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Retinoic acid induces leukemia cell G₁ arrest and transition into differentiation by inhibiting cyclin-dependent kinase-activating kinase binding and phosphorylation of PML/RAR

Jian-guang Wang^*, Lora W. Barsky, Elai Davicioni, Kenneth I. Weinberg^,, Timothy J. Triche^*,, Xiao-kun Zhang and Lingtao Wu^*,,1

^* Department of Pathology,

Division of Research Immunology/BMT, Childrens Hospital Los Angeles Saban Research Institute, Los Angeles, California, USA;

The Burnham Institute Cancer Center, La Jolla, California, USA;

University of Southern California Keck School of Medicine, Los Angeles, California, USA

¹Correspondence: Department of Pathology, MS# 103, Childrens Hospital Los Angeles, University of Southern California Keck School of Medicine, 4650 Sunset Blvd., Los Angeles, CA 90027 USA. E-mail: lingtaow{at}usc.edu

SPECIFIC AIMS

Acute promyelocytic leukemia (APL) cells express promyelocytic leukemia/retinoic acid receptor alpha (PML/RAR) fusion protein, which leads to the blocking of APL cell differentiation. Treatment of APL with all-trans-retinoic acid (ATRA) induces disease remission by in vivo differentiation of APL cells. Differentiation requires cell cycle exit, yet how ATRA couples cell cycle exit to differentiation of APL remains largely unknown. We previously found that ATRA-induced cell differentiation accompanies ubiquitination-proteolysis of ménage à trois 1 (MAT1), an assembly factor and targeting subunit of cyclin-dependent kinase (CDK)-activating kinase (CAK) that regulates G₁ exit. This finding led us to study the effect of ATRA:PML/RAR binding in modulating CAK-dependent G₁ exit and cell differentiation. Our studies reveal a novel ATRA-signaling in mediating APL cell proliferation/differentiation (P/D) transition, in which ATRA coordinates G₁ arrest and transition into differentiation by inducing MAT1 degradation and PML/RAR hypophosphorylation through disrupting PML/RAR binding and phosphorylation by CAK.

PRINCIPAL FINDINGS

1. Binding of PML/RAR with CAK
In vitro translated CAK subunits, in vitro assembled CAK, or cellular CAK complexes precipitated from NB4 cells were incubated with glutathione S-transferase (GST)-PML/RAR proteins. Using either GST-pull-down assay or immunoprecipitation-dependent protein binding assay, we found that GST-PML/RAR interacted with CAK and its different subunits. To further test whether PML/RAR binds to CAK in vivo, the putative CAK-PML/RAR complexes were coprecipitated from NB4 cells using anti-PML, anti-MAT1, or anti-CDK7 antibody (Ab). The complexed precipitates were then immunoblotted with anti-RAR and anti-MAT1 antibodies. The results demonstrate that PML/RAR interacts with CAK in vivo.

2. CAK-PML/RAR dissociation precedes MAT1 degradation and reduction of CAK abundance in ATRA-induced P/D transition
CAK-PML/RAR complexes were precipitated with anti-CDK7 Ab from NB4 cells with different periods of ATRA treatment. The resulting precipitates were immunoblotted with anti-RAR and anti-MAT1 antibodies. The levels of PML/RAR bound to CAK were reduced within 12 h of ATRA treatment in the presence of unchanged levels of MAT1 and CAK abundance (Fig. 1 A). Thereafter, the levels of PML/RAR bound to CAK remain at constant low levels, whereas CAK abundance represented by MAT1 levels was decreased after 48 h of ATRA treatment (Fig. 1B ). We then examined how temporal changes in CAK-PML/RAR dissociation and MAT1-dependent reduction of CAK abundance were correlated to the development of ATRA-induced NB4 cell P/D transition. The results showed that ATRA-induced CAK-PML/RAR dissociation leads to MAT1 degradation and decreased CAK abundance. Reduction of MAT1 is then followed by proliferation inhibition, decreased hyperphosphorylated form of pRb, nuclear segmentation, and CD11b induction (Fig. 1, C-F ).

View larger version (25K): [in this window] [in a new window]   Figure 1. CAK-PML/RAR dissociation precedes MAT1 degradation and reduction of CAK abundance in ATRA-induced P/D transition. A and B) PML/RAR was precipitated by anti-CDK7 Ab from NB4 cells exposed to ATRA for up to 12 h (A) or for up to 120 h (B). The precipitates were then probed with anti-RAR and anti-MAT1 antibodies. C) Either 1 or 5 µM of ATRA showed similar inhibitory effect on NB4 cell proliferation. The growth curves represent the means ± SD from the cells of triplicate wells. D) pRb expression and phosphorylation status were depicted by Western blot analysis in ATRA-induced P/D transition. P-pRb, hyperphosphorylated form of pRb; pRb, hypophosphorylated form of pRb. E) nuclear segmentation of NB4 cells treated with ATRA. F) CD11b expression following ATRA stimuli.

3. ATRA selectively induces MAT1 degradation in G₁ arresting cells that commit to differentiation
NB4 cells were arrested in G₁ phase with different periods of ATRA treatment. In parallel, NB4 cells were synchronized at the G₁/S boundary using thymidine/aphidicolin double block or in prometaphase with nocodazole treatment. The synchronized cells were then released from cell cycle block at different time points crossing a full cell cycle. The results showed that over 80% of cells were arrested in G₁ phase after 72 h of ATRA treatment. In parallel, 70% of cells were blocked in G₁ phase with thymidine/aphidicolin treatment or progressed into G₁ phase between 3 to 9 h after released from nocodazole arrest. In duplicate cultures, we determined the total protein levels of MAT1 using Western blot analysis. The results showed that MAT1 was significantly decreased in ATRA-induced G₁ arresting cells after 48 h of ATRA treatment, which was followed by the occurrence of ATRA-induced G₁ arrest after 48–72 h of ATRA treatment. In contrast, the levels of MAT1 remained unchanged in the cells that were synchronized in G₁/S boundary with thymidine/aphidicolin or released into G₁ phase from nocodazole block.

4. Decreased CAK phosphorylation of PML/RAR occurs only when MAT1 levels are reduced
CAK complexed-PML/RAR was precipitated by CDK7 Ab from NB4 cells treated with ATRA. Using GST-PML/RAR as an additional substrate in an in vitro kinase assay, CAK activity in the precipitates was assessed by its phosphorylation of both GST-PML/RAR and CAK-bound PML/RAR simultaneously in the presence of [³²P]-ATP. The results (Fig. 2 ) showed that the phosphorylation of CAK-bound PML/RAR was inhibited to undetectable levels after 48 h of ATRA treatment, whereas GST-PML/RAR phosphorylation was progressively diminished after 48 h of ATRA treatment, and it then became undetectable after 72 h of ATRA treatment. Such decreased phosphorylation of both GST-PML/RAR and CAK-bound PML/RAR corresponded with decreased CAK activity, as shown by autohypophosphorylated [³²P]-CDK7 after 48 h of ATRA treatment. By immunoblotting the autoradiography blot with anti-RAR, anti-CDK7, and anti-MAT1 antibodies, we found that decreased CAK phosphorylation of either GST-PML/RAR or CAK-bound PML/RAR occurred when MAT1 levels were decreased after 48 h of ATRA treatment.

View larger version (38K): [in this window] [in a new window]   Figure 2. Decreased CAK phosphorylation of PML/RAR occurs only when MAT1 levels are reduced. Autoradiography showed that, in the presence of constant levels of GST-PML/RAR, CAK activity in phosphorylation of GST-PML/RAR was inhibited when MAT1 levels were reduced after 48 h of ATRA treatment. The phosphorylation of CAK-bound PML/RAR in the precipitates was also similarly diminished on ATRA-induced MAT1 reduction. The autoradiography blot was probed with different antibodies as indicated. GST-PML/RAR (lane 9) was used as a positive control in Western blot analysis. P-K7, hyperphosphorylated form of CDK7; K7, hypophosphorylated form of CDK7.

5. Decreased CAK phosphorylation of PML/RAR accompanies progressive degradation of MAT1 in vivo
NB4 cells following different periods of ATRA exposure were in vivo labeled with [³²P]-orthophosphate. CAK and PML/RAR were then coprecipitated using anti-CDK7 or anti-MAT1 Ab. The results showed that CAK phosphorylation of PML/RAR was inhibited in differentiating cells, and that decreased MAT1 levels were associated with reduced CAK phosphorylation of PML/RAR.

6. ATRA selectively decreases CAK activity in G₁ arresting cells committed to differentiation
NB4 cells were either arrested in G₁ phase with ATRA treatment or released into G₁ stage from nocodazole-block. CAK complexes were precipitated by MAT1 antibody from these different G₁ cells, and then histone H1 was used as substrate to assess CAK activities in the precipitates by using in vitro kinase assay. The results showed that CAK phosphorylation of histone H1 was decreased in ATRA-induced G₁ arresting cells undergoing differentiation but not in the synchronized G₁ cells that do not differentiate.

CONCLUSIONS AND SIGNIFICANCE

We investigated the relationship between MAT1-dependent CAK activity and PML/RAR phosphorylation in ATRA-induced NB4 cell P/D transition. We find that CAK binds to and hyperphosphorylates PML/RAR in actively proliferating cells. ATRA dissociates PML/RAR from CAK, leading to MAT1 degradation, G₁ arrest, and decreased CAK phosphorylation of PML/RAR. CAK phosphorylation of PML/RAR is inhibited when MAT1 levels are reduced, and PML/RAR hypophosphorylation accompanies cell differentiation. These results therefore present evidence that, following ATRA-induced CAK-PML/RAR dissociation, reduction of MAT1 simultaneously induces CAK-dependent G₁ arrest and decreases CAK phosphorylation of PML/RAR, leading to NB4 cell differentiation. Thus, PML/RAR hypophosphorylation may modulate the effect of ATRA in transcriptional control of cell differentiation by transactivating differentiation-responsive genes in CAK-dependent G₁ arresting cells (Fig. 3 ).

View larger version (25K): [in this window] [in a new window]   Figure 3. ATRA may modulate CAK-dependent P/D transition by disrupting PML/RAR binding and phosphorylation by CAK.

The effect of ATRA:PML/RAR binding on cell differentiation is generally considered to act in a transcription-dependent mechanism. Our studies here reveal that ATRA may induce MAT1 degradation by dissociating PML/RAR from CAK to coordinate G₁ arrest and transition into differentiation. Hence, the complex formation of PML/RAR with CAK and its regulation by ligand represent a novel mechanism of ATRA-signaling in mediating cell differentiation. We anticipate that the detailed molecular mechanisms of ATRA-action in posttranslational regulation of MAT1 degradation and PML/RAR hypophosphorylation will present new idea in the design of improved intervention strategies against leukemia.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.06-5900fje

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This Article Abstract Full Text Full Text (PDF) All Versions of this Article: fj.06-5900fjev1 20/12/2142    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Wang, J.-g. Articles by Wu, L. Search for Related Content PubMed PubMed Citation Articles by Wang, J.-g. Articles by Wu, L.