APPROACHES TO DETERMINE THE SPECIFIC ROLE OF THE DELTA-ISOFORM OF PROTEIN-KINASE-C

Two dimensional gel electrophoresis of proteins from HL-60 human leuka emia cells treated with bistratene A, a specific activator of protein kinase C (PKC)delta, was performed in conjunction with sequencing in o rder to identify components of the signal transduction pathway of this isoform of PKC, Stathmin (oncoprotein 18) was identified in this way and the phosphorylation of this protein after treatment with bistraten e A, was confirmed by Western blotting of 2D gels. Since stathmin has phosphorylation sites for mitogen activated protein (MAP) kinases, cyc lin dependent kinases and calcium/calmodulin dependent protein kinases , it is assumed that one of these enzymes, acting downstream from PKC delta, is responsible for the phosphorylation. Another approach to det ermining the role of PKC delta involves the identification of interact ing proteins using the yeast two hybrid screen. The sequence of nine o ut of ten independently isolated clones from a two hybrid screen showe d perfect homology to human ribosomal protein L8. This protein has pre viously been shown to exist in complexes with ribosomal RNA, aminoacyl -tRNA and elongation factor-1 alpha, a known substrate of PKC delta, s uggesting a role for PKC delta in protein synthesis regulation. (C) 19 97 Elsevier Science B.V.