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Retinoid-modulated MAT1 ubiquitination and CAK activity

QIAOJUN HE, HUI PENG, STEVEN J. COLLINS, TIMOTHY J. TRICHE^*, and LINGTAO WU^*,,1

^* Department of Pathology, University of Southern California Keck School of Medicine, Los Angeles, California, USA;
Childrens Hospital Los Angeles Saban Research Institute, Los Angeles, California, USA; and
Human Biology Division, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA

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

SPECIFIC AIMS

Human cyclin-dependent kinase (CDK)-activating kinase (CAK) consists of CDK7, cyclin H, and MAT1 (ménage à trois 1). CAK cross-regulates cell cycle and differentiation whereas MAT1 assembles CAK and determines CAK’s substrate specificity. How intracellular regulation of MAT1 controls CAK activity is unknown. Recent studies show that retinoic acid (RA)-induced HL60 cell proliferation/differentiation (P/D) transition is associated with MAT1 degradation and decreased CAK phosphorylation of retinoic acid receptor (RARa). Here we demonstrate that RA-induced MAT1 ubiquitination switches CAK hyperphosphorylation of RARa in proliferating cells to CAK hypophosphorylation of RARa in differentiating cells.

PRINCIPAL FINDINGS

1. RA induces ubiquitination-proteolysis of MAT1
HL60 and other RA-sensitive cells were exposed to RA for different periods with or without MG132 in the last 8 h of incubation. RA specifically induces ubiquitination-proteolysis of MAT1 in G₁ arresting and differentiating cells (Fig. 1 ).

View larger version (50K): [in this window] [in a new window]   Figure 1. RA induces ubiquitination-proteolysis of MAT1. A) ATRA induced MAT1 ubiquitination in HL60 cells. Ubiquitin-MAT1 conjugates and MAT1 were precipitated with MAT1 antibodies and separated on SDS-PAGE. The generated blot was analyzed by immunoblotting with anti-ubiquitin and anti-MAT1 antibodies. Addition of MG132 inhibited MAT1 degradation and allowed ubiquitinated MAT1 to accumulate to detectable levels (lanes 4–7 vs. 8–11). Accumulation of ubiquitinated MAT1 and the inhibition of MAT1 degradation were enhanced by increased doses of MG132 (lanes 14–16). PI, preimmune IgG; IP, immunoprecipitation; WB, Western blot. B) ATRA induced ubiquitinating enzyme activities as evidenced by the ladder of ubiquitinated general proteins in the precipitates (lanes 4–7), which corresponded well with the pattern of ubiquitinated MAT1 as shown in panel A, lanes 4–7. C) No CDK7 ubiquitination was detected in the precipitates. D, E) As described above (A), RA induced ubiquitination-proteolysis of MAT1 was also detected in NB4 (D) and CHP126 cells (E).

2. MAT1 is a substrate for ubiquitination
The purified recombinant MAT1 (rMAT1) was ubiquitinated in HL60 cell lysate. This rMAT1 ubiquitination was enhanced upon addition of RA. Furthermore, rMAT1 was ubiquitinated in rabbit reticulocyte lysate (RRL), an established source of ubiquitinating enzymes and proteasome complexes. rMAT1 ubiquitination in the RRL was enhanced by extended incubation time.

3. CAK-free MAT1 is a substrate for ubiquitination
MAT1 exists as either CAK-free or CAK-complexed MAT1. Ubiquitinated MAT1 was detected in precipitates containing CAK-free MAT1, which was precipitated by anti-MAT1 antibodies. In contrast, MAT1 ubiquitination was not detected in the precipitates containing only CAK-complexed MAT1 but without CAK-free MAT1, precipitated by CDK7 antibodies.

4. MAT1 ubiquitination decreases CAK abundance and inhibits CAK activity
MAT1 was decreased with RA treatment at both total protein levels and in the complexed precipitates. RA-induced MAT1 ubiquitination reduces CAK abundance, decreases CAK-bound RAR, and induces CAK hypophosphorylation of RAR. Addition of MG132 in the presence of RA for the last 8 h of incubation reverses these RA actions (Fig. 2 ).

View larger version (39K): [in this window] [in a new window]   Figure 2. Ubiquitination-proteolysis of MAT1 decreases CAK abundance and inhibits CAK phosphorylation of RAR. A) In contrast to steady levels of total CDK7, total MAT1 levels declined with ATRA treatment but were restored by addition of MG132 (lanes 5–7 vs. 2–4). Actin served as a loading control. The levels of CAK-complexed CDK7 resulting from anti-MAT1 precipitation represented a net CAK abundance in precipitates (lanes 11–16). Levels of both MAT1 and CDK7 in the precipitates declined upon ATRA treatment but were recovered by addition of MG132 (lanes 14–16 vs. 11–13). B) ATRA-induced MAT1 reduction and CAK hypophosphorylation of RAR (lane 3) were reversed by addition of MG132 (lane 4). MAT1 reduction was associated with decreased CAK-bound RAR (lane 3). C) Total phosphorylated RAR was decreased with ATRA treatment but restored by the addition of MG132. Total CDK7 levels were not affected by ATRA/MG132 treatment.

CONCLUSIONS AND SIGNIFICANCE

It was a surprise when constant CAK activity, initially expected to be cell cycle regulated, was later discovered throughout the cell cycle. We recently find that, in an identified RA-induced P/D transition characterized by a coordination of G₁ arrest and differentiation in HL60 cells, CAK hyperphosphorylation of RAR is switched to CAK hypophosphorylation of RAR. Since switch of CAK activity is accompanied by decreasing MAT1 expression and MAT1 fragmentation to a 30 kDa fragment (M30), this suggests that CAK activity may be modulated at P/D transition by RA-induced MAT1 degradation. Here we demonstrate that by using the characterized model system of HL60 cells undergoing RA-induced P/D transition, RA degrades MAT1 via the ubiquitin-proteasome pathway. In contrast to levels of cyclin H and CDK7, which remain steady at total protein levels but decrease in the complexed precipitates, MAT1 levels are decreased both at total protein levels and in the complexed precipitates. This indicates that RA-induced MAT1 ubiquitination may lead to a declined CAK assembly. Indeed, RA-induced ubiquitination-proteolysis of CAK-free MAT1 decreases CAK abundance, which in turn reduces the levels of CAK-bound RAR and decreases CAK phosphorylation of RAR. Inhibition of MAT1 ubiquitination by addition of MG132 restores levels of MAT1, CAK abundance, CAK-bound RAR, and CAK hyperphosphorylation of RAR. Previous studies demonstrate that 1) pRb is best known as a proliferation suppressor and a differentiation enhancer; 2) MAT1 reduction induces CAK hypophosphorylation of pRb and G₁ arrest; and 3) pRb hypophosphorylation is associated with decreased both MAT1 levels and CAK activity in RA-mediated P/D transition. Those data together with our studies presented here suggest that MAT1 ubiquitination may be causal to decreasing CAK phosphorylation of key differentiation regulators pRb and RAR in RA-mediated P/D transition (Fig. 3 ).

View larger version (19K): [in this window] [in a new window]   Figure 3. Schematic diagram. Ubiquitination-proteolysis of MAT1 modulates the effect of RA in decreasing CAK phosphorylation of differentiation regulators and inducing P/D transition.

How RA induces differentiation in proliferating cells remains largely unknown, even though these compounds are used clinically to induce cancer cell differentiation. Our findings here represent a novel mechanism of RA action by which MAT1 ubiquitination may modulate the effect of RA in decreasing CAK activity and inducing a switch from proliferation to differentiation. To further advance our understanding of MAT1 ubiquitination modification of CAK phosphorylation of RAR mediated by RA, it is necessary to identify ubiquitin ligase targeting MAT1 and determine how hypophosphorylated RAR modulates the effect of RA in inducing MAT1 ubiquitination.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-2182fje;

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