Characterization of the differential roles of the twin C1a and C1b domains of protein kinase C-delta.
Classic and novel protein kinase C (PKC) isozymes contain two zinc finger motifs, designated "C1a" and "C1b" domains, which constitute the recognition modules for the second messenger diacylglycerol (DAG) or the phorbol esters. However, the individual contributions of these tandem C1 domains to PKC function and, reciprocally, the influence of protein context on their function remain uncertain. In the present study, we prepared PKCdelta constructs in which the individual C1a and C1b domains were deleted, swapped, or substituted for one another to explore these issues. As isolated fragments, both the deltaC1a and deltaC1b domains potently bound phorbol esters, but the binding of [(3)H]phorbol 12,13-dibutyrate ([(3)H]PDBu) by the deltaC1a domain depended much more on the presence of phosphatidylserine than did that of the deltaC1b domain. In intact PKCdelta, the deltaC1b domain played the dominant role in [(3)H]PDBu binding, membrane translocation, and down-regulation. A contribution from the deltaC1a domain was nonetheless evident, as shown by retention of [(3)H]PDBu binding at reduced affinity, by increased [(3)H]PDBu affinity upon expression of a second deltaC1a domain substituting for the deltaC1b domain, and by loss of persistent plasma membrane translocation for PKCdelta expressing only the deltaC1b domain, but its contribution was less than predicted from the activity of the isolated domain. Switching the position of the deltaC1b domain to the normal position of the deltaC1a domain (or vice versa) had no apparent effect on the response to phorbol esters, suggesting that the specific position of the C1 domain within PKCdelta was not the primary determinant of its activity.
Pu
Yongmei
Y
Molecular Mechanisms of Tumor Promotion Section, Laboratory of Cancer Biology and Genetics, NCI, National Institutes of Health, Bethesda, Maryland 20892, USA.
Garfield
Susan H
SH
Kedei
Noemi
N
Blumberg
Peter M
PM
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D015536:Down-Regulation; D006801:Humans; D008024:Ligands; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D010703:Phorbol Esters / Q000378:metabolism; D011485:Protein Binding; D051745:Protein Kinase C-delta / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D013379:Substrate Specificity; D016335:Zinc Fingers
D008024:Ligands; D010703:Phorbol Esters; D051745:Protein Kinase C-delta