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The Cyclin H/Cdk7/Mat1 Kinase Activity Is Regulated by CK2 Phosphorylation of Cyclin H

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The Cyclin H/Cdk7/Mat1 Kinase Activity Is Regulated by CK2 Phosphorylation of Cyclin H Oncogene (2002) 21, 5031 – 5037 ª 2002 Nature Publishing Group All rights reserved 0950 – 9232/02 $25.00 www.nature.com/onc ORIGINAL PAPERS The cyclin H/cdk7/Mat1 kinase activity is regulated by CK2 phosphorylation of cyclin H Eberhard Schneider1,2, Sabine Kartarius1, Norbert Schuster1,3 and Mathias Montenarh*,1 1Medical Biochemistry and Molecular Biology, University of the Saarland, Building 44, D-66424, Homburg, Germany Cyclin dependent kinases are regulated by phosphoryla- an initiation of mitosis. A third phosphorylation at tion and dephosphorylation of the catalytic cdk subunits, Thr-160 is also essential for the activity of p34cdc2 and by assembly with specific cyclins and by specific inhibitor this phosphorylation is achieved by another cyclin molecules. Recently, it turned out that cyclins are also dependent kinase complex which is known as cdk phosphoproteins, which means that they are also activating kinase CAK. This kinase complex consists of potential targets for a regulation by phosphorylation three subunits: the regulatory subunit cyclin H, the and dephosphorylation. Here, we show that cyclin H was catalytic subunit cdk7 and a third protein factor, phosphorylated by protein kinase CK2. Like most other named Mat1. This third factor, which is unusual for CK2 substrates cyclin H was much better phosphorylated other cdk/cyclin-complexes is an assembly factor for by the CK2 holoenzyme than by the a-subunit alone. By the cyclin H/cdk7/Mat1 complex (Johnson and Walker, using point mutants derived from the cyclin H sequence 1999) and seems to be implicated in the regulation of we mapped the CK2 phosphorylation site at threonine substrate specificity. 315 at the C-terminal end of cyclin H. Phosphorylation In contrast to other cyclin dependent kinases levels of at this position had no influence on the assembly of the cyclin H, cdk7 and Mat1 subunits as well as the resulting cyclin H/cdk7/Mat1 complex. However, phosphorylation kinase activity do not greatly vary over the eukaryotic at amino acid 315 of cyclin H turned out to be critical cell cycle (Adamczewski et al., 1996; Fisher and for a full cyclin H/cdk7/Mat1 kinase activity when the Morgan, 1994, Fisher et al, 1995). The cyclin H/cdk7/ CTD peptide of RNA polymerase II or cdk2 was used as Mat1 complex is found either in a free state (Morgan, a substrate. 1995) or as a part of the transcription factor complex Oncogene (2002) 21, 5031 – 5037. doi:10.1038/sj.onc. TFIIH (Svejstrup et al., 1996). This TFIIH complex has 1205690 a central role in two processes: first as a basal factor in the regulation of the transcription of RNA polymerase Keywords: protein kinase CK2; cyclin dependent II and second as an essential component of the DNA kinases; phosphorylation regulation repair complex which is responsible for the nucleotide excision repair (Schaefer et al., 1993). Two classes of substrates for the cyclin H/cdk7/Mat1 complexes are Introduction known, namely downstream cyclin dependent kinases as well as components of the transcription machinery of Cell cycle progression is tightly regulated by cyclin the cell including the C-terminal domain (CTD) of the dependent kinases (cdks). The activity of these kinases large subunit of RNA polymerase II (Feaver et al., 1994) is controlled by phosphorylation and dephosphoryla- and the growth suppressor protein p53 (Ko et al., 1997; tion events, by binding of the regulatory subunits, Lu et al., 1997). Human cyclin H is a polypeptide of 323 namely the cyclins to the catalytic cdk-subunits, as well amino acids with a molecular weight of 38 kDa. When as by the interaction of inhibitor molecules with the cyclin H assembles with cdk7 the kinase is activated and cyclin/cdk-complexes (Kaldis, 1999). Cyclin dependent the activity is further enhanced by the third factor Mat1. kinases are phosphorylated on several sites including So far, only little is known about the regulation of cyclin two inhibitory and one activating site. The inhibitory H/cdk7/Mat1 activity. phosphorylation sites on human cdc2, Thr-14 and Tyr- We have recently shown that wild-type p53 binds to 15, keep the p34cdc2 kinase silent until dephosphoryla- cyclin H and this binding leads to a down-regulation of tion by cdc25C activates p34cdc2, which goes along with the kinase activity in vitro and in vivo (Schneider et al., 1998). p53 is a growth suppressor and a regulator of cell cycle progression. Upon DNA damage and after *Correspondence: M Montenarh; E-mail: [email protected] reduction of the nucleotide pool p53 transactivates a Current addresses: 2Phylos GmbH, Industrial Park Hoechst, Building number of different genes including the p21WAF1 gene. G 830, 65926 Frankfurt, Germany; E-mail: Eberhard.Schneider@x- WAF1 3 Elevated transcription of the p21 leads to an zillion.com; Ludwig Institute for Cancer Research, Box 595, 75124 WAF1 Uppsala, Sweden increase in the concentration of the p21 protein Received 17 January 2002; revised 12 May 2002; accepted 20 May which is an efficient inhibitor of cyclin dependent 2002 kinases (El-Deiry et al., 1993). Thus, p53 regulates CK2 phosphorylation of cyclin H E Schneider et al 5032 cyclin dependent kinases indirectly by transactivating strates the amount of cyclin H which was present in an inhibitor molecule for these kinases. An alternative each lane of Figure 1a. As shown in Figure 1a (lane 1) way to explain a p53 dependent cell cycle arrest cyclin H was efficiently phosphorylated by the CK2 independent of p21 might exist in form of the observed holoenzyme and weaker phosphorylated by the CK2 a- p53/CAK interaction. subunit alone (lane 3). Phosphorylation of cyclin H by Recently, it was shown that cyclin H is a the CK2 holoenzyme as well as the CK2 a-subunit was phosphoprotein and one of the kinases that phosphor- inhibited by heparin (lanes 2 and 4). This shows that ylate cyclin H was identified as cyclin C/cdk8. This cyclin H is in fact a substrate of CK2 and behaves very enzyme phosphorylates cyclin H at residues Ser-5 and similar to the majority of other CK2 substrates. Lanes Ser-304 both in vitro and in vivo (Akoulitchev et al., 5 and 6 are controls where cyclin H was incubated with 2000). These two phosphorylation sites are in the [g-32P]ATP in the absence of added CK2 in order to vicinity of N- and C-terminal helices which are demonstrate the absence of a protein kinase in the uniquely orientated in cyclin H as compared with cyclin H preparation. As a control for the correct other cyclins (Andersen et al., 1997). Phosphorylation separation of cyclin H by SDS polyacrylamide gel of cyclin H in the TFIIH complex results in inhibition electrophoresis we used bacterially expressed recombi- of its transcription activity whereas when Ser-5 and nant cyclin H which was phosphorylated with protein Ser-304 were substituted by alanine transcription was kinase A (heart muscle kinase, Sigma) within an not inhibited. Here, we show that cyclin H is a artificial PKA-site at the N-terminus of the cyclin H substrate for another protein kinase namely protein (lane 7). kinase CK2. Furthermore, we identified the phosphor- By scanning the polypeptide chain of cyclin H for ylation site for CK2 at position 315 on the polypeptide potential phosphorylation sites five different sequences chain of cyclin H and analysed the influence of this were found as putative CK2 phosphorylation sites phosphorylation on the activity of the cyclin H/cdk7/ (marked by P in Figure 2). Taking into consideration Mat1 complex. Using point mutants of cyclin H the consensus sequence for CK2 phosphorylation and (T315D and T315A) mimicking phosphorylated or the fact that the presence of additional Asp or Glu unphosphorylated cyclin H, respectively, we analysed residues at positions 73,+1,+2,+4,+5 or +7 the kinase activity of the cyclin H/cdk7/Mat1 complex. relative to the Ser or Thr residues which are Based on these data we concluded that the phosphor- phosphorylated improves phosphorylation efficacy ylation of cyclin H at Thr-315 by CK2 seems to be (Marin et al., 1994; Meggio et al., 1994) all points to necessary for full kinase activity of the cyclin H/cdk7/ T315 as the preferred target site for phosphorylation. Mat1 complex with respect to the phosphorylation of Threonine 315 is exceptional out of the five putative the CTD peptide derived from the C-terminal domain CK2 sites because it is immersed within an acidic of RNA polymerase II as well as the phosphorylation of cdk2. Results Human cyclin H is a phosphoprotein of 323 amino acids with a molecular weight of 38 kDa. Analysis of the primary sequence of cyclin H revealed the existence of several potential phosphorylation sites for protein kinase CK2. Protein kinase CK2 is a holoenzyme composed of two regulatory b-subunits and two catalytic a-ora’-subunits. Most of the CK2 substrates are efficiently phosphorylated only by the CK2 holoenzyme whereas a few substrates are phosphory- lated by the catalytic a-subunit alone (for review see: Guerra and Issinger, 1999b). Therefore, we first analysed whether cyclin H is in vitro a substrate of CK2 and second whether cyclin H is phoshorylated by the a-subunit alone or by the holoenzyme composed of Figure 1 Cyclin H is phosphorylated by the catalytic a-subunit a-andb-subunits. Bacterially expressed recombinant of CK2 and by the holoenzyme. (a) Purified cyclin H was incu- cyclin H was purified and then incubated with the a- bated with the CK2 holoenzyme (1,2) consisting of a- and b-sub- units or with the a-subunit of CK2 (3,4) and with [g-32P]ATP in subunit and [g-32P]ATP or with the holoenzyme and 32 (b), (c) the presence (even lanes) or absence (odd lanes) of [g- P]ATP in the absence or presence of heparin, an 0.5 mg heparin.
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