Fig. 5.

PKCα is a signaling intermediate linking EGFR to mTOR in glioma. (A) Analysis of different p-PKC isoforms showed an increase in both p-PKCα and p-PKCδ in response to treatment with either EGF (50 ng/ml) or PMA (100 nM) and inhibition of the increase in phosphorylation of these PKC isoforms by EGF in response to erlotinib (5 μM). (B) Knockdown of PKCα by shRNA led to decreased abundance of total PKCα,p-PKCα, and p-rpS6K by immunoblot, consistent with a pathway linking EGFR, PKCα, and mTOR. (C) In cells transduced with a dominant-active allele of PKCα (PKCα-Cat) (14) erlotinib (5 μM) has reduced ability to block rpS6. Immunoblot indicates that the abundance of p-PKCα-Cat was comparable to that of endogenous p-PKCα. Erlotinib (5 μM) decreased the abundance of p-rpS6 in control cells, but was less effective in cells transduced with PKCα-Cat. The band at ~60 kDa is a nonspecific contaminant. A blot representative of three independent experiments is shown. (D)By flow cytometry, PKCα-Cat abrogated the antiproliferative activity of erlotinib (5 μM). (P < 0.0001 by Student's t test for vector-transduced cells versus PKCα-Cat-transduced cells in response to erlotinib. Data shown are means ± SDs for triplicate measurements). In (A) to (D), LN229:EGFR cells were used.