Functional carboxyl terminal deletion map of protein kinase C alpha.
The phorbol ester receptor protein kinase C (PKC) gene family encodes essential mediators of various eukaryotic cellular signals. Based on the predicted amino acid (aa) sequence homology of more than ten distinct PKC gene coding sequences, four highly conserved regions C1-C4 and five variable regions V1-V5 have been defined for the different PKC subtypes. Some of these regions, such as C1 and C3/V4/C4, have been correlated with specific PKC functions, such as activator binding and enzymatic activity, respectively, while the biological role of others is unknown. The biological significance of the PKC carboxyl terminus is unclear and the predicted boundary of the catalytic C4 region is controversial due to different interpretations of aa sequence comparisons. We explored the PKC alpha carboxyl terminal requirement for basic PKC function and mapped the boundary of the sequences essential for enzymatic activity based on functional criteria. cDNAs encoding normal and random carboxyl terminal truncations of bovine PKC alpha were introduced into Saccharomyces cerevisiae, allowing its rapid functional expression and characterization for catalytic as well as biological activity. We found that deletion of up to 11 carboxyl terminal aa still results in a phorbol ester-responsive, biologically active enzyme in vivo which is dependent on calcium and phospholipids for catalytic activation in vitro. Deletion of 15 and 23 aa results in marginal and total loss of catalytic activity, respectively, and in complete loss of biological activity for both truncations.(ABSTRACT TRUNCATED AT 250 WORDS)
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Joslin Diabetes Center, Department of Medicine, Brigham and Women's Hospital, Boston, Massachusetts 02215.
Parissenti
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Riedel
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D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D029742:Caenorhabditis elegans Proteins; D002118:Calcium / Q000378:metabolism; D002352:Carrier Proteins; D002417:Cattle; D002874:Chromosome Mapping; D018076:DNA, Complementary / Q000235:genetics; D007527:Isoenzymes / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010703:Phorbol Esters / Q000378:metabolism; D011493:Protein Kinase C / Q000235:genetics* / Q000378:metabolism; D051571:Protein Kinase C-alpha; D011955:Receptors, Drug / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion
D029742:Caenorhabditis elegans Proteins; D002352:Carrier Proteins; D018076:DNA, Complementary; D007527:Isoenzymes; D010703:Phorbol Esters; D011955:Receptors, Drug; C083141:phorbol ester binding protein; C027776:phorbol ester receptor; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D002118:Calcium