RESUMEN
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.
Asunto(s)
Difosfatos/farmacología , Penaeidae/metabolismo , Proteína Quinasa C/metabolismo , Receptores de Superficie Celular/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , ADN Complementario/genética , Ojo/enzimología , Datos de Secuencia Molecular , Paclitaxel/antagonistas & inhibidores , Paclitaxel/farmacología , Penaeidae/enzimología , Penaeidae/genética , Fosforilación , Fosfatos de Poliisoprenilo/farmacología , Unión Proteica/efectos de los fármacos , Proteína Quinasa C/aislamiento & purificación , Receptores de Cinasa C Activada , Receptores de Superficie Celular/química , Receptores de Superficie Celular/genética , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Sesquiterpenos , Tubulina (Proteína)/metabolismoRESUMEN
Beta-tubulin cDNA from the shrimp Penaeus japonicus was isolated by homology cloning. Expression of cDNA in Escherichia coli yielded a 55 kDa polypeptide, positive for monoclonal antibodies against mammalian beta-tubulin. Autoradiography demonstrated the bacterially expressed hepatopancreas beta-tubulin of P. japonicus is specifically phosphorylated by the delta isoenzyme of protein kinase C (PKC-delta) purified from the plasma membrane of the shrimp heart, in the presence of the receptor for activated PKC (RACK), but not in its absence. Purified shrimp heart PKC-delta is able to phosphorylate bacterially expressed shrimp beta-tubulin without the presence of Ca(++), but requires Mg(++). The kinase activity of purified PKC-delta on bacterially expressed beta-tubulin was enhanced by incubation with PEP(taxol), a synthetic peptide encoding the taxol-binding region of beta-tubulin. In other words, PEP(taxol) modulates the kinase activity of PKC-delta through RACK.