Casein Kinase 2 Interacts with Cyclin-dependent Kinase 11 (CDK11) in Vivo and Phosphorylates Both the RNA Polymerase II Carboxyl-terminal Domain and CDK11 in Vitro

Abstract

The PITSLRE protein kinases, hereafter referred to as cyclin-dependent kinase 11 (CDK11) due to their association with cyclin L, are part of large molecular weight protein complexes that contain RNA polymerase II (RNAP II) as well as numerous transcription and RNA processing factors. Data presented here demonstrate that the influence of CDK11 p110 on transcription and splicing does not involve phosphorylation of the RNAP II carboxyl-terminal domain by CDK11 p110 . We have isolated a DRB-and heparin-sensitive protein kinase activity that co-purifies with CDK11 p110 after ion exchange and affinity purification chromatography. This protein kinase was identified as casein kinase 2 (CK2) by immunoblot and mass spectrometry analyses. In addition to the RNAP II carboxyl-terminal domain, CK2 phosphorylates the CDK11 p110 amino-terminal domain. These data suggest that CDK11 p110 isoforms participate in signaling pathways that include CK2 and that its function may help to coordinate the regulation of RNA transcription and processing events. Future experiments will determine how phosphorylation of CDK11 p110 by CK2 specifically affects RNA transcription and/or processing events.The complex biochemical events of transcription and RNA processing, resulting in the production of mature RNA transcripts, are now understood to be highly integrated and co-dependent processes (1). It is hypothesized that regulation of these events occurs through the active exchange of associated factors with the RNAP II 1 complex (2-4). This hypothesis is based upon identification of numerous positive and negative regulatory factors/complexes, influencing both transcription and RNA processing enzymes, in physical association with RNAP II. Many of these complexes exert their effects directly or indirectly through association with the RNAP II CTD. In mammals, the RNAP II CTD is composed of 52 heptapeptide repeats with the consensus sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser (YSPTSPS), which are essential for viability (5).The RNAP II CTD is heavily phosphorylated in vivo, and it is likely that sequential phosphorylation events, as well as phosphorylation of specific residues by specific protein kinases, help regulate transcript production. This model appears to fit much of the data coming from numerous laboratories and was recently proposed as the most likely means of coordinating the various steps of transcription, RNA processing, and mRNA export (6). Many protein kinases modify the RNAP II CTD. Several of the CTD kinases identified thus far are from the cyclin-dependent kinase family (CDKs) and include CDK1, CDK7, CDK8, and CDK9. In addition, another regulator of cell cycle events, casein kinase 2 (CK2), is known to phosphorylate a number of transcriptional proteins, including the RNAP II CTD and the RAP74 subunit of TFIIF (7, 8).Data from this laboratory and others demonstrate that the CDK11 p110 (PITSLRE) protein kinases associate with the cyclin L regulatory protein, bind directly to various splicing factors, and play a role in pre-mRNA splicing (9 -1...

CDK11 Complexes Promote Pre-mRNA Splicing

“…1C). This result is consistent with previous analyses of CDK11 p110 functional domains, which indicate that this same region of the amino-terminal domain is necessary for the majority of the protein-protein interactions between this protein kinase and other proteins (16,17,21), whereas the carboxylterminal half of the protein encodes the catalytic domain of the protein kinase that is most homologous to CDK2 (18).…”

“…The inclusion of the kinase-inactive form of the CDK11 p110 protein kinase in the in vitro kinase assay was essential for this experiment, since we have previously shown that the casein kinase 2 protein kinase interacts and co-purifies with CDK11 p110 (21). The inclusion of a kinaseinactive form of CDK11 p110 also eliminates the possibility that another co-purifying or contaminating protein kinase is responsible for the in vitro phosphorylation of the 9G8 substrate.…”

“…Previous work as well as unpublished work 3 has demonstrated that the CDK11 p110 protein kinase is essential for cell survival (16,17,21); thus, RNAi (22) was the method chosen due to the greater possibility so that the levels of CDK11 p110 could be reduced transiently in HeLa cells long enough to perform the phospholabeling experiments involving 9G8. As shown in Fig.…”

Orphan kinases turn eccentric

“…54 Thus, this class of cyclin/CDK pairs appears to be regulated by complex formation-induced stabilization of subunits. In contrast to most of the CDKs, which require phosphorylation of a conserved threonine in the T-loop, 42,50,55 CDK8 is proposed to be solely activated by interaction with its cyclin. 39 Additional mechanisms of regulation of this subclass of CDKs include autophosphorylation (CDK9, 11) 41 and activation-dependent dimerization (CDK11).…”

“…2,49 Cyclin-CDK pairs involved in transcriptional control and mRNA splicing, e.g., cyclin C/CDK8 and cyclin L/CDK11, appear to be mainly regulated by their assembly into large multi-protein complexes. [50][51][52] Via their C-terminal cyclin fold, "transcriptional" cyclins make additional interactions with the catalytic subunit, resulting in a more open conformation suitable to accommodate additional proteins. 41,42 The formation of these complexes usually stabilizes one of the partners, like the CDK subunit in the cyclin T1/CDK9 53 or the cyclin subunit in the cyclin C/CDK8 complex.…”