PKCδ Regulates Translation Initiation through PKR and eIF2α in Response to Retinoic Acid in Acute Myeloid Leukemia Cells

Figure 5

PKCδ regulates the phosphorylation (Ser51) of eIF2α by ATRA and ATO in APL cells. (a) NB4 cells were left untreated or incubated with ATRA (1 μM) or ATO (0.4 μM), for the indicated time periods. Equal amounts of total cell lysate were analyzed by SDS-PAGE and immunoblotted with specific antibodies against PKCδ or p-eIF2α. β-Actin was used as a loading control. (b) ATRA (1 μM) induces phosphorylation (Thr505) of PKCδ at the activation domain detected by Western blot in NB4 cells. (c) ATRA (1 μM) induces PKCδ mRNA expression. After 24 h of ATRA treatment, NB4 cells were collected and total cellular RNA was extracted to detect PKC by RT-PCR using specific primers. The reaction products were analyzed on 2% agarose gels. cDNA synthesis and equal loading were verified by detection of the β-actin transcript. (d) ATRA (1 μM) induces phosphorylation of eIF2α in U937 (M4/M5-AML) cells. (e) Inhibition of PKCδ by rottlerin inhibits basal and ATRA-induced phosphorylation (Ser51) of eIF2α. Exponentially growing NB4 cells were collected and pretreated with specific PKCδ inhibitor rottlerin (4 μM) for 4 h before adding ATRA (1 μM) into the culture medium. Equal amount of total cell lysates were analyzed by SDS-PAGE and immunoblotted with specific antibodies against p-eIF2α, as described in Section 2.