PKCbetaII modulates translation independently from mTOR and through RACK1.
RACK1 (receptor for activated C kinase 1) is an abundant scaffolding protein, which binds active PKCbetaII (protein kinase C betaII) increasing its activity in vitro. RACK1 has also been described as a component of the small ribosomal subunit, in proximity to the mRNA exit channel. In the present study we tested the hypothesis that PKCbetaII plays a specific role in translational control and verified whether it may associate with the ribosomal machinery. We find that specific inhibition of PKCbetaI/II reduces translation as well as global PKC inhibition, but without affecting phosphorylation of mTOR (mammalian target of rapamycin) targets. These results suggest that PKCbetaII acts as a specific PKC isoform affecting translation in an mTOR-independent fashion, possibly close to the ribosomal machinery. Using far-Western analysis, we found that PKCbetaII binds ribosomes in vitro. Co-immunoprecipitation studies indicate that a small but reproducible pool of PKCbetaII is associated with membranes containing ribosomes, suggesting that in vivo PKCbetaII may also physically interact with the ribosomal machinery. Polysomal profiles show that stimulation of PKC results in an increased polysomes/80S ratio, associated with a shift of PKCbetaII to the heavier part of the gradient. A RACK1-derived peptide that inhibits the binding of active PKCbetaII to RACK1 reduces the polysomes/80S ratio and methionine incorporation, suggesting that binding of PKCbetaII to RACK1 is important for PKC-mediated translational control. Finally, down-regulation of RACK1 by siRNA (small interfering RNA) impairs the PKC-mediated increase of translation. Taken together the results of the present study show that PKCbetaII can act as a specific PKC isoform regulating translation, in an mTOR-independent fashion, possibly close to the ribosomal machinery.
Molecular Histology and Cell Growth, HSR, 20132 Milan, Italy.
Stefano
S
Grosso
Viviana
V
Volta
Leonardo A
LA
Sala
Marina
M
Vietri
Pier Carlo
PC
Marchisio
Dorit
D
Ron
Stefano
S
Biffo
D000818:Animals; D006367:HeLa Cells; D006801:Humans; D008297:Male; D051379:Mice; D011132:Polyribosomes / Q000378:metabolism; D014176:Protein Biosynthesis / Q000187:drug effects*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000502:physiology*; D064546:Protein Kinase C beta; D011494:Protein Kinases / Q000502:physiology*; D011753:Pyrones / Q000494:pharmacology; D000075370:Receptors for Activated C Kinase; D011956:Receptors, Cell Surface / Q000502:physiology*; D012270:Ribosomes / Q000378:metabolism; D058570:TOR Serine-Threonine Kinases
D011753:Pyrones; D000075370:Receptors for Activated C Kinase; D011956:Receptors, Cell Surface; D011494:Protein Kinases; C546842:MTOR protein, human; C546843:mTOR protein, mouse; D058570:TOR Serine-Threonine Kinases; D011493:Protein Kinase C; D064546:Protein Kinase C beta; C007946:hispidin