Phospholipid functional groups involved in protein kinase C activation, phorbol ester binding, and binding to mixed micelles.
The specificity of the phospholipid cofactor requirement of rat brain protein kinase C was investigated using Triton X-100 mixed micellar methods. Sixteen analogues of phosphatidylserine were prepared and tested for their ability to support protein kinase C activity, [3H]phorbol 12,13-dibutyrate binding, and protein kinase C binding to mixed micelles. Phosphatidylserinol, -L-serine methyl ester, -N-acetyl-L-serine, -2-hydroxyacetate, -3-hydroxypropionate, and -4-hydroxybutyrate did not activate protein kinase C in mixed micelles containing 2 mol % of sn-1,2-dioleoylglycerol. This indicates that both the carboxyl and amino moieties are important for activation. Phosphatidyl-D-serine and -L-homoserine were incapable of supporting full activation; this demonstrates stereospecificity and the importance of the distance between the phosphate and carboxyl and amino moieties. Since 1,2-rac-phosphatidyl-L-serine and 1,3-phosphatidyl-L-serine fully supported protein kinase C activity, the stereochemistry within the glycerol backbone at the interface was not necessary for maximal activation. Neither lysophosphatidyl-L-serine nor 1-oleoyl-2-acetyl-sn-glycero-3-phospho-L-serine supported protein kinase C activity implying that the interfacial conformation is critical to the activation process. The phospholipid dependencies of [3H]phorbol 12,13-dibutyrate binding and of protein kinase C binding to mixed micelles containing sn-1,2-dioleoylglycerol did not mirror those for activation. The data demonstrate that protein kinase C possesses a high degree of specificity with respect to phospholipid activation and implicate several functional groups within the phospho-L-serine polar head group in binding and activation.
Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710.
M H
MH
Lee
R M
RM
Bell
D000818:Animals; D029742:Caenorhabditis elegans Proteins; D002352:Carrier Proteins; D003102:Colloids; D008823:Micelles; D017830:Octoxynol; D015240:Phorbol 12,13-Dibutyrate / Q000378:metabolism*; D010712:Phosphatidic Acids / Q000502:physiology; D010714:Phosphatidylethanolamines / Q000502:physiology; D010718:Phosphatidylserines / Q000502:physiology; D010743:Phospholipids / Q000502:physiology*; D011092:Polyethylene Glycols; D011493:Protein Kinase C / Q000378:metabolism*; D051381:Rats; D011955:Receptors, Drug / Q000032:analysis*; D013329:Structure-Activity Relationship
D029742:Caenorhabditis elegans Proteins; D002352:Carrier Proteins; D003102:Colloids; D008823:Micelles; D010712:Phosphatidic Acids; D010714:Phosphatidylethanolamines; D010718:Phosphatidylserines; D010743:Phospholipids; D011955:Receptors, Drug; C083141:phorbol ester binding protein; C027776:phorbol ester receptor; D015240:Phorbol 12,13-Dibutyrate; D011092:Polyethylene Glycols; D017830:Octoxynol; D011493:Protein Kinase C