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

Background: The protein kinase C (PKC) family has been implicated in the co ntrol of many cellular functions. Although PKC isotypes are characterized b y their allosteric activation, phosphorylation also plays a key role in con trolling activity. In classical PKC isotypes, one of the three critical sit es is a carboxyterminal hydrophobic site also conserved in other AGC kinase subfamily members. Although this site is crucial to the control of this cl ass of enzymes, the upstream kinase(s) has not been identified. Results: A membrane-associated kinase activity that phosphorylates the hydr ophobic site in PKC alpha was detected. This activity was suppressed when c ells were pretreated with the immunosuppresant drug rapamycin or the phosph oinositide (PI) 3-kinase inhibitor LY294002. These pretreatments also block ed specifically the serum-induced phosphorylation of the hydrophobic site i n PKC delta in vivo. The most highly purified hydrophobic site kinase prepa rations (similar to 10,000-fold) reacted with antibodies to PKC zeta/(l). C onsistent with this, rapamycin and LY294002 reduced the recovery of PKC zet a from the membrane fraction of transfected cells. An activated mutant of P KC zeta, but not wild-type PKC zeta, induced phosphorylation of the PKC del ta hydrophobic site in a rapamycin-independent manner, whereas a kinase-dea d PKC zeta mutant suppressed this serum-induced phosphorylation. The immuno purified, activated mutant of PKC zeta could phosphorylate the PKC delta hy drophobic site in vitro, whereas wild-type PKC zeta could not. Conclusions: PKC zeta is identified as a component of the upstream kinase r esponsible for the phosphorylation of the PKC delta hydrophobic site in vit ro and in vivo. PKC zeta can therefore control the phosphorylation of this PKC delta site, antagonizing a rapamycin-sensitive pathway. (C) Elsevier Sc ience Ltd ISSN 0960-9822.