A critical intramolecular interaction for protein kinase Cepsilon translocation.
Disruption of intramolecular interactions, translocation from one intracellular compartment to another, and binding to isozyme-specific anchoring proteins termed RACKs, accompany protein kinase C (PKC) activation. We hypothesized that in inactive epsilonPKC, the RACK-binding site is engaged in an intramolecular interaction with a sequence resembling its RACK, termed psiepsilonRACK. An amino acid difference between the psiepsilonRACK sequence in epsilonPKC and its homologous sequence in epsilonRACK constitutes a change from a polar non-charged amino acid (asparagine) in epsilonRACK to a polar charged amino acid (aspartate) in epsilonPKC. Here we show that mutating the aspartate to asparagine in epsilonPKC increased intramolecular interaction as indicated by increased resistance to proteolysis, and slower hormone- or PMA-induced translocation in cells. Substituting aspartate for a non-polar amino acid (alanine) resulted in binding to epsilonRACK without activators, in vitro, and increased translocation rate upon activation in cells. Mathematical modeling suggests that translocation is at least a two-step process. Together our data suggest that intramolecular interaction between the psiepsilonRACK site and RACK-binding site within epsilonPKC is critical and rate limiting in the process of PKC translocation.
Schechtman
Deborah
D
Department of Molecular Pharmacology, Stanford University School of Medicine, Stanford, California 94305, USA.
Craske
Madeleine L
ML
Kheifets
Viktoria
V
Meyer
Tobias
T
Schechtman
Jack
J
Mochly-Rosen
Daria
D
D019943:Amino Acid Substitution; D000818:Animals; D001224:Aspartic Acid; D001665:Binding Sites / Q000235:genetics; D016466:CHO Cells; D006224:Cricetinae; D004789:Enzyme Activation / Q000235:genetics; D006801:Humans; D008956:Models, Chemical; D011485:Protein Binding; D011493:Protein Kinase C / Q000737:chemistry* / Q000378:metabolism; D051744:Protein Kinase C-epsilon; D021381:Protein Transport / Q000235:genetics; D011956:Receptors, Cell Surface / Q000378:metabolism; D015398:Signal Transduction; D013329:Structure-Activity Relationship
D011956:Receptors, Cell Surface; D001224:Aspartic Acid; C497255:PRKCE protein, human; D011493:Protein Kinase C; D051744:Protein Kinase C-epsilon