CrkRS (Cdc2-related kinase Arg/Ser) or cyclin-dependent kinase 12 (CKD12) Ibutamoren (MK-677)

CrkRS (Cdc2-related kinase Arg/Ser) or cyclin-dependent kinase 12 (CKD12) Ibutamoren (MK-677) is a serine/threonine kinase thought to coordinate transcription and RNA splicing. CDK12/CrkRS but usually do not contain CDK9 a presumed partner of cyclin K. Evaluation of comprehensive RNA-Seq data implies that the 65-kDa cyclin K1 isoform may be the mostly expressed type across numerous tissues types. We also demonstrate that CDK12/CrkRS would depend on cyclin K1 because of its kinase activity which little interfering RNA (siRNA) knockdown of CDK12/CrkRS or cyclin K1 provides similar effects over the expression of the luciferase reporter gene. Our data claim that cyclin K1 is the main cyclin partner Ibutamoren (MK-677) for CDK12/CrkRS and that cyclin K1 is required to activate CDK12/CrkRS to phosphorylate the CTD of RNA Pol II. These properties are consistent with a role of CDK12/CrkRS in regulating gene expression through phosphorylation of RNA Pol II. INTRODUCTION Cyclin-dependent kinases (CDKs) regulate a variety of cellular processes including cell cycle progression transcription and RNA processing (52). Their activity requires heterodimeric interactions with specific cyclins where the CDK is the catalytic subunit and the cyclin is the regulatory subunit (52). CDKs/cyclins typically are the nexus of signal transduction pathways for the regulation of central cellular processes. Many of the ~20 human CDKs such as CDK1 -2 -3 -4 -5 -6 -10 and -11p58 which partner with users of the cyclin A B C D and E families are involved in cell cycle progression (3 43 52 Cyclins A B D and E which interact with CDK1 -2 -3 and -6 show fluctuating expression levels during different phases of the cell cycle (25). CDK7 -8 -9 and -11 are involved in transcriptional regulation (20). CDK9 CDK11p110 CDK12/CrkRS (Cdc2-related kinase Arg/Ser) and CDK13/CDC2L5 have been reported to have functions in both transcription and pre-mRNA processing (4 6 9 10 19 26 37 38 The multiple functions of CDKs are due in part to their interactions with different cyclin partners which modulate their localization and activity (15 42 67 Previous studies have exhibited a link between transcription and pre-mRNA processing (29 45 47 The multisubunit RNA polymerase II (RNA Pol II) links transcription and cotranscriptional maturation of pre-mRNA through differential recruitment of transcription and RNA processing factors to the C-terminal domain name (CTD) of its largest subunit Rbp1 (24 37 The integration of these two processes is usually tightly regulated by posttranslational modification of the CTD. CDK7 -8 and -9 differentially phosphorylate the amino acid residues in the heptad repeat of the CTD to regulate the activation of elongation during transcription. The CTD which in humans consists of 52 tandem heptaresidue repeats with a consensus sequence of Tyr1Ser2Pro3Thr4Ser5Pro6Ser7 serves as the acknowledgement platform for conversation with a variety of transcription factors cofactors and pre-mRNA processing factors (18 37 In mammals positive transcription elongation factor b (P-TEFb) complexes are composed of CDK9 as the catalytic subunit and one of the cyclins T1 T2a T2b or K (48). The ratio of two CDK9 isoforms CDK9p42 and CDK9p55 generated through alternate transcription start sites (56 57 is different in various tissues and changes when cells Ibutamoren (MK-677) are challenged by different stimuli (40 56 Both CDK9 isoforms interact Ibutamoren (MK-677) with cyclins T1 T2a/b and K; these different CDK9/cyclin heterodimers add cellular functional complexity to P-TEFb complexes (15 23 53 58 65 67 For example conversation of CDK9p42/cyclin T1 with the viral Tat protein facilitates the transactivation of the long terminal repeat of human PLA2G4F/Z immunodeficiency computer virus type 1 (HIV-1) and HIV-2 and is essential for productive viral replication (22). Recently cyclin K was found to inhibit HIV-1 gene expression and replication through its conversation with HIV-1 Nef (32). CDK9/cyclin T2 complexes interact with PKNα and Rho-activated Ser/Thr kinase and are involved in regulating terminal differentiation in muscle mass cells through activation of Myo-D Ibutamoren (MK-677) (12 58 CDK9/cyclin K complexes have also been proposed to function in replication stress response to maintain genome integrity (65). Thus the conversation of CDK9 with specific cyclins modulates Ibutamoren (MK-677) CDK9 kinase activity to effect different cellular outcomes (15 65 67 Cyclin K was originally identified as a 357-residue protein that restores cell cycle progression and rescues the lethality of deletions of the G1 cyclins CLN1 CLN2 and CLN3 in (17). Cyclin K was independently identified in a yeast two-hybrid screen for interactors of human CDK9 (23)..