Formation of a phorbol ester-binding fragment from protein kinase C by proteolytic digestion.
When washed human platelets were disrupted by sonication in the presence of ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, both the catalytic and [3H]phorbol-12,13-dibutyrate (PDBu)-binding activities of protein kinase C were recovered in the soluble fraction and were not separable from each other upon several column chromatographies. Platelet protein kinase C required diacylglycerol, Ca2+, and phospholipid for its activation and showed a molecular weight of about 87,000 as estimated by gel filtration analysis. However, when platelets were first incubated with 2 microM Ca2+-ionophore A23187 for 5 min at 37 degrees C in the medium containing 3 mM CaCl2 and then disrupted under the same conditions, the catalytic and [3H]phorbol-12,13-dibutyrate-binding activities were separately recovered in the soluble and particulate fractions, respectively; moreover, the catalytic activity recovered in the soluble fraction became independent of diacylglycerol, Ca2+, and phospholipid, and showed a molecular weight of about 50,000 as estimated by gel filtration analysis. The kinetic properties of this Mr 50,000 enzyme were similar to those of the catalytic fragment of rat brain protein kinase C described previously. In a cell-free system, digestion with trypsin of protein kinase C highly purified from rat brain caused the generation of a fragment which had no catalytic activity but showed full [3H]phorbol-12,13-dibutyrate-binding activity. The molecular weight of this fragment was estimated to be about 35,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These results indicate that protein kinase C consists of at least two functionally different domains, a hydrophobic phorbol ester- or diacylglycerol-binding and hydrophilic catalytic domains.
Hoshijima
M
M
Kikuchi
A
A
Tanimoto
T
T
Kaibuchi
K
K
Takai
Y
Y
D001665:Binding Sites; D001792:Blood Platelets / Q000378:metabolism; D029742:Caenorhabditis elegans Proteins; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000494:pharmacology; D002352:Carrier Proteins; D002474:Cell-Free System; D004075:Diglycerides / Q000494:pharmacology; D006801:Humans; D066298:In Vitro Techniques; D008970:Molecular Weight; D010446:Peptide Fragments / Q000032:analysis* / Q000378:metabolism; D010447:Peptide Hydrolases / Q000494:pharmacology; D015240:Phorbol 12,13-Dibutyrate; D010703:Phorbol Esters / Q000378:metabolism; D010743:Phospholipids / Q000494:pharmacology; D011493:Protein Kinase C / Q000032:analysis* / Q000378:metabolism; D011955:Receptors, Drug; D011971:Receptors, Immunologic / Q000032:analysis*; D014316:Tritium
D029742:Caenorhabditis elegans Proteins; D002352:Carrier Proteins; D004075:Diglycerides; D010446:Peptide Fragments; D010703:Phorbol Esters; D010743:Phospholipids; D011955:Receptors, Drug; D011971:Receptors, Immunologic; C083141:phorbol ester binding protein; C027776:phorbol ester receptor; D014316:Tritium; D015240:Phorbol 12,13-Dibutyrate; D000001:Calcimycin; D011493:Protein Kinase C; D010447:Peptide Hydrolases; D002118:Calcium; C013965:diolein