Posttranslational regulation of Myc by promyelocytic leukemia zinc finger protein

Abstract

The promyelocytic leukemia zinc finger (PLZF) protein, a transcriptional repressor, induces cellular resistance to oncogenic transformation by diverse oncoproteins. Two point mutants of PLZF that have lost the antioncogenic activity of the wild-type protein are oncogenic in chicken embryo fibroblasts; this activity is correlated with differential effects on Myc. Wild-type PLZF represses Myc transcription without affecting total Myc protein levels and reduces the levels of phosphorylated Myc. The PLZF mutants do not alter Myc transcription or protein expression but increase the levels of phosphorylated Myc. These modifications of Myc are correlated with PLZF-induced changes in Akt and the mitogen-activated protein kinase (MAPK) pathway. Wild-type PLZF downregulates the MAPK pathway and activates Akt, resulting in reduced phosphorylation on serine 62 of Myc by Erk and on threonine 58 by glycogen synthase kinase 3beta. The mutants fail to activate Akt and only slightly downregulate phospho-Erk. We postulate that the 2 PLZF mutants are oncogenic, because they function as dominant negatives of wild-type PLZF, enhancing Myc phosphorylation and increasing Myc transcriptional and oncogenic activity. In support of this suggestion, a specific inhibitor of Myc is able to revert the transformed phenotype of PLZF mutant-expressing cells.

Fig. 1

Wild-type PLZF interferes with oncogenic transformation. Equal numbers of CEF transfected with RCAS-PLZF or vector only were seeded onto 35-mm wells and challenged with transforming viruses encoding myr-P3k, myr-Akt and Myc. Numbers on the top of wells indicate the log10 of viral dilutions.

Fig. 2

Expression of mutant PLZF induces oncogenic transformation in CEF. (A) Schematic structure of PLZF protein. POZ/BTB: N-terminal repression domain; RD2: A second repression domain; ZF: Nine C2H2 zinc-finger-like domain. The box represents the Akt consensus phosphorylation motif; numbers above the drawing refer to the amino acid position. (B) Cellular transformation induced by mutant PLZF. CEF were transfected with RCAS vectors or vectors encoding wt or mutant PLZF. The cultures were overlaid with nutrient agar and stained with crystal violet on day 14.

Fig. 3

Mutant PLZF stimulates growth of CEF. CEF were stably transfected with RCAS vectors encoding wt PLZF, mutant PLZF or with vector only and cultured for 10 days. Cell numbers were determined on the indicated days.

Fig. 4

Wild-type PLZF decreases the level of Myc mRNA and Myc phosphorylation. Mutant PLZF restores Myc mRNA to control levels and increases the levels of phosphorylated Myc protein. (A) Northern blot. GAPDH was used as a loading control. (B) Western blot. CEF were stably transfected with RCAS expressing various PLZF proteins or RCAS only. Protein extracts were prepared from CEF stimulated with insulin following serum starvation. CEF transformed by overexpressed Myc were used as a positive control for total Myc and phospho-Myc.

Fig. 5

An inhibitor of Myc, 10058-F4 ((Z,E)-5-(4-Ethylbenzylidine)-2-thioxothiazolidin-4-one), reverses mutant PLZF and Myc induced oncogenic transformation. CEF were stably transfected with RCAS vector encoding S615A, S615D, v-Src, or Myc and cultured for 14 days resulting in complete oncogenic transformation of the cultures. Then, equal numbers of cells were seeded onto 35-mm wells and overlaid with nutrient agar containing the Myc inhibitor, 10058-F4 at 10µM concentration. The change of cellular phenotype is from rounded and refractile to a more polygonal, flattened shape.

Fig. 6

Wild-type and mutant PLZF affect the Akt and MAPK pathways in opposite ways, and the regulation of Akt activity by PLZF is independent of PI3K activity. (A) Wt PLZF increase Akt phosphorylation and downregulates the phosphorylation of Erk1/2. The PLZF mutants show the opposite effect on the phosphorylation of these proteins. CEF stably transfected with wt, mutant PLZF or vector only were serum starved for 42 h and subsequently stimulated with insulin for 30 min. Cells were then lysed, and proteins were separated on a 4–20% gradient SDS-/polyacrylamide gel. The transferred blots were probed with antibodies directed against total protein or antibodies recognizing phosphorylated protein. (B) The PLZF-mediated phosphorylation of Akt is independent of PI3K activity. Stably PLZF-transfected CEF were treated with or without LY294002 for 30 min during the stimulation of CEF with insulin after serum starvation. Akt phosphorylation was determined by Western blotting. PLZF was probed with anti-Flag antibody.

Figure 7

A schematic, partially hypothetical summary of PLZF-Myc interactions. Wild-type PLZF (wtPLZF) inhibits the transcription of Myc, it also activates Akt, resulting in enhanced Akt-dependent phosphorylation and hence inhibition of GSK3β. As a result, phosphorylation of Myc at T58 is reduced. wtPLZF also downregulates the MAP kinase pathway leading to reduced phosphorylation of Myc at S62. Under-phosphorylated Myc shows diminished activity as a transcriptional regulator. In contrast, PLZF mutated at position 615 (mutPLZF) has no significant inhibitory effect on the MAP kinase pathway but reduces the phosphorylation and hence the activity of Akt. The result is enhanced phosphorylation of Myc and increased transcriptional activity of Myc. Transcription from the Myc promoter is also increased. (Reduced activity is indicated by light gray symbols.) In summary, wtPLZF induces a loss of function of Myc, mutPLZF causes a gain of function in Myc.