Molecular cloning and characterization of the acidic 80-kDa protein kinase C substrate from rat brain. Identification as a glycoprotein.
The complete amino acid sequence of 80 K, the major acidic protein kinase C (PKC) substrate of rat brain, was deduced from a cDNA nucleotide sequence. An open reading frame of 927 bases predicted a protein of 309 amino acid residues (Mr = 29,796, pI = 4.06). 58% of the deduced protein sequence was confirmed by Edman degradation of peptides generated by proteolysis of purified 80 K. The absence of internal methionine residues in the deduced amino acid sequence was confirmed by the inability to cleave 80 K with CNBr. Antiserum raised against a synthetic peptide corresponding to residues 298-309 of the predicted amino acid sequence recognizes the 80-kDa polypeptide in Western blots. The protein shows 65% sequence identity with a closely related PKC substrate from bovine brain. Genomic Southern blot analysis using a probe corresponding to a segment of the 80 K gene devoid of introns showed one major band. Northern blot analysis of rat brain RNA reveals a prominent transcript of 2.2 kilobases which hybridizes to 80 K cDNA. The amino acid composition and hydropathicity plot suggest an extended structure with no hydrophobic domains. The amino acid sequence showed many short repeats as well as several potential phosphorylation sites, five of which were for PKC, one was for both PKC and cyclic AMP-dependent protein kinase, and one for casein kinase II, and potential glycosylation sites. Indeed, carbohydrate moieties were detected on electroblots of purified 80 K using both a specific glycan stain and Galanthus nivalis plant lectin which binds to terminal D-mannose in the glycan moiety. This is the first time that this major PKC substrate has been identified as a glycoprotein.
Erusalimsky
J D
JD
Imperial Cancer Research Fund, Lincoln's Inn Fields, London, United Kingdom.
Brooks
S F
SF
Herget
T
T
Morris
C
C
Rozengurt
E
E
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015139:Blotting, Southern; D001921:Brain / Q000378:metabolism*; D002417:Cattle; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D027801:Galanthus; D006023:Glycoproteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D007106:Immune Sera; D037102:Lectins; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D015345:Oligonucleotide Probes; D037121:Plant Lectins; D011487:Protein Conformation; D011493:Protein Kinase C / Q000378:metabolism*; D012313:RNA / Q000235:genetics / Q000302:isolation & purification; D051381:Rats; D012689:Sequence Homology, Nucleic Acid; D013379:Substrate Specificity
D006023:Glycoproteins; D007106:Immune Sera; D037102:Lectins; D009419:Nerve Tissue Proteins; D015345:Oligonucleotide Probes; D037121:Plant Lectins; D012313:RNA; D004247:DNA; D011493:Protein Kinase C