Identification of a Cyclin T-Binding Domain in Hexim1 and Biochemical Analysis of Its Binding Competition with HIV-1 Tat

Schulte, Antje; Czudnochowski, Nadine; Barborič, Matjaž; Schönichen, André; Blažek, Dalibor; Peterlin, B. Matija; Geyer, Matthias

doi:10.1074/jbc.m501431200

Cited by 100 publications

(129 citation statements)

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“…This result differs from studies of budding yeast cyclins 52 or the Cdk2/ CycA complex and the substrate recognition site on CycA, which was identified to interact with the RxL motif of the substrate Cdc6 about 20 residues downstream of the phosphorylation site 22 . The corresponding surface patch on the first cyclin box repeat of CycT1 is covered both by P-TEFb-activating factors, such as Tat 33,53 , and by inhibitory factors, such as Hexim 30,31 , which do not directly contribute to substrate binding as shown here. However, co-factors of P-TEFb as shown for AFF4 of the super elongation complex could stimulate its catalytic activity or might potentially even change its substrate recognition profile.…”

Section: Y S P T S P S Y S P T S P S Y S P T S P Smentioning

confidence: 70%

“…7a). This effect is supposed to occur by displacement of Hexim1 from its binding site on CycT1, as no ternary complex between these two P-TEFb regulating proteins with CycT1 is formed 30,31 . An indication for a change or weakening of the P-TEFb substrate specificity towards both Ser2 and Ser5 phosphorylation 32,33 , for example, by means of increasing numbers of phosphorylation, was not observed.…”

Section: Hiv-1 Tat/tar Does Not Change P-tefb Phosphorylationsmentioning

confidence: 99%

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Serine-7 but not serine-5 phosphorylation primes RNA polymerase II CTD for P-TEFb recognition

2012

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Phosphorylation of RnA polymerase II carboxy-terminal domain (CTD) in hepta-repeats YsPTsPs regulates eukaryotic transcription. Whereas ser5 is phosphorylated in the initiation phase, ser2 phosphorylation marks the elongation state. Here we show that the positive transcription elongation factor P-TEFb is a ser5 CTD kinase that is unable to create ser2/ser5 double phosphorylations, while it exhibits fourfold higher activity on a CTD substrate prephosphorylated at ser7 compared with the consensus hepta-repeat or the YsPTsPK variant. mass spectrometry reveals an equal number of phosphorylations to the number of heptarepeats provided, yet the mechanism of phosphorylation is distributive despite the repetitive nature of the substrate. Inhibition of P-TEFb activity is mediated by two regions in Hexim1 that act synergistically on Cdk9 and Cyclin T1. HIV-1 Tat/TAR abrogates Hexim1 inhibition to stimulate transcription of viral genes but does not change the substrate specificity. Together, these results provide insight into the multifaceted pattern of CTD phosphorylation.

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Section: Y S P T S P S Y S P T S P S Y S P T S P Smentioning

confidence: 70%

Section: Hiv-1 Tat/tar Does Not Change P-tefb Phosphorylationsmentioning

confidence: 99%

Serine-7 but not serine-5 phosphorylation primes RNA polymerase II CTD for P-TEFb recognition

2012

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“…HEXIM1 and Tat interact with CycT1 in a region that is immediately C-terminal to the cyclin box (24)(25)(26). Previous studies have shown that the interactions of Tat and HEXIM1 with CycT1 are mutually exclusive (20).…”

Section: Aff1-p-tefb Interaction Facilitatesmentioning

confidence: 99%

AFF1 is a ubiquitous P-TEFb partner to enable Tat extraction of P-TEFb from 7SK snRNP and formation of SECs for HIV transactivation

Xue

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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The positive transcription elongation factor b (P-TEFb) stimulates RNA polymerase elongation by inducing the transition of promoter proximally paused polymerase II into a productively elongating state. P-TEFb itself is regulated by reversible association with various transcription factors/cofactors to form several multisubunit complexes [e.g., the 7SK small nuclear ribonucleoprotein particle (7SK snRNP), the super elongation complexes (SECs), and the bromodomain protein 4 (Brd4)-P-TEFb complex] that constitute a P-TEFb network controlling cellular and HIV transcription. These complexes have been thought to share no components other than the core P-TEFb subunits cyclin-dependent kinase 9 (CDK9) and cyclin T (CycT, T1, T2a, and T2b). Here we show that the AF4/FMR2 family member 1 (AFF1) is bound to CDK9-CycT and is present in all major P-TEFb complexes and that the tripartite CDK9-CycT-AFF1 complex is transferred as a single unit within the P-TEFb network. By increasing the affinity of the HIV-encoded transactivating (Tat) protein for CycT1, AFF1 facilitates Tat's extraction of P-TEFb from 7SK snRNP and the formation of Tat-SECs for HIV transcription. Our data identify AFF1 as a ubiquitous P-TEFb partner and demonstrate that full Tat transactivation requires the complete SEC.T ranscription by RNA polymerase II (Pol II) is a dynamic process consisting of several distinct but interconnected stages. Over the past three decades, much attention has been focused on the initiation and preinitiation stages of the transcription cycle, because they had been thought to be the principal points at which transcription is controlled (1, 2). Since 2007, however, accumulating evidence has revealed that promoter-proximal pausing of Pol II during early elongation is much more prevalent than previously thought, suggesting that intricate control of gene expression can occur frequently at this stage also (3, 4). Indeed, the importance of controlled pause and release of Pol II is illustrated by the observations that this process plays a prominent role in regulating cell growth, renewal, and differentiation (5, 6).Transcription of the integrated HIV-1 proviral genome is hypersensitive to elongation defects, thus making it an ideal model for elucidating the mechanism and factors that control elongation. It has long been known that in the absence of the HIVencoded transactivating protein (Tat), Pol II can initiate transcription from the viral promoter efficiently but pauses soon after the synthesis of a short RNA segment that folds into a stem-loop structure termed the "transactivation-response" (TAR) element. Tat overcomes Pol II pausing by recruiting the host positive transcription elongation factor b (P-TEFb) to the newly formed TAR RNA to stimulate the production of full-length HIV transcripts (7,8). Containing cyclin-dependent kinase 9 (CDK9) and cyclin T1 (CycT1), P-TEFb triggers the release of paused Pol II by phosphorylating and thereby antagonizing the inhibitory actions of two negative elongation factors, DSIF and NELF (5, 6). P-...

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“…HEXIM1 along with 7SK RNA was shown to be essential for inactivation of the P-TEFb complex (26,27). Reportedly, the HEXIM1-7SK RNA complex physically competes with Tat for binding to P-TEFb (28). Recently, CycK has been also shown to inhibit viral replication in a T-cell line by inhibiting LTR-mediated transcription (29), although the mechanism of inhibition remains to be elucidated.…”

mentioning

confidence: 99%

Cyclin K Inhibits HIV-1 Gene Expression and Replication by Interfering with Cyclin-dependent Kinase 9 (CDK9)-Cyclin T1 Interaction in Nef-dependent Manner

Khan

Mitra

2011

Journal of Biological Chemistry

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Human immunodeficiency virus type-1 (HIV-1) perpetuates its survival in the host by utilizing multiple strategies such as irreversible integration of its DNA into the host cell genome to establish the provirus (1), diversification of its genome to escape or tolerate adaptive immune responses (2), and use of the accessory genes (nef, vif, vpu, and vpr) to modulate host cell immune responses further (3-5). These viral accessory genes are frequently seen as dispensable in many in vitro cell culture systems but seem to be indispensable, in the context of natural infections in vivo. Among these accessory genes, pleiotropic functions of Nef have been studied extensively. It is a 27-30-kDa, N-terminally myristoylated protein having a well characterized CD4 down-regulatory activity (6). Nef does not possess any enzymatic activity, but it modulates diverse signaling pathways and the gene expression of host cell proteins (7,8). Furthermore, Nef influences the activation state of the host cell by bringing together different host cell proteins, protein kinases, and components of the endocytic machinery and making the cells permissible to the virus (9, 10). All these functions of Nef are manifested by a number of events such as activation of signaling molecules, modulation of apoptosis, activation of transcription factors, mitigation of transcriptional repressors, and an increase in the infectivity of virions (11). Although these functions of Nef have been well studied, controversy exists on its role in viral infectivity and replication as both negative (12, 13) and positive (14, 15) roles have been attributed in the literature. Although a number of reports show that Nef increases viral replication by activating T-cells, the molecular basis of Nef-induced viral gene expression and replication remains to be clearly elucidated. Nef was also shown to physically interact with Tat and augment viral gene expression (16), although availability of Tat is critical for the elongation step of long terminal repeat (LTR) 2 -driven transcription by RNA polymerase II. Several cellular factors have been identified for their roles in this process, including positive transcription elongation factor b (P-TEFb) complex. Different P-TEFb complexes isolated from mammalian cells contain a common catalytic subunit, cyclin-dependent kinase 9 (CDK9), and one of the regulatory cyclins, CycT1, CycT2a, CycT2b,. However, CycT1-containing P-TEFb complex is the key complex responsible for HIV-1 transcription elongation (19). Tat is expressed early in the replicative cycle of HIV and is essential for viral gene expression from the LTR promoter. It recognizes the 5Ј-bulge in the transactivation response stem loop RNA, which is located at the 5Ј-end of all viral transcripts. Tat binds to CycT1, and together they form the combinatorial surface that interacts with the transactivation response RNA with high affinity and specificity. The obligate partner of CycT1, CDK9, then hyperphosphorylates the C-terminal domain (CTD) of RNA polymerase II, resulting in incre...

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Identification of a Cyclin T-Binding Domain in Hexim1 and Biochemical Analysis of Its Binding Competition with HIV-1 Tat

Cited by 100 publications

References 36 publications

Serine-7 but not serine-5 phosphorylation primes RNA polymerase II CTD for P-TEFb recognition

Serine-7 but not serine-5 phosphorylation primes RNA polymerase II CTD for P-TEFb recognition

AFF1 is a ubiquitous P-TEFb partner to enable Tat extraction of P-TEFb from 7SK snRNP and formation of SECs for HIV transactivation

Cyclin K Inhibits HIV-1 Gene Expression and Replication by Interfering with Cyclin-dependent Kinase 9 (CDK9)-Cyclin T1 Interaction in Nef-dependent Manner

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