Journal of Cell Science, Supplem ent 16, 77-85 (1992) Printed in G reat B ritain © T he Com pany o f Biologists Lim ited 1992
77
Cyclin A recruits p33cdk2Xo the cellular transcription factor DRTF1
LASANTHA R. BANDARA1, JORG P. ADAMCZEWSKI2, MARYAM ZAMANIAN1, RANDY Y. C. POON2, TIM HUNT2 and NICHOLAS B. LA THANGUE1-* 1Laboratory o f Eukaryotic M olecular Genetics, M R C National Institute fo r M edical Research, The Ridgeway, M ill Hill, London, NW 7 1AA, UK 2Im perial Cancer Research Fund, Clare H all Laboratories, Blanche Lane, South Mimms, Potters Bar, Herts., EN6 3LD, UK ♦Corresponding author
Summary Cyclins are regulatory molecules that undergo periodic accumulation and destruction during each cell cycle. By activating p34cdc2 and related kinase subunits they con trol important events required for normal cell cycle progression. Cyclin A, for example, regulates at least two distinct kinase subunits, the mitotic kinase subunit p34cdc2 and related subunit p33cdk2, and is widely believed to be necessary for progression through S phase. However, cyclin A also forms a stable complex with the cellular transcription factor DRTF1 and thus may perform other functions during S phase. DRTF1, in addition, associates with the tumour suppressor retinoblastoma (Rb) gene product and the Rb-related protein p l0 7 . We now show, using biologically active fusion proteins, that cyclin A can direct the binding of
the cdc2-like kinase subunit, p33cdk2, to complexed DRTF1, containing either Rb or p l07, as well as acti vate its histone H I kinase activity. Cyclin A cannot, however, direct p34cdc2 to the DRTF1 complex and we present evidence suggesting that the stability of the cyclin \- p 3 3 cdk2 complex is influenced by DRTF1 or an associated protein. Cyclin A, therefore, serves as an acti vating and targeting subunit of p33cdk2. The ability of cyclin A to activate and recruit p33cdk2 to DRTF1 may play an important role in regulating cell cycle progres sion and moreover defines a mechanism for coupling cell-cycle events to transcriptional initiation.
Introduction
progression through S phase. In Xenopus egg extracts, p33«/a or a ciosejy related kinase subunit is required for DNA replication (Fang and Newport, 1991) and is believed to fulfill a similar role in other types of cells (Pines and Hunter, 1991). Moreover, inactivation of cyclin A, either through antibody or antisense approaches, prevents cells from completing S phase (Girard et al., 1991). It is likely, therefore, that cyclin A regulates the kinase activity of p33Citt2 which in turn modifies protein substrates that are necessary for cell-cycle progression. The retinoblastoma (Rb) gene product is a negative reg ulator of the cell cycle that acts by controlling progression through Gi (Goodrich et al., 1991). This effect is widely believed to result from the activity of the un- or under-phosphorylated Rb protein, because this form of the protein pre dominates during Gi (Buchkovich et al., 1989; DeCaprio et al., 1989). Certain viral oncoproteins, such as adenovirus E l a, SV40 large T antigen and HPV E7 bind the Rb pro tein (Whyte et al., 1988; DeCaprio et al., 1988; Dyson et al., 1989) and the Rb gene is frequently mutated in tumour cells (Hu et al., 1990; Huang et al., 1990). Both of these processes are thought to inactivate the growth suppressing properties of the Rb protein by preventing it from regulat ing cellular targets. Moreover, the Rb-related protein, pl07,
Cyclins are an evolutionarily conserved group of regulatory molecules that undergo periodic accumulation and destruc tion during the cell cycle and are required for normal cell cycle progression (Hunt, 1989). They were initially identi fied in marine invertebrates (Evans et al., 1983), and act by binding to and regulating the activity o f the mitotic kinase catalytic subunit p34c* 2 or related kinase subunits (Pines and Hunter, 1990a). Cyclins, which have been isolated and characterised from a variety of sources, are grouped into several classes. Cyclins of the B class, which function at mitosis, peak towards M phase and are responsible for acti vating the mitotic kinase subunit p34e* 2 (Draetta et al., 1989; Minshull et al., 1990). Cyclin A, however, accumu lates somewhat earlier (Giordano et al., 1989; Minshull et al., 1990; Pines and Hunter, 1990b) and in addition to p34cdc2 activates the related kinase subunit p33alk2 (Pines arid Hunter, 1990b; Tsai et al., 1991). Other cyclins have recently been defined which are thought to regulate pro gression through earlier phases of the cell cycle (Matsushime et al., 1991; Xiong et al., 1991; Lew et al., 1991; Koff et al., 1991). Several reports have suggested that cyclin A regulates
Key words: cyclin, transcription, retinoblastoma, CDK, tumour suppressor.
78
L. R. B andara a nd others
is also sequestered by adenovirus E la and SV40 large T antigen (Whyte et al., 1988; DeCaprio et al., 1988) and, although there is no detailed information available yet on its regulation during the cell cycle, is likely to have an important role in cell-cycle control (Ewen et al., 1991). It is thought that the Rb protein mediates its growth reg ulating properties by modulating the activity of molecules that regulate progression through the cell cycle, but only recently have potential candidates been identified (Bandara and La Thangue, 1991; Bagchi et al., 1991; Chellapan et al., 1991; Chittenden et al., 1991; Defeo-Jones et al., 1991). A particularly good candidate is the cellular transcription factor DRTF1 which is found in complexes that contain the Rb protein (Bandara and La Thangue, 1991). DRTF1, first defined in F9 embryonal carcinoma (EC) stem cells as a differentiation-regulated transcription factor (La Thangue and Rigby, 1987), is mostly uncomplexed in F9 EC cells, but in other cell-types forms a stable complex with the Rb protein (Bandara and La Thangue, 1991). The Rb-DRTFl complex is dissociated by Ela, large T antigen and E7 pro teins (Bandara and La Thangue, 1991; Bandara and La Thangue, data not shown), which releases the transcrip tionally active protein, the form that predominates in stem cells. Moreover, the protein products of all naturally occur ring mutant Rb alleles so far studied fail to bind stably to DRTF1 (Bandara et al., 1991; Bandara and La Thangue, data not shown), underscoring the potential importance of DRTF1 in controlling cellular proliferation. Recent experiments have shown that the Rb protein can specifically repress the transcription of promoters driven by DRTF1, whereas proteins encoded by some naturallyoccurring mutant Rb alleles, which fail to bind DRTF1 in vitro, cannot. Furthermore, Rb-mediated transcriptional repression is relieved by the adenovirus E la protein (Zamanian and La Thangue, 1992). The Rb protein, there fore, represses the transcriptional activity of DRTF1 which thus provides a potential mechanistic explanation for how the Rb protein exerts its growth regulating properties, given that DRTF1 binding sites occur in the promoters of genes that are necessary for cell cycle progression (Blake and Azizkhan, 1989; Pearson et al., 1991). DRTF1 binds to a similar DNA sequence to that of the HeLa cell transcription factor E2F, and although the exact relationship between these two transcription factors has yet to be established, they are likely to be related because sim ilar interactions have been defined for E2F (Bagchi et al., 1990; Chellappan et al., 1991). Cyclin A also binds to DRTF1 (Bandara et al., 1991) and previously we have suggested that this allows cell cycle events to be coupled to transcription. We have developed an in vitro assay in which cyclin A efficiently binds to DRTF1 and established that cyclin A can direct the cdc2like kinase subunit, p33c
77
Cyclin A recruits p33cdk2Xo the cellular transcription factor DRTF1
LASANTHA R. BANDARA1, JORG P. ADAMCZEWSKI2, MARYAM ZAMANIAN1, RANDY Y. C. POON2, TIM HUNT2 and NICHOLAS B. LA THANGUE1-* 1Laboratory o f Eukaryotic M olecular Genetics, M R C National Institute fo r M edical Research, The Ridgeway, M ill Hill, London, NW 7 1AA, UK 2Im perial Cancer Research Fund, Clare H all Laboratories, Blanche Lane, South Mimms, Potters Bar, Herts., EN6 3LD, UK ♦Corresponding author
Summary Cyclins are regulatory molecules that undergo periodic accumulation and destruction during each cell cycle. By activating p34cdc2 and related kinase subunits they con trol important events required for normal cell cycle progression. Cyclin A, for example, regulates at least two distinct kinase subunits, the mitotic kinase subunit p34cdc2 and related subunit p33cdk2, and is widely believed to be necessary for progression through S phase. However, cyclin A also forms a stable complex with the cellular transcription factor DRTF1 and thus may perform other functions during S phase. DRTF1, in addition, associates with the tumour suppressor retinoblastoma (Rb) gene product and the Rb-related protein p l0 7 . We now show, using biologically active fusion proteins, that cyclin A can direct the binding of
the cdc2-like kinase subunit, p33cdk2, to complexed DRTF1, containing either Rb or p l07, as well as acti vate its histone H I kinase activity. Cyclin A cannot, however, direct p34cdc2 to the DRTF1 complex and we present evidence suggesting that the stability of the cyclin \- p 3 3 cdk2 complex is influenced by DRTF1 or an associated protein. Cyclin A, therefore, serves as an acti vating and targeting subunit of p33cdk2. The ability of cyclin A to activate and recruit p33cdk2 to DRTF1 may play an important role in regulating cell cycle progres sion and moreover defines a mechanism for coupling cell-cycle events to transcriptional initiation.
Introduction
progression through S phase. In Xenopus egg extracts, p33«/a or a ciosejy related kinase subunit is required for DNA replication (Fang and Newport, 1991) and is believed to fulfill a similar role in other types of cells (Pines and Hunter, 1991). Moreover, inactivation of cyclin A, either through antibody or antisense approaches, prevents cells from completing S phase (Girard et al., 1991). It is likely, therefore, that cyclin A regulates the kinase activity of p33Citt2 which in turn modifies protein substrates that are necessary for cell-cycle progression. The retinoblastoma (Rb) gene product is a negative reg ulator of the cell cycle that acts by controlling progression through Gi (Goodrich et al., 1991). This effect is widely believed to result from the activity of the un- or under-phosphorylated Rb protein, because this form of the protein pre dominates during Gi (Buchkovich et al., 1989; DeCaprio et al., 1989). Certain viral oncoproteins, such as adenovirus E l a, SV40 large T antigen and HPV E7 bind the Rb pro tein (Whyte et al., 1988; DeCaprio et al., 1988; Dyson et al., 1989) and the Rb gene is frequently mutated in tumour cells (Hu et al., 1990; Huang et al., 1990). Both of these processes are thought to inactivate the growth suppressing properties of the Rb protein by preventing it from regulat ing cellular targets. Moreover, the Rb-related protein, pl07,
Cyclins are an evolutionarily conserved group of regulatory molecules that undergo periodic accumulation and destruc tion during the cell cycle and are required for normal cell cycle progression (Hunt, 1989). They were initially identi fied in marine invertebrates (Evans et al., 1983), and act by binding to and regulating the activity o f the mitotic kinase catalytic subunit p34c* 2 or related kinase subunits (Pines and Hunter, 1990a). Cyclins, which have been isolated and characterised from a variety of sources, are grouped into several classes. Cyclins of the B class, which function at mitosis, peak towards M phase and are responsible for acti vating the mitotic kinase subunit p34e* 2 (Draetta et al., 1989; Minshull et al., 1990). Cyclin A, however, accumu lates somewhat earlier (Giordano et al., 1989; Minshull et al., 1990; Pines and Hunter, 1990b) and in addition to p34cdc2 activates the related kinase subunit p33alk2 (Pines arid Hunter, 1990b; Tsai et al., 1991). Other cyclins have recently been defined which are thought to regulate pro gression through earlier phases of the cell cycle (Matsushime et al., 1991; Xiong et al., 1991; Lew et al., 1991; Koff et al., 1991). Several reports have suggested that cyclin A regulates
Key words: cyclin, transcription, retinoblastoma, CDK, tumour suppressor.
78
L. R. B andara a nd others
is also sequestered by adenovirus E la and SV40 large T antigen (Whyte et al., 1988; DeCaprio et al., 1988) and, although there is no detailed information available yet on its regulation during the cell cycle, is likely to have an important role in cell-cycle control (Ewen et al., 1991). It is thought that the Rb protein mediates its growth reg ulating properties by modulating the activity of molecules that regulate progression through the cell cycle, but only recently have potential candidates been identified (Bandara and La Thangue, 1991; Bagchi et al., 1991; Chellapan et al., 1991; Chittenden et al., 1991; Defeo-Jones et al., 1991). A particularly good candidate is the cellular transcription factor DRTF1 which is found in complexes that contain the Rb protein (Bandara and La Thangue, 1991). DRTF1, first defined in F9 embryonal carcinoma (EC) stem cells as a differentiation-regulated transcription factor (La Thangue and Rigby, 1987), is mostly uncomplexed in F9 EC cells, but in other cell-types forms a stable complex with the Rb protein (Bandara and La Thangue, 1991). The Rb-DRTFl complex is dissociated by Ela, large T antigen and E7 pro teins (Bandara and La Thangue, 1991; Bandara and La Thangue, data not shown), which releases the transcrip tionally active protein, the form that predominates in stem cells. Moreover, the protein products of all naturally occur ring mutant Rb alleles so far studied fail to bind stably to DRTF1 (Bandara et al., 1991; Bandara and La Thangue, data not shown), underscoring the potential importance of DRTF1 in controlling cellular proliferation. Recent experiments have shown that the Rb protein can specifically repress the transcription of promoters driven by DRTF1, whereas proteins encoded by some naturallyoccurring mutant Rb alleles, which fail to bind DRTF1 in vitro, cannot. Furthermore, Rb-mediated transcriptional repression is relieved by the adenovirus E la protein (Zamanian and La Thangue, 1992). The Rb protein, there fore, represses the transcriptional activity of DRTF1 which thus provides a potential mechanistic explanation for how the Rb protein exerts its growth regulating properties, given that DRTF1 binding sites occur in the promoters of genes that are necessary for cell cycle progression (Blake and Azizkhan, 1989; Pearson et al., 1991). DRTF1 binds to a similar DNA sequence to that of the HeLa cell transcription factor E2F, and although the exact relationship between these two transcription factors has yet to be established, they are likely to be related because sim ilar interactions have been defined for E2F (Bagchi et al., 1990; Chellappan et al., 1991). Cyclin A also binds to DRTF1 (Bandara et al., 1991) and previously we have suggested that this allows cell cycle events to be coupled to transcription. We have developed an in vitro assay in which cyclin A efficiently binds to DRTF1 and established that cyclin A can direct the cdc2like kinase subunit, p33c
