CHARACTERIZATION OF ACTIVATORS AND INHIBITORS OF PROTEIN-KINASE C-MU

In order to investigate regulatory mechanisms and to identify potentia l substrates of a novel member of the protein kinase C (PKC) family, P KC mu, specific antibodies have been raised against unique amino- and carboxy-terminal regions. PKC mu kinase activity was studied upon immu noprecipitation from stably transfected cell lines as well as from the A549 carcinoma cell line expressing the endogeneous PKC mu gene. Cell fractionation revealed that PKC mu is predominantly found in the part iculate fraction, suggesting an association with the membrane or membr ane-bound structures. In vitro kinase assays with immuno-precipitated PKC mu demonstrated a Ca2+ independent enhancement of constitutive aut ophosphorylation 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 substrat e phosphorylation of myelin basic protein and histone III were enhance d under these conditions. However, long-term treatment with the phorbo l ester did not result in downregulation of PKC mu protein levels and kinase activity. Studies with several protein kinase inhibitors reveal ed a novel sensitivity profile of PKC mu, with no inhibition by calpho stin C, reduced sensitivity to staurosporine but, compared to other PK Cs, an approximately 60-fold higher sensitivity to the selective PKA i nhibitor H89. Together, the data presented here show that localization of PKC mu and regulation of its kinase activity differ from that of o ther PKCs suggesting a novel function of PKC mu in intracellular signa l pathways.