title: CDK2 regulates HIV-1 transcription by phosphorylation of CDK9 on serine 90.
abstract: BACKGROUND
  HIV-1 transcription is activated by the viral Tat protein that recruits host positive transcription elongation factor-b (P-TEFb) containing CDK9/cyclin T1 to the HIV-1 promoter. P-TEFb in the cells exists as a lower molecular weight CDK9/cyclin T1 dimer and a high molecular weight complex of 7SK RNA, CDK9/cyclin T1, HEXIM1 dimer and several additional proteins. Our previous studies implicated CDK2 in HIV-1 transcription regulation. We also found that inhibition of CDK2 by iron chelators leads to the inhibition of CDK9 activity, suggesting a functional link between CDK2 and CDK9. Here, we investigate whether CDK2 phosphorylates CDK9 and regulates its activity.
  
  
  RESULTS
  The siRNA-mediated knockdown of CDK2 inhibited CDK9 kinase activity and reduced CDK9 phosphorylation. Stable shRNA-mediated CDK2 knockdown inhibited HIV-1 transcription, but also increased the overall level of 7SK RNA. CDK9 contains a motif (90SPYNR94) that is consensus CDK2 phosphorylation site. CDK9 was phosphorylated on Ser90 by CDK2 in vitro. In cultured cells, CDK9 phosphorylation was reduced when Ser90 was mutated to an Ala. Phosphorylation of CDK9 on Ser90 was also detected with phospho-specific antibodies and it was reduced after the knockdown of CDK2. CDK9 expression decreased in the large complex for the CDK9-S90A mutant and was correlated with a reduced activity and an inhibition of HIV-1 transcription. In contrast, the CDK9-S90D mutant showed a slight decrease in CDK9 expression in both the large and small complexes but induced Tat-dependent HIV-1 transcription. Molecular modeling showed that Ser 90 of CDK9 is located on a flexible loop exposed to solvent, suggesting its availability for phosphorylation.
  
  
  CONCLUSIONS
  Our data indicate that CDK2 phosphorylates CDK9 on Ser 90 and thereby contributes to HIV-1 transcription. The phosphorylation of Ser90 by CDK2 represents a novel mechanism of HIV-1 regulated transcription and provides a new strategy for activation of latent HIV-1 provirus.
authors.affiliation: Center for Sickle Cell Disease, Department of Medicine, Howard University, 1840 7th Street, N.W. HURB1, Suite 202, Washington, DC 20001, USA.
authors.forename: Denitra
authors.initials: D
authors.lastname: Breuer
authors.forename: Alexander
authors.initials: A
authors.lastname: Kotelkin
authors.forename: Tatiana
authors.initials: T
authors.lastname: Ammosova
authors.forename: Namita
authors.initials: N
authors.lastname: Kumari
authors.forename: Andrey
authors.initials: A
authors.lastname: Ivanov
authors.forename: Andrey V
authors.initials: AV
authors.lastname: Ilatovskiy
authors.forename: Monique
authors.initials: M
authors.lastname: Beullens
authors.forename: Philip R
authors.initials: PR
authors.lastname: Roane
authors.forename: Mathieu
authors.initials: M
authors.lastname: Bollen
authors.forename: Michael G
authors.initials: MG
authors.lastname: Petukhov
authors.forename: Fatah
authors.initials: F
authors.lastname: Kashanchi
authors.forename: Sergei
authors.initials: S
authors.lastname: Nekhai
mesh_terms: D002460:Cell Line; D051357:Cyclin-Dependent Kinase 2 / Q000235:genetics / Q000378:metabolism*; D042863:Cyclin-Dependent Kinase 9 / Q000737:chemistry / Q000378:metabolism*; D004789:Enzyme Activation / Q000235:genetics; D015967:Gene Expression Regulation, Viral; D020868:Gene Silencing; D015497:HIV-1 / Q000235:genetics*; D006801:Humans; D008958:Models, Molecular; D009154:Mutation; D010766:Phosphorylation; D042925:Positive Transcriptional Elongation Factor B / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D034622:RNA Interference; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism; D012694:Serine / Q000737:chemistry; D014158:Transcription, Genetic
chemical_list: D012367:RNA, Viral; D012694:Serine; D042925:Positive Transcriptional Elongation Factor B; D051357:Cyclin-Dependent Kinase 2; D042863:Cyclin-Dependent Kinase 9