Retinoid-induced G1 Arrest and Differentiation Activation Are Associated with a Switch to Cyclin-dependent Kinase-activating Kinase Hypophosphorylation of Retinoic Acid Receptor α

Abstract

Cell cycle G 1 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 G 1 exit are presently unclear. We previously showed that MAT1 (mé nage à trois 1), an assembly factor and targeting subunit of the cyclin-dependent kinase (CDK)-activating kinase (CAK), modulates CAK activities to regulate G 1 exit. Here we find that the retinoid-induced G 1 arrest and differentiation activation of cultured human leukemic cells are associated with a switch to CAK hypophosphorylation of retinoic acid receptor ␣ (RAR␣) from CAK hyperphosphorylation of RAR␣. The switch to CAK hypophosphorylation of RAR␣ 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 RAR␣ do not demonstrate any changes in MAT1 levels or CAK phosphorylation of RAR␣ following ATRA stimuli, these biochemical changes appear to be mediated directly through RAR␣. These studies indicate that significant changes in MAT1 levels and CAK activities on RAR␣ phosphorylation accompany the ATRA-induced G 1 arrest and differentiation activation, which provide new insights to explore the inversely coordinated P/D transition at G 1 exit.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 G 1 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 G 1 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 ␣ (RAR␣) (23).Among the above substrates of CAK, RAR␣ is involved mainly in differentiation regulation. RAR␣ belongs to the superfamily of...

Retinoid-suppressed phosphorylation of RARα mediates the differentiation pathway of osteosarcoma cells

“…Similar to the observation made by the other group, OPG mRNA was inhibited within 24 h (Figure 2f) in the presence of RA (Jacobson et al, 2004). As RARa is a substrate for CAK Wang et al, 2002) and a transcription factor activated by RA, these results suggest that RA-suppressed CAK phosphorylation of RARa links CAK-dependent G 1 arrest to osteoblastic differentiation through transactivation of RA target genes.…”

“…RA-suppressed RARa phosphorylation is associated with decreased levels of CDK7 and increased expression of differentiation regulators Previous studies have shown that differentiating osteoblastic cells serve as a niche (microenvironment) to mediate granulopoiesis (Taichman and Emerson, 1994;Taichman et al, 1996;Mayack and Wagers, 2008), whereas RA-induced loss of CAK phosphorylation of RARa has a pivotal role in granulocytic differentiation of hematopoietic precursors (Wang et al, 2002(Wang et al, , 2006He et al, 2004;Luo et al, 2007). We therefore examined whether such CAK-RARa signaling also mediates osteoblastic differentiation.…”

“…It is known that cancer cell differentiation requires cell-cycle exit, by which at least two waves of signaling events are involved: induction of cell-cycle arrest and transition into transactivation of target genes that regulates differentiation (Studzinski and Harrison, 1999;Zhu and Skoultchi, 2001). As RAinduced cancer cell differentiation usually takes 3-7 days depending on the different cell types (Taneja et al, 1997;Wang et al, 2002Wang et al, , 2006Zhang et al, 2004), we assessed changes in gene expression from the induction of osteosarcoma cell-cycle G 1 arrest to differentiation induced by RA or S77A (Figures 1-3) in parallel. Therefore, instead of using one time point of duplicate RNA samples in microarray analysis, a single RNA sample was used for each progressive time point at 3 and 7 days, respectively; blank U2OS cells were used as control.…”

“…Proliferation and cell-cycle analysis Cell replication (Luo et al, 2007) and cell-cycle analysis (Wang et al, 2002) were performed as described before.…”

“…These studies suggest that changes in CAK phosphorylation of different substrates modulate cell-cycle G 1 exit and transcriptional control. Indeed, CAK hyperphosphorylates RARa in proliferating cancer cells; however, RA decreases CAK activity, induces cell-cycle G 1 arrest and suppresses CAK phosphorylation of RARa, leading to a transition from inhibition of proliferation to differentiation of several types of cancer cells (Crowe and Kim, 2002;Wang et al, 2002;He et al, 2004;Zhang et al, 2004). These studies indicate that RA links cell-cycle G 1 exit to transcriptional control of cell differentiation by inhibiting CAK phosphorylation of RARa in cancer cells.…”