In vitro activation and substrates of recombinant, baculovirus expressed human protein kinase C mu.
To study enzymatic activity and activation conditions of the recently identified novel protein kinase C mu (PKC mu) subtype, epitope tagged PKC mu was propagated in the baculovirus expression system and was purified to homogeneity. PKC mu displays high affinity phorbol ester binding (Kd=7 nM) resulting in enhanced phosphatidylserine-dependent kinase activity. From various lipid second messengers known to activate PKCs only diacylglycerol and PtdIns-4,5-P2, were found to promote PKC mu kinase activity. Two peptides derived from the glycogen synthase, GS-peptide and syntide 2, were found to be phosphorylated efficiently in vitro. MARCKS (myristoylated alanine-rich C-kinase substrate) served as an in vitro substrate for PKC mu too. However, in contrast to other PKCs, a peptide derived from the MARCKS phosphorylation domain is phosphorylated only at serine 156, and not at serines 152 and 163, implicating a differential regulation by PKC mu.
Dieterich
S
S
Institute of Cell Biology and Immunology, University of Stuttgart, Germany.
Herget
T
T
Link
G
G
Böttinger
H
H
Pfizenmaier
K
K
Johannes
F J
FJ
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D005822:Genetic Vectors; D006801:Humans; D007313:Insecta; D047908:Intracellular Signaling Peptides and Proteins; D007527:Isoenzymes / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D008970:Molecular Weight; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D009842:Oligopeptides / Q000378:metabolism; D015240:Phorbol 12,13-Dibutyrate / Q000378:metabolism; D010766:Phosphorylation; D011493:Protein Kinase C / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D014162:Transfection
D047908:Intracellular Signaling Peptides and Proteins; D007527:Isoenzymes; C000619710:MARCKS protein, human; D008565:Membrane Proteins; D009842:Oligopeptides; D011506:Proteins; D011994:Recombinant Proteins; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D015240:Phorbol 12,13-Dibutyrate; C037642:kemptide; D011493:Protein Kinase C