Effects of prenyl pyrophosphates on the binding of PKCgamma with RACK1.
Receptors for activated C kinase (RACKs) are a group of PKC binding proteins that have been shown to mediate isoform-selective functions of PKC and to be crucial in the translocation and subsequent functioning of the PKC isoenzymes on activation. RACK1 cDNA from the shrimp Penaeus japonicus was isolated by homology cloning. The hepatopancreas cDNA from this shrimp was found to encode a 318-residue polypeptide whose predicted amino acid sequence shared 91% homology with human G(beta2)-like proteins. Expression of the cDNA of shrimp RACK1 in vitro yielded a 45-kDa polypeptide with positive reactivity toward the monoclonal antibodies against RACK1 of mammals. The shrimp RACK1 was biotinylated and used to compare the effects of geranylgeranyl pyrophosphate and farnesyl pyrophosphate on its binding with PKCgamma in anti-biotin-IgG precipitates. PKCgammas were isolated from shrimp eyes and mouse brains. Both enzyme preparations were able to inhibit taxol-induced tubulin polymerization. Interestingly, when either geranylgeranyl pyrophosphate or farnesyl pyrophosphate was reduced to the submicrogram level, the recruitment activity of RACK1 with purified PKCgamma was found to increase dramatically. The activation is especially significant for RACK1 and PKCgamma from different species. The observation implies that the deprivation of prenyl pyrophosphate might function as a signal for RACK1 to switch the binding from the conventional isoenzymes of PKC (cPKC) to the novel isoenzymes of PKC (nPKC). A hydrophobic binding pocket for geranylgeranyl pyrophosphate in RACK1 is further revealed via prenylation with protein geranylgeranyl transferase I of shrimp P. japonicus.
Chen
Yu-Hsun
YH
Division of Biochemistry and Molecular Science, Institute of Zoology, Academia Sinica, Nankang 11529, Taipei, Taiwan.
Wang
Han-Chung
HC
Lin
Ching-Yu
CY
Chuang
Nin-Nin
NN
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D018076:DNA, Complementary / Q000235:genetics; D011756:Diphosphates / Q000494:pharmacology*; D005123:Eye / Q000201:enzymology; D008969:Molecular Sequence Data; D017239:Paclitaxel / Q000037:antagonists & inhibitors / Q000494:pharmacology; D033561:Penaeidae / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011106:Polyisoprenyl Phosphates / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects; D011493:Protein Kinase C / Q000302:isolation & purification / Q000378:metabolism*; D000075370:Receptors for Activated C Kinase; D011956:Receptors, Cell Surface / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D012717:Sesquiterpenes; D014404:Tubulin / Q000378:metabolism
D018076:DNA, Complementary; D011756:Diphosphates; D011106:Polyisoprenyl Phosphates; D000075370:Receptors for Activated C Kinase; D011956:Receptors, Cell Surface; D012717:Sesquiterpenes; D014404:Tubulin; C004808:farnesyl pyrophosphate; C087857:protein kinase C gamma; D011493:Protein Kinase C; C002963:geranylgeranyl pyrophosphate; D017239:Paclitaxel