Rapamycin-sensitive phosphorylation of PKC on a carboxy-terminal site by an atypical PKC complex

AbstractBackground: The protein kinase C (PKC) family has been implicated in the control of many cellular functions. Although PKC isotypes are characterized by their allosteric activation, phosphorylation also plays a key role in controlling activity. In classical PKC isotypes, one of the three critical sites is a carboxy-terminal hydrophobic site also conserved in other AGC kinase subfamily members. Although this site is crucial to the control of this class of enzymes, the upstream kinase(s) has not been identified.Results: A membrane-associated kinase activity that phosphorylates the hydrophobic site in PKCα was detected. This activity was suppressed when cells were pretreated with the immunosuppresant drug rapamycin or the phosphoinositide (Pl) 3-kinase inhibitor LY294002. These pretreatments also blocked specifically the serum-induced phosphorylation of the hydrophobic site in PKCδ in vivo. The most highly purified hydrophobic site kinase preparations (∼10,000-fold) reacted with antibodies to PKCζ/ι. Consistent with this, rapamycin and LY294002 reduced the recovery of PKCζ from the membrane fraction of transfected cells. An activated mutant of PKCζ, but not wild-type PKCζ, induced phosphorylation of the PKCδ hydrophobic site in a rapamycin-independent manner, whereas a kinase-dead PKCζ mutant suppressed this serum-induced phosphorylation. The immunopurified, activated mutant of PKCζ could phosphorylate the PKCδ hydrophobic site in vitro, whereas wild-type PKCζ could not.Conclusions: PKCζ is identified as a component of the upstream kinase responsible for the phosphorylation of the PKCζ hydrophobic site in vitro and in vivo. PKCζ can therefore control the phosphorylation of this PKCδ site, antagonizing a rapamycin-sensitive pathway