Enzymatic properties of a novel phorbol ester receptor/protein kinase, nPKC.
A protein kinase C-related cDNA encodes a novel phorbol ester receptor/protein kinase, nPKC epsilon, clearly distinct from the four "conventional" PKCs [Ohno, S., Akita, Y., Konno, Y., Imajoh, S., & Suzuki, K. (1988) Cell 53, 731-741]. We purified nPKC epsilon from COS cells transfected with nPKC cDNA and compared its enzymatic properties with a conventional PKC, PKC alpha. nPKC epsilon was eluted from a hydroxyapatite column at a position coincident with type II PKC and thus was separated from type III PKC (PKC alpha), the only PKC expressed in COS cells. The protein kinase activity of nPKC epsilon is activated by phospholipids and diacylglycerols (or phorbol esters) in a manner similar to conventional PKCs. However, the cofactor dependencies and substrate specificities were clearly different from PKC alpha. A phospholipid, cardiolipin, enhances the kinase activity three- to fourfold compared with phosphatidylserine. The optimum Mg2+ concentration (3 mM) is clearly different from those of conventional PKCs (10-20 mM). The activation of nPKC epsilon by these cofactors is totally independent of Ca2+. Similar to conventional PKCs, nPKC epsilon autophosphorylates serine and threonine residues, indicating the specificity of the kinase to these amino acid residues. However, it shows a clearly different substrate specificity against exogenous substrates in that myelin basic proteins rather than histone are good substrates. These properties of nPKC epsilon permit clear discrimination of nPKC epsilon from conventional PKCs.
Konno
Y
Y
Department of Molecular Biology, Tokyo Metropolitan Institute of Medical Science.
Ohno
S
S
Akita
Y
Y
Kawasaki
H
H
Suzuki
K
K
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D015153:Blotting, Western; D029742:Caenorhabditis elegans Proteins; D002352:Carrier Proteins; D002478:Cells, Cultured; D002852:Chromatography, Ion Exchange; D015870:Gene Expression; D006882:Hydroxyapatites; D015240:Phorbol 12,13-Dibutyrate / Q000378:metabolism; D010743:Phospholipids / Q000032:analysis; D011493:Protein Kinase C / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011817:Rabbits; D011955:Receptors, Drug / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity
D000596:Amino Acids; D029742:Caenorhabditis elegans Proteins; D002352:Carrier Proteins; D006882:Hydroxyapatites; D010743:Phospholipids; D011955:Receptors, Drug; C083141:phorbol ester binding protein; C027776:phorbol ester receptor; D015240:Phorbol 12,13-Dibutyrate; D011493:Protein Kinase C