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Originally published In Press as doi:10.1074/jbc.M206792200 on September 3, 2002

J. Biol. Chem., Vol. 277, Issue 45, 43369-43376, November 8, 2002
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Retinoid-induced G1 Arrest and Differentiation Activation Are Associated with a Switch to Cyclin-dependent Kinase-activating Kinase Hypophosphorylation of Retinoic Acid Receptor alpha *

Jiwei WangDagger , Lora W. Barsky§, Chung H. Shum, Ambrose Jong||, Kenneth I. Weinberg§, Steven J. Collins**, Timothy J. TricheDagger , and Lingtao WuDagger DaggerDagger

From the Dagger  Department of Pathology, § Division of Research Immunology/Bone Marrow Transplant, and || Division of Hematology-Oncology, Childrens Hospital Los Angeles Research Institute, Los Angeles, California 90027, the ** Human Biology Division, Fred Hutchinson Cancer Research Center, Seattle, Washington 98109, and the  University of Southern California Keck School of Medicine, Los Angeles, California 90033

Received for publication, July 8, 2002, and in revised form, September 3, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cell cycle G1 exit is a critical stage where cells commonly commit to proliferate or to differentiate, but the biochemical events that regulate the proliferation/differentiation (P/D) transition at G1 exit are presently unclear. We previously showed that MAT1 (ménage à trois 1), an assembly factor and targeting subunit of the cyclin-dependent kinase (CDK)-activating kinase (CAK), modulates CAK activities to regulate G1 exit. Here we find that the retinoid-induced G1 arrest and differentiation activation of cultured human leukemic cells are associated with a switch to CAK hypophosphorylation of retinoic acid receptor alpha  (RARalpha ) from CAK hyperphosphorylation of RARalpha . The switch to CAK hypophosphorylation of RARalpha is accompanied by decreased MAT1 expression and MAT1 fragmentation that occurs in the differentiating cells through the all-trans-retinoic acid (ATRA)-mediated proteasome degradation pathway. Because HL60R cells that harbor a truncated ligand-dependent AF-2 domain of RARalpha do not demonstrate any changes in MAT1 levels or CAK phosphorylation of RARalpha following ATRA stimuli, these biochemical changes appear to be mediated directly through RARalpha . These studies indicate that significant changes in MAT1 levels and CAK activities on RARalpha phosphorylation accompany the ATRA-induced G1 arrest and differentiation activation, which provide new insights to explore the inversely coordinated P/D transition at G1 exit.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The cyclin-dependent kinase (CDK)1-activating kinase (CAK), a trimeric CDK7-cyclin H-MAT1 (ménage à trois 1) complex, was originally implicated in cell cycle control by its ability to phosphorylate and activate CDKs (1, 2). Previous studies demonstrated that CAK regulates cell cycle G1 exit both by phosphorylation activation of cyclin D-CDK complexes (3-7) and by phosphorylation inactivation of retinoblastoma tumor suppressor protein (pRb) (8). Also, CAK is a subcomplex of transcription factor IIH (TFIIH) (9-12) and a kinase of TFIIH that phosphorylates the COOH-terminal domain of the largest subunit of RNA polymerase II for transcription initiation (9, 13-15). Thus, CAK is considered a cross-road regulator in linking cell cycle control with transcription. Recently, distinct regions of MAT1 have been shown to regulate CAK kinase and TFIIH transcription activities (16). To date, comprehensive studies demonstrate that MAT1 regulates CAK substrate specificity and protein-protein interactions, i.e. MAT1 mediates the association of CAK with core TFIIH and shifts CAK substrate preference from CDK2 to the COOH-terminal domain (12, 14, 17, 18). Mice lacking MAT1 are unable to enter S phase and are defective in RNA polymerase II phosphorylation (19). Antisense abrogation of MAT1 induces cell cycle G1 arrest (20); and MAT1 regulates the interaction and phosphorylation of CAK with tumor suppressor p53 (21), octamer transcription factors (22), pRb (8), and retinoic acid receptor alpha  (RARalpha ) (23).

Among the above substrates of CAK, RARalpha is involved mainly in differentiation regulation. RARalpha belongs to the superfamily of nuclear ligand-activated transcriptional regulators, the retinoic acid receptors. RARalpha is a phosphoprotein (23-26) and mediates the action of retinoids in myeloid differentiation (27-29). In HL60 leukemic cells, the all-trans retinoic acid (ATRA)-induced differentiation is mediated directly through the RARalpha (30, 31). Indeed, a subclone of HL60 (designated HL60R) harbors a truncated AF-2 domain of RARalpha (RARalpha Delta AF-2) and is resistant to differentiation induction by ATRA. However, introducing a normal RARalpha cDNA into these cells restores their differentiating response to ATRA (30-32). Both the ligand-dependent transcriptional activation function AF-2 (located in the RARalpha E region) and the ligand-independent transcriptional activation function AF-1 (located in the RARalpha A/B region) are involved in cell differentiation (33, 34). In vitro studies show that CAK phosphorylates RARalpha at both the A/B and F regions (23). However, the precise molecular mechanisms whereby CAK phosphorylates RARalpha and its functional consequences remain unknown.

The decision of cells to differentiate is commonly made in cell cycle G1 phase, and differentiation induction requires cell cycle arrest (35-38), but little is known about how the cell cycle machinery coordinates cell cycle arrest with differentiation activation. Given that CAK regulates cell cycle G1 exit for S-phase entry (3-8, 20) and that RARalpha , a key player in myeloid differentiation (36, 39, 40), is a substrate for CAK in Cos-1 cells (23), we investigated whether there was any correlation between CAK-RARalpha signaling and ATRA-induced differentiation in cultured human leukemic cells. We found that ATRA-induced MAT1 reduction and CAK hypophosphorylation of RARalpha are RARalpha -dependent and are associated with G1 arrest and differentiation activation.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cell Culture-- Human leukemic cells AML1 and Nalm6 (provided by Dr. Kohn), NB4 (provided by Dr. Lanotte), HL60, HL60R, REH, Jurkat, lymphoma U937, and human osteosarcoma U2OS cells were cultured in RPMI 1640 plus 10% fetal bovine serum. Human diploid fibroblast IMR90 and human transformed embryonal kidney 293 cells were cultured in minimal essential medium (Eagle) supplemented with 10% fetal bovine serum. Human Ewing's sarcoma TC71, TC32, human rhabdomyosarcoma (RD), and human Ewing's/pPNET CHP100L cells are USC pathology cell lines and were cultured in RPMI 1640 containing 10% fetal bovine serum. Cell lines were purchased from ATCC unless otherwise specified. ATRA and protease inhibitor MG-132 were from Sigma. Either 1 or 5 µM of ATRA were used to treat the cells, and similar effects were observed between the two concentrations.

In Vivo Phosphorylation and Immunoprecipitation-- Immediately before labeling, subconfluent cells (5 × 105/ml) were adjusted to 1 × 106/ml and cultured in phosphate-free complete medium for 1 h. Then, 1 × 106/ml cells were incubated with 125 µCi of [32P]orthophosphate (ICN) in the same medium for 2 h at 37 °C. Cells were washed and harvested in ice-cold phosphate-buffered saline. Nuclear protein extraction was performed at 4 °C using a modified high salt extraction buffer (41). The same amounts of nuclear proteins from each sample were used for immunoprecipitation as described (8). The resulting immunoprecipitates were resolved by SDS-PAGE, electrotransferred onto polyvinylidene difluoride membrane, and autoradiographed. All anti-human polyclonal and monoclonal antibodies used in immunoprecipitation were purchased from Santa Cruz Biotechnology.

Western Blotting and Cell Proliferation Analyses-- Western blotting was performed as described previously (8). All anti-human polyclonal and monoclonal antibodies were purchased from Santa Cruz Biotechnology. The rate of cell duplication determined by cell counting was described before (20).

Analysis of Cell Cycle Profile and Detection of Cytodifferentiation Antigen-- Cell cycle profile was analyzed as described before (20). A direct immunofluorescence staining technique was applied to analyze myeloid differentiation marker CD11b. Cells were exposed to phosphatidylethanolamine-conjugated CD11b antibodies at 4 °C for 30 min and fixed with fresh 1% paraformaldehyde. The antigens were then determined by a FACScan flow cytometer (BD Biosciences). The percentage of positive cells and the mean associated fluorescence were quantitated using a FACScan analyzer (CellQuest software V3.2). Control studies were performed with fluorescein isothiocyanate-conjugated and phosphatidylethanolamine-conjugated antihuman CD45, and fluorescein isothiocyanate-conjugated and phosphatidylethanolamine-conjugated non-binding mouse gamma 1 (IgG1). All antibodies and mouse IgG were purchased from BD Biosciences.

Characterization of Nuclear Segmentation-- Subconfluent cells with or without ATRA treatment were fixed by methanol and stained with Wright-Giemsa (Sigma). The mature nuclear segmentation of leukemic cells was evaluated under a Zeiss Axioplan microscope. Images were color balanced in Adobe Photoshop.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

CAK Hypophosphorylation of RARalpha Accompanies the ATRA-induced G1 Arrest and Differentiation Activation-- Since CAK interacts with and phosphorylates RARalpha in vitro (23), we wanted to determine whether the ATRA-induced terminal differentiation of HL60 cells was associated with any changes of CAK-RARalpha signaling. HL60 cells following different periods of ATRA exposure were in vivo labeled with [32P]orthophosphate. We used CDK7 antibody to immunoprecipitate the CAK-bound RARalpha from the labeled cells for visualizing CAK-RARalpha signaling by autoradiography. As we expected, CAK interacted with and phosphorylated RARalpha in vivo because CDK7 antibody brought down phosphorylated RARalpha and autophosphorylated CDK7 simultaneously (Fig. 1A). We found that CAK hyperphosphorylated RARalpha in proliferating cells. However, CAK hyperphosphorylation of RARalpha was inhibited about 40% after 24 h of ATRA stimuli and then diminished to over 90% after 96 h of ATRA stimuli (densitometer results not shown) (Fig. 1A). CAK activity as represented by CDK7 autophosphorylation also decreased significantly, showing a correspondence to decreased RARalpha phosphorylation (Fig. 1A). Western analyses of CAK subunits and their associated RARalpha were performed by using this same blot. The results showed that RARalpha and CDK7 antibodies recognized RARalpha and CDK7, respectively, at the corresponding molecular weight positions of their phosphorylations (Fig. 1A). CDK7 polyclonal antibodies distinguished both forms of the autohyperphosphorylated CDK7 (P-K7) and the autohypophosphorylated CDK7 (K7). Following ATRA stimulation, the P-K7 diminished gradually, whereas the levels of K7 correspondingly increased (Fig. 1A). The dynamic levels of P-K7 and K7 corresponded well with the dynamic status of RARalpha hyperphosphorylation and RARalpha hypophosphorylation (Fig. 1A). Hence, these results show that ATRA induces a switch to CAK hypophosphorylation of RARalpha in differentiating cells from CAK hyperphosphorylation of RARalpha in proliferating cells and that this reduced CAK phosphorylation of RARalpha is associated with a decreased CAK activity.


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  		Fig. 1.   In vivo CAK hypophosphorylation of RARalpha in HL60 cells correlates with P/D transition. A, in vivo phosphorylated 32P-labeled RARalpha and CDK7 were immunoprecipitated with CDK7 antibodies. Further, this same blot was used for Western analyses of CDK7, MAT1, RARalpha , and cyclin H. A MAT1-associated M30 fragment was recognized by MAT1 polyclonal antibodies. P-K7, autohyperphosphorylated form of CDK7; K7, autohypophosphorylated form of CDK7; WB, Western blot; IP, immunoprecipitate. B, Western detection of in vivo phosphorylation status of pRb. P-pRb, hyperphosphorylated form of pRb; pRb, hypophosphorylated form of pRb. C, progressively developed G1 arrest under ATRA stimuli. D, cell proliferation under ATRA stimuli. The growth curves represent the mean ± S.D. from the cells of triplicate wells. E, analysis of differentiation antigen CD11b. Samples I-IV minus ATRA were stained with mouse IgG, CD45, and CD11b antibodies as indicated for controls. Sample V was stimulated with ATRA for 120 h and then stained with CD11b antibodies. 40% of cells were positive of CD11b expression in sample V. F, nuclear segmentation was markedly evident after 120 h of ATRA stimuli.

Surprisingly, we observed a 30-kDa MAT1-associated fragment (M30) that was immunoprecipitated by CDK7 antibodies and recognized by MAT1 polyclonal antibodies in Western blotting (Fig. 1A). This indicated that M30, together with MAT1, was within the CAK complex. We found that gradual diminution of M30 paralleled the developments of both RARalpha hypophosphorylation and CDK7 hypophosphorylation, whereas the levels of both cyclin H and RARalpha appear unrelated to this dynamic phosphorylation pattern (Fig. 1A). The results suggest that ATRA-induced diminution of M30 within the CAK complex might be associated with the dynamic changes of CAK phosphorylation of RARalpha .

We further monitored, in parallel, the relationship between CAK-RARalpha signaling and P/D transition. We found that the reduction in RARalpha phosphorylation was associated with the occurrence of pRb hypophosphorylation and G1 arrest after 48 h of ATRA stimuli (Fig. 1, A-C). Proliferation was halted after 72 h of either 1 or 5 µM of ATRA treatment, and then cell numbers remained stable (Fig. 1D). During this period, differentiation proceeded as shown by CD11b expression and nuclear segmentation (Fig. 1, E and F). Interestingly, although we found pRb hypophosphorylation under ATRA stimuli (Fig. 1B), there was no change in either cyclin D1 expression or CDK4 phosphorylation by Western analyses (data not shown). Hence, these results indicate that ATRA concurrently induces CAK hypophosphorylation of RARalpha and cyclin D/CDK4-independent pRb hypophosphorylation. The dynamic switch to CAK hypophosphorylation of RARalpha was associated with the transition from actively proliferating cells to G1 arrest that accompanies the terminal myeloid differentiation.

MAT1 Expression and the Origin of M30-- The aforementioned results show that MAT1-associated M30 exists within CAK complex and that ATRA-induced diminution of M30 is associated with reduced CAK phosphorylation of RARalpha . Therefore we explored the following: (a) the relationship between MAT1 and M30; and (b) the origin of M30. By Western analyses of leukemic HL60 and NB4 cells, we found that M30 always was associated with MAT1 in proliferating cells. Total cellular MAT1 expression was inhibited about 50% after 48 h of ATRA stimuli (Fig. 2, A and B). M30 was decreased significantly more, approaching 90-100% reduction after 48-72 h of ATRA stimuli (Fig. 2, A and B), which corresponded to the reduced levels of M30 within the CAK complex (Fig. 1A). In contrast to the reduction of MAT1/M30 by ATRA, the total CDK7 protein level remained unchanged (Fig. 2, A and B, densitometer results not shown). To investigate whether M30 is associated with MAT1 in other tumor cells, we analyzed several solid tumor and leukemic cell lines using Western blotting. We found that MAT1 antibodies recognized several fragments ranging from about 20 to 30 kDa in certain tumor cell lines. Further, not only was M30 consistently present but also MAT1 expression was enhanced in these tumor cells compared with normal IMR90 cells (Fig. 2, C and D). Because ATRA-induced diminution of MAT1/M30 is associated with G1 arrest and differentiation activation in differentiating cells (Figs. 1 and 2, A and B), these results suggest that the overexpressed MAT1 and the high levels of M30 in these tumor cells may be related to uncontrolled cell proliferation.


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  		Fig. 2.   MAT1 expression and the origin of M30. A and B, Western analyses of MAT1 expression and M30 formation following ATRA stimuli of HL60 and NB4 cells. Actin was used as a loading control. C and D, Western analysis of MAT1/M30 in tumor cells and normal IMR90 cells. E, M30 is likely a COOH-terminal deleted MAT1 fragment. Lanes labeled 1 were blotted with full-length anti-MAT1 antibodies, lanes labeled 2 with N termini anti-MAT1 (against about 1-30 amino acids), and lanes labeled 3 with C termini anti-MAT1 (against approximately 280-309 amino acids).

We anticipated that M30 could be one of the following: (a) a degraded MAT1 fragment; (b) a splicing variant of the MAT1 gene; or (c) a new gene of sequence homology with MAT1. To address these issues we used multiple pairs of specific and degenerate MAT1 primers for reverse transcription-PCR detection of possible M30 mRNA. A splice variant appeared unlikely because we could not detect any fragments of different sizes that might correspond to this possible MAT1 splice product (data not shown). We also used several MAT1 fragments encompassing the MAT1 coding region to screen a cDNA library with low stringency hybridization. All positive clones retrieved from the screening were MAT1 (data not shown). We performed Western analysis using three different MAT1 antibodies, recognizing full-length MAT1, NH2-terminal MAT1, and COOH-terminal MAT1. We found that whereas the full-length and NH2-terminal antibodies recognized M30, the COOH terminus antibody did not (Fig. 2E, lanes 3), indicating that M30 is likely a COOH-terminal truncated MAT1. Recently, Egly's group identified a "minimal" MAT1 fragment that remains within the CAK complex after its spontaneous degradation (16), suggesting that the degraded MAT1 fragment may form Delta CAK in cells along with the wild type CAK complex in these leukemic cells.

ATRA Induces Proteasome-dependent Degradation of MAT1/M30-- Previous studies have demonstrated that ATRA induces a proteasome-dependent degradation of retinoic acid receptors (42, 43). Thus, we wanted to determine whether ATRA-induced reduction of MAT1/M30 was similarly related to enhanced protein degradation. To test this, HL60 cells were incubated with or without ATRA for 48 h, and then cells were exposed either to vehicle or to protease inhibitor MG-132 for an additional 24 h before harvest. Since ATRA stimuli lead to RARalpha degradation but have no effect on the levels of CDK7 and cyclin H, we used RARalpha as a positive control, whereas CDK7 and cyclin H were used as negative controls in parallel. We found that the levels of MAT1, M30, and RARalpha decreased with ATRA stimuli (Fig. 3A, lanes 2) but then could be overcome by the addition of MG-132 (Fig. 3A, lanes 3). In contrast, neither CDK7 nor cyclin H was affected by ATRA stimuli or MG-132 treatment (Fig. 3B, lanes 2 and 3). In an extension of this approach, we blocked the proteasome pathway first by treating HL60 cells with MG-132 for 8 h. We then added ATRA for an additional incubation of 60 h. Compared with the cells with ATRA stimuli alone (Fig. 3C, lane 2), the cells with pretreatment of proteasome inhibitor blocked ATRA-induced MAT1/M30 degradation (Fig. 3C, lane 3). These results demonstrate that ATRA inhibits MAT1 expression and M30 formation via an ATRA-induced proteasome pathway. Since CAK activity is MAT1 dose-dependent (8, 18, 22, 44) and the reduction of MAT1/M30 is associated with decreased CAK phosphorylation of RARalpha (Fig. 1A), these results suggest that ATRA-induced diminution of MAT1/M30 via the protease pathway may inhibit CAK activities on RARalpha phosphorylation.


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  		Fig. 3.   ATRA inhibits M30 and MAT1 via an ATRA-activated protease pathway. A, Western analyses showed that MG-132 overcomes the ATRA-induced degradation of MAT1/M30 and RARalpha . Lanes labeled 1 were treated with vehicles only. Lanes labeled 2 were treated with ATRA (1 µM) for 72 h. Lanes labeled 3 were treated with ATRA for 48 h first, and then 0.2 µM MG-132 was added for an additional incubation of 24 h. MG-132 was dissolved in Me2SO (DMSO) and ATRA in ethanol. B, neither ATRA nor MG-132 affects the levels of CDK7 and cyclin H. The sample order is the same as described in the A section. C, Western analyses showed that MG-132 prevents the ATRA-induced degradation of MAT1/M30. Lane 1 was with vehicles only. Lane 2 was treated with ATRA for 68 h. Lane 3 was treated with MG-132 (0.3 µM) first for 8 h and then added ATRA for an additional incubation of 60 h.

RARalpha Activation by ATRA Is Required for Both the Reduction of MAT1/M30 and the Switch to CAK Hypophosphorylation of RARalpha -- ATRA, signaling via the ligand-dependent AF-2 domain of RARalpha in HL60 cells containing a wild type RARalpha (RARalpha WT) (30-32), inhibits MAT1/M30 through a proteasome degradation pathway (Fig. 3) and induces CAK hypophosphorylation of RARalpha in ATRA-induced P/D transition (Fig. 1). Thus, we utilized HL60R cells, which harbor RARalpha Delta AF-2 and are resistant to differentiation by ATRA (30-32), to explore the relationship of ATRA-induced P/D transition with RARalpha activation, the dynamic changes of MAT1/M30 levels, and the CAK activities on RARalpha phosphorylation. We first tested whether MAT1 expression and M30 formation would be inhibited by ATRA-activated proteasome pathway in HL60R cells. We treated HL60 and HL60R cells with ATRA for 48 h and then added MG-132 for an additional incubation of 24 h before harvest. Western analyses showed that in contrast to HL60 cells showing a reduction of MAT1/M30 by ATRA (Fig. 4A, lane 2) but an overcoming by MG-132 (Fig. 4A, lane 3), there was virtually no change of MAT1/M30 in HL60R cells (Fig. 4A, lanes 5 and 6). Second, we compared MAT1 expression and M30 formation in HL60 and HL60R cells by Western analyses. In contrast to HL60 cells, HL60R cells retained high levels of MAT1/M30 under ATRA stimuli (Fig. 4B). These results show that RARalpha activation by ATRA is required for inhibition of MAT1 expression and M30 formation.


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  		Fig. 4.   In vivo CAK hyperphosphorylation of RARalpha correlates with the inhibition of P/D transition. A, Western analyses of MAT1/M30 degradation by ATRA in both HL60 and HL60R cells. Lanes 1 and 4 were treated with vehicles only. Lanes 2 and 5 were treated with ATRA (1 µM) for 48 h. Lanes 3 and 6 were treated with ATRA first for 24 h, and then 0.3 µM MG-132 was added for an additional incubation of 24 h. DMSO, dimethyl sulfoxide. B, Western analysis of MAT1 expression and M30 formation in both HL60 and HL60R cells. C, in vivo phosphorylated 32P-labelled RARalpha and CDK7 in HL60R cells under ATRA stimuli were immunoprecipitated with CDK7 antibodies. Further, the same blot was used for analyzing MAT1 and CDK7 by Western blotting (WB). PI, pre-immune IgG. IP, immunoprecipitate. D-G, HL60R cells following ATRA exposure displayed persistent cell cycling (D), continuous proliferation (E), no detectable CD11b expression (F, V), and no detectable nuclear segmentation (G).

Next, we examined the relationship between the levels of MAT1/M30, CAK phosphorylation of RARalpha , and G1 arrest/differentiation activation in HL60R cells. Similarly in studies of CAK-RARalpha signaling as performed in HL60 cells (Fig. 1), we labeled HL60R cells in vivo with [32P]orthophosphate and immunoprecipitated CAK-bound RARalpha using CDK7 antibodies. We found no change in either CDK7 autophosphorylation or CAK phosphorylation of RARalpha following ATRA stimuli of the HL60R cells (Fig. 4C). Further, Western analyses of this same blot showed that high levels of MAT1/M30 were retained in the CAK complexes and corresponded well to both CDK7 auto-hyperphosphorylation and RARalpha hyperphosphorylation (Fig. 4C). Also, as we expected that whereas CAK hyperphosphorylation of RARalpha was retained in HL60R cells, the cells remained virtually continuous cycling (Fig. 4D) and proliferating (Fig. 4E) without detectable CD11b expression (Fig. 4F) or morphology change (Fig. 4G) following ATRA stimuli. Thus, these results indicate that ATRA-induced RARalpha activation via the AF-2 domain of RARalpha is critical to the changes in MAT1/M30 levels and CAK activities on RARalpha phosphorylation that occur in the differentiating HL60 cells (Fig. 1). Moreover, in the ATRA-treated HL60R cells the lack of changes in MAT1/M30 levels or CAK activities on RARalpha phosphorylation is correlated with the absence of both G1 arrest and differentiation activation (Fig. 4). Hence, these results strengthen the observed associations between MAT1/M30 levels, CAK phosphorylation of RARalpha , and terminal myeloid differentiation.

CAK Phosphorylation Regulation of RARalpha Is Independent of RARalpha Degradation-- Because ATRA induces RARalpha degradation (42, 43) (Fig. 3A), we investigated whether the reduction in CAK phosphorylation of RARalpha was related to the diminished levels of RARalpha substrate resulting from RARalpha degradation. Using Western analyses, we first monitored ATRA-induced RARalpha degradation in HL60 cells from 1 h to 8 days. We found that the onset of RARalpha degradation was evident after 7 h of ATRA stimuli, but afterward the RARalpha levels remained relatively stable for up to 8 days (Fig. 5A). RARalpha also maintained a relatively steady level within the CAK complex after its onset of degradation (Fig. 1A). In contrast to this pattern of RARalpha degradation, the reduction in CAK phosphorylation of RARalpha was not observed until 24 h after ATRA stimuli; it then diminished about 70% to more than 90% after 48-96 h of ATRA stimuli (Figs. 5B and 1A). These marked differences in the kinetics of degradation versus the pattern of CAK phosphorylation of RARalpha suggest that CAK phosphorylation regulation of RARalpha under ATRA stimuli is independent of RARalpha degradation.


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  		Fig. 5.   CAK phosphorylation of RARalpha is independent of RARalpha -degradation. A, Western analyses of RARalpha -degradation. B, HL60 cells were treated with ATRA for the indicated period of time. In vivo phosphorylated 32P-labeled RARalpha was immunoprecipitated with CDK7 antibodies. Further, this same blot was used for analyzing MAT1 by Western blotting. IP, immunoprecipitate; PI, pre-immune IgG; WB, Western blot.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In a variety of different tissues P/D transition is a process of coordinating cell cycle arrest with differentiation activation. However, the physiological phenomena and the regulatory mechanisms that are involved in coordinating these inverse molecular events remain unclear. We present evidence to show an ATRA-induced transition from proliferating to differentiating cells, where the ATRA-mediated MAT1 reduction and CAK hypophosphorylation of RARalpha may be involved in coordination of G1 arrest and differentiation activation of these cultured leukemic cells.

CAK-RARalpha Signaling May Be Instrumental in Coordinating G1 Arrest and Differentiation Activation-- CAK regulates cell cycle G1 exit for S-phase entry (3-8, 20), and RARalpha is involved in regulating the terminal differentiation of different cell types (27-29, 36, 39, 40). The fact that CAK interacts with and phosphorylates RARalpha in vitro (23) led us to determine whether there was any in vivo association between CAK phosphorylation of RARalpha and ATRA-induced differentiation of HL60 cells. Since RARalpha is also phosphorylated by protein kinase A and a variety of proline-directed protein kinases (23, 26, 45), immunoprecipitation of in vivo phosphorylated RARalpha by RARalpha antibodies cannot distinguish the signaling specificity of RARalpha by CAK. Thus, our experimental strategy was to immunoprecipitate in vivo CAK-bound RARalpha using CDK7 antibodies, which not only ensures the specificity of RARalpha phosphorylation by CAK but also allows us to visualize RARalpha phosphorylation status and CAK activities simultaneously. By analyzing these immunoprecipitates, we observe that CAK interacts with and phosphorylates RARalpha in vivo (Figs. 1A, 4C, and 5B). ATRA-induced G1 arrest and differentiation activation are associated with markedly decreased CAK activities and CAK phosphorylation of RARalpha (Fig. 1). Similar events in P/D transition also were observed in ATRA-sensitive NB4 cells (data not shown). In contrast, we did not observe any such reduced CAK phosphorylation of RARalpha as well as its association with G1 arrest and differentiation activation in ATRA-treated HL60R cells that harbor RARalpha Delta AF-2 (Fig. 4, C-G). Hence, our data indicate that ATRA-induced diminution of CAK activities on RARalpha phosphorylation is mediated directly via the RARalpha and suggest that in HL60 cells containing RARalpha WT, ATRA-induced CAK hypophosphorylation of RARalpha may coordinate G1 arrest with differentiation activation.

MAT1-dependent CAK Activities-- What are the factors that mediate the switch to CAK hypophosphorylation of RARalpha in ATRA-induced differentiation of leukemic cells? Previous studies demonstrate that MAT1 determines CAK substrate specificity and further enhances CAK activities in either a dose-dependent manner or via MAT1-mediated protein-protein interactions (8, 14, 18, 19, 21-23, 44). MAT1 mRNA is overexpressed in multiple tumor cell lines (46), and differentiation induction by ATRA in NB4 cells is associated with reduction of MAT1 mRNA (47). Also, MAT1 protein is overexpressed in multiple solid tumor cell lines and leukemic cell lines (Fig. 2, C and D). These data therefore indicate that high levels of both MAT1 mRNA and MAT1 protein are associated with enhanced cell proliferation. Importantly, we consistently observe that a unique M30, likely derived from cleavage of MAT1, was produced along with MAT1 overexpression in tumor cell lines (Fig. 2, C and D). MAT1/M30 existing in proliferating cells but not in differentiating cells (Figs. 1 and 2, A and B) are immunoprecipitated together by CDK7 antibodies (Figs. 1A, 4C, and 5B). Thus, M30 may form a Delta CAK together with the exceeded CAK complexes formed by overexpressed MAT1 to alter CAK substrate specificity on RARalpha phosphorylation in these leukemic cells. Indeed, these high levels of MAT1/M30 were consistently associated with CAK hyperphosphorylation of RARalpha in the actively proliferating HL60 cells, but these levels were markedly diminished by ATRA stimuli and thus were associated with the reduced CAK phosphorylation of RARalpha in the differentiating cells (Figs. 1, and 2, A and B, and Fig. 5B). In HL60 cells containing RARalpha WT, MAT1 overexpression and in particular the M30 formation are markedly diminished by ATRA (Figs. 1A, 2A, 2B, and 5B) through a proteasome degradation pathway (Fig. 3). However, in clear contrast, there is no change of MAT1/M30 in the HL60R cells harboring RARalpha Delta AF-2 following exposure to ATRA (Fig. 4, A-C). Thus, ATRA-activated RARalpha appears to modulate these dynamic changes of MAT1/M30 levels directly. These observations therefore suggest that the RARalpha -dependent MAT1 levels, and in particular the levels of the M30, might be important in regulation of CAK activities on RARalpha phosphorylation and that the ATRA-mediated proteasome degradation of MAT1/M30 may be a critical event in down-regulation of CAK-RARalpha signaling that is associated with G1 arrest and differentiation activation of these leukemic cells.

Although ATRA-induced differentiation of leukemic cells is associated with degradation of RARalpha , we note that the pattern of RARalpha degradation either within or outside the CAK complex does not match the substrate stoichiometry of the gradually developing CAK hypophosphorylation of RARalpha (Figs. 1A and 5). In contrast, gradually inhibited MAT1/M30 by ATRA parallel gradually developed CAK hypophosphorylation of RARalpha (Figs. 1A and 5B). As CAK activities are known to be MAT1 dose-dependent (8, 18, 22, 44), these results therefore suggest that MAT1 reduction, rather than RARalpha degradation, may be a main factor to modulate CAK phosphorylation of RARalpha in ATRA-induced P/D transition.

The Significance of CAK Hypophosphorylation of RARalpha -- What is the significance of the diminished CAK phosphorylation of RARalpha that accompanies the ATRA-induced P/D transition in HL60 cells? Such decreased RARalpha phosphorylation might reflect a generalized decrease in CAK activity because the ATRA-induced cells proceed from actively proliferating to terminally differentiating. Indeed, as discussed above, the markedly decreased MAT1/M30 levels via the ATRA-activated protease pathway may reduce CAK activities on RARalpha phosphorylation. Alternatively, the decreased RARalpha phosphorylation might be more directly involved in regulating the molecular events that accompany terminal myeloid differentiation. This latter hypothesis might appear counterintuitive, because a previous study in Cos-1 cells indicated that CDK7 phosphorylation of RARalpha was associated with enhanced RARalpha transcriptional activity (23), and thus reduced phosphorylation of RARalpha predicts reduced RARalpha activity. However, such reduced activity might indeed occur as a negative feedback mechanism during terminal myeloid differentiation. Alternatively, the functional significance of RARalpha phosphorylation might be markedly different in hematopoietic cells versus Cos-1 cells.

In summary, we find that ATRA-induced cell cycle G1 arrest and differentiation activation of HL60 cells are associated with a markedly decreased CAK phosphorylation of RARalpha . An accompanying event is the ATRA-mediated degradation of MAT1/M30 via the proteasome pathway, which might play a critical role in modulating this decreased RARalpha phosphorylation by CAK. The potential role of MAT1/M30 in regulating CAK activity on RARalpha phosphorylation as well as the functional significance of the switch to CAK hypophosphorylation of RARalpha in coordinating G1 arrest and differentiation activation are currently being explored in our laboratory. The detailed molecular and biochemical pathways regarding MAT1-mediated CAK-RARalpha signaling in control of the transition from actively proliferating to terminally differentiating cells might provide a mechanistic insight into new approaches for leukemia therapy.

    ACKNOWLEDGEMENTS

We thank Drs. D. Kohn and M. Lanotte for providing the cell lines. We thank Dr. G. McNamara for assistance with digital microscopy.

    FOOTNOTES

* This work was supported by a research scholar grant from the American Cancer Society.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Dagger To whom correspondence should be addressed: Dept. of Pathology, Mail stop 103 Children's Hospital Los Angeles, University of Southern California Keck School of Medicine, 4650 Sunset Blvd., Los Angeles, CA 90027. Tel.: 323-660-2450 (Ext. 6318); Fax: 323-671-3669; E-mail: lingtaow@usc.edu.

Published, JBC Papers in Press, September 3, 2002, DOI 10.1074/jbc.M206792200

    ABBREVIATIONS

The abbreviations used are: CDK, cyclin-dependent kinase; P/D, proliferation/differentiation; CAK, cyclin-dependent kinase (CDK)-activating kinase; MAT1, ménage à trois 1; RARalpha , retinoic acid receptor alpha ; ATRA, all-trans retinoic acid; RARalpha Delta AF-2, truncated ligand-dependent AF-2 domain of RARalpha ; RARalpha WT, wild type RARalpha ; pRb, retinoblastoma tumor suppressor protein; TFIIH, transcription factor IIH.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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