Characterization of activators and inhibitors of protein kinase C mu.
In order to investigate regulatory mechanisms and to identify potential substrates of a novel member of the protein kinase C (PKC) family, PKC mu, specific antibodies have been raised against unique amino- and carboxy-terminal regions. PKC mu kinase activity was studied upon immunoprecipitation from stably transfected cell lines as well as from the A549 carcinoma cell line expressing the endogenous PKC mu gene. Cell fractionation revealed that PKC mu is predominantly found in the particulate fraction, suggesting an association with the membrane or membrane-bound structures. In vitro kinase assays with immunoprecipitated PKC mu demonstrated a Ca2+ independent enhancement of constitutive autophosphorylation activity by phosphatidylserine. Despite a limited in vitro phorbol ester response, an apparent phorbol ester activation of PKC mu was observed when cell cultures, instead of immunoprecipitated enzyme, were treated with either phorbol 12-myristate 13-acetate or 1,2 dioleoyl-sn-glycerol. Both in vitro autophosphorylation and substrate phosphorylation of myelin basic protein and histone III were enhanced under these conditions. However, long-term treatment with the phorbol ester did not result in downregulation of PKC mu protein levels and kinase activity. Studies with several protein kinase inhibitors revealed a novel sensitivity profile of PKC mu, with no inhibition by calphostin C, reduced sensitivity to staurosporine but, compared to other PKCs, an approximately 60-fold higher sensitivity to the selective PKA inhibitor H89. Together, the data presented here show that localization of PKC mu and regulation of its kinase activity differ from that of other PKCs suggesting a novel function of PKC mu in intracellular signal pathways.
Institute of Cell Biology and Immunology, University of Stuttgart, Germany.
F J
FJ
Johannes
J
J
Prestle
S
S
Dieterich
P
P
Oberhagemann
G
G
Link
K
K
Pfizenmaier
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D002460:Cell Line; D004789:Enzyme Activation; D006801:Humans; D007527:Isoenzymes / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors* / Q000276:immunology / Q000378:metabolism*; D013347:Subcellular Fractions / Q000201:enzymology; D014407:Tumor Cells, Cultured
D000906:Antibodies; D007527:Isoenzymes; D011493:Protein Kinase C