mr 80,000, the 80K protein, is a substrate for protein kinase C in fibroblasts and epidermal carcinoma cells. We purified the 80K protein from human squamous carcinoma Ca9-22 cells and fractionated it into two distinct molecular species, designated the 80K-L and 80K-H proteins. The amino acid sequences of the NH2-terminal region and cyanogen bromide-cleaved fragments of the 80K-H protein were determined and a corresponding oligonucleotide sequence was synthesized. Using this as a probe, two cDNA clones, lambda 80H-1 and lambda 80H-2, were selected from a lambda gt10 cDNA library from human A431 cells. The nucleotide sequence has an open reading frame of 1581 nucleotides encoding a protein of 527 amino acids. The deduced amino acid sequence revealed an extremely Glu-rich region. RNA blot analysis with the lambda 80H-1 cDNA clone detected two polyadenylated transcripts of 2.3 and 3.5 kb in Ca9-22 cells. Spot blot hybridization using flow-sorted human chromosomes provided evidence that the gene (G19P1) encoding 80K-H protein maps to human chromosome 19.