Cyclin K functions as a CDK9 regulatory subunit and participates in RNA polymerase II transcription.
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Important progress in the understanding of elongation control by RNA polymerase II (RNAPII) has come from the recent identification of the positive transcription elongation factor b (P-TEFb) and the demonstration that this factor is a protein kinase that phosphorylates the carboxyl-terminal domain (CTD) of the RNAPII largest subunit. The P-TEFb complex isolated from mammalian cells contains a catalytic subunit (CDK9), a cyclin subunit (cyclin T1 or cyclin T2), and additional, yet unidentified, polypeptides of unknown function. To identify additional factors involved in P-TEFb function we performed a yeast two-hybrid screen using CDK9 as bait and found that cyclin K interacts with CDK9 in vivo. Biochemical analyses indicate that cyclin K functions as a regulatory subunit of CDK9. The CDK9-cyclin K complex phosphorylated the CTD of RNAPII and functionally substituted for P-TEFb comprised of CDK9 and cyclin T in in vitro transcription reactions. (+info)
A binding site for the transcription factor Grainyhead/Nuclear transcription factor-1 contributes to regulation of the Drosophila proliferating cell nuclear antigen gene promoter.
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The Drosophila proliferating cell nuclear antigen promoter contains multiple transcriptional regulatory elements, including upstream regulatory element (URE), DNA replication-related element, E2F recognition sites, and three common regulatory factor for DNA replication and DNA replication-related element-binding factor genes recognition sites. In nuclear extracts of Drosophila embryos, we detected a protein factor, the URE-binding factor (UREF), that recognizes the nucleotide sequence 5'-AAACCAGTTGGCA located within URE. Analyses in Drosophila Kc cells and transgenic flies revealed that the UREF-binding site plays an important role in promoter activity both in cultured cells and in living flies. A yeast one-hybrid screen using URE as a bait allowed isolation of a cDNA encoding a transcription factor, Grainyhead/nuclear transcription factor-1 (GRH/NTF-1). The nucleotide sequence required for binding to GRH was indistinguishable from that for UREF detected in embryo nuclear extracts. Furthermore, a specific antibody to GRH reacted with UREF in embryo nuclear extracts. From these results we conclude that GRH is identical to UREF. Although GRH has been thought to be involved in regulation of differentiation-related genes, this study demonstrates, for the first time, involvement of a GRH-binding site in regulation of the DNA replication-related proliferating cell nuclear antigen gene. (+info)
Interaction of c-Jun amino-terminal kinase interacting protein-1 with p190 rhoGEF and its localization in differentiated neurons.
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c-Jun amino-terminal kinase (JNK) interacting protein-1 (JIP-1) was originally identified as a cytoplasmic inhibitor of JNK. More recently, JIP-1 was proposed to function as a scaffold protein by complexing specific components of the JNK signaling pathway, namely JNK, mitogen-activated protein kinase kinase 7, and mixed lineage kinase 3. We have identified the human homologue of JIP-1 that contains a phosphotyrosine binding (PTB) domain in addition to a JNK binding domain and an Src homology 3 domain. To identify binding targets for the hJIP-1 PTB domain, a mouse embryo cDNA library was screened using the yeast two-hybrid system. One clone encoded a 191-amino acid region of the neuronal protein rhoGEF, an exchange factor for rhoA. Overexpression of rhoGEF promotes cytoskeletal rearrangement and cell rounding in NIE-115 neuronal cells. The interaction of JIP-1 with rhoGEF was confirmed by coimmunoprecipitation of these proteins from lysates of transiently transfected HEK 293 cells. Using glutathione S-transferase rhoGEF fusion proteins containing deletion or point mutations, we identified a putative PTB binding site within rhoGEF. This binding site does not contain tyrosine, indicating that the JIP PTB domain, like that of Xll alpha and Numb, binds independently of phosphotyrosine. Several forms of endogenous JIP-1 protein can be detected in neuronal cell lines. Indirect immunofluorescence analysis localized endogenous JIP-1 to the tip of the neurites in differentiated NIE-115 and PC12 cells. The interaction of JIP-1 with rhoGEF and its subcellular localization suggests that JIP-1 may function to specifically localize a signaling complex in neuronal cells. (+info)
c-Ski acts as a transcriptional co-repressor in transforming growth factor-beta signaling through interaction with smads.
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Smads are intracellular signaling mediators of the transforming growth factor-beta (TGF-beta) superfamily that regulates a wide variety of biological processes. Among them, Smads 2 and 3 are activated specifically by TGF-beta. We identified c-Ski as a Smad2 interacting protein. c-Ski is the cellular homologue of the v-ski oncogene product and has been shown to repress transcription by recruiting histone deacetylase (HDAC). Smad2/3 interacts with c-Ski through its C-terminal MH2 domain in a TGF-beta-dependent manner. c-Ski contains two distinct Smad-binding sites with different binding properties. c-Ski strongly inhibits transactivation of various reporter genes by TGF-beta. c-Ski is incorporated in the Smad DNA binding complex, interferes with the interaction of Smad3 with a transcriptional co-activator, p300, and in turn recruits HDAC. c-Ski is thus a transcriptional co-repressor that links Smads to HDAC in TGF-beta signaling. (+info)
Regulation of CDK4 activity by a novel CDK4-binding protein, p34(SEI-1).
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The p16(INK4a) tumor suppressor inhibits cyclin-dependent kinases (CDK4 and CDK6). Here we report the isolation of a novel gene, SEI-1, whose product (p34(SEI-1)) appears to antagonize the function of p16(INK4a). Addition of p34(SEI-1) to cyclin D1-CDK4 renders the complex resistant to inhibition by p16(INK4a). Expression of SEI-1 is rapidly induced on addition of serum to quiescent fibroblasts, and ectopic expression of p34(SEI-1) enables fibroblasts to proliferate even in low serum concentrations. p34(SEI-1) seems to act as a growth factor sensor and may facilitate the formation and activation of cyclin D-CDK complexes in the face of inhibitory levels of INK4 proteins. (+info)
Gal83 mediates the interaction of the Snf1 kinase complex with the transcription activator Sip4.
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The Snf1/AMPK protein kinase family is widely conserved in eukaryotes. In Saccharomyces cerevisiae, the Snf1 kinase is an essential element of the glucose response pathway and has diverse regulatory roles. The Snf1 complex contains one of the related proteins Sip1, Sip2 and Gal83, which are also conserved in higher eukaryotes. Previous studies showed that the Sip1/Sip2/Gal83 component plays a structural role in the complex. We present evidence that this component also mediates the interaction of the Snf1 kinase complex with specific targets. We show that Gal83 mediates the association of the kinase with Sip4, a Snf1-regulated transcription activator of gluconeogenic genes. Gal83 interacts with Sip4 in two-hybrid assays in vivo, and bacterially expressed proteins bind in vitro. Moreover, Gal83 is required for the two-hybrid interaction of Sip4 with the Snf1 kinase. Gal83 also facilitates the rapid Snf1-dependent phosphorylation and activation of Sip4 in response to glucose limitation, indicating that Gal83 mediates the functional interaction of Snf1 with Sip4. Evidence indicates that Sip1 and Sip2 do not interact with Sip4. We propose that members of the Sip1/Sip2/Gal83 family confer specificity to the kinase complex in its interactions with target proteins. (+info)
NF-kappaB activation by a signaling complex containing TRAF2, TANK and TBK1, a novel IKK-related kinase.
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The activation of NF-kappaB by receptors in the tumor necrosis factor (TNF) receptor and Toll/interleukin-1 (IL-1) receptor families requires the TRAF family of adaptor proteins. Receptor oligomerization causes the recruitment of TRAFs to the receptor complex, followed by the activation of a kinase cascade that results in the phosphorylation of IkappaB. TANK is a TRAF-binding protein that can inhibit the binding of TRAFs to receptor tails and can also inhibit NF-kappaB activation by these receptors. However, TANK also displays the ability to stimulate TRAF-mediated NF-kappaB activation. In this report, we investigate the mechanism of the stimulatory activity of TANK. We find that TANK interacts with TBK1 (TANK-binding kinase 1), a novel IKK-related kinase that can activate NF-kappaB in a kinase-dependent manner. TBK1, TANK and TRAF2 can form a ternary complex, and complex formation appears to be required for TBK1 activity. Kinase-inactive TBK1 inhibits TANK-mediated NF-kappaB activation but does not block the activation mediated by TNF-alpha, IL-1 or CD40. The TBK1-TANK-TRAF2 signaling complex functions upstream of NIK and the IKK complex and represents an alternative to the receptor signaling complex for TRAF-mediated activation of NF-kappaB. (+info)
The E2-E3 interaction in the N-end rule pathway: the RING-H2 finger of E3 is required for the synthesis of multiubiquitin chain.
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We dissected physical and functional interactions between the ubiquitin-conjugating (E2) enzyme Ubc2p and Ubr1p, the E3 component of the N-end rule pathway in Saccharomyces cerevisiae. The binding of the 20 kDa Ubc2p by the 225 kDa Ubr1p is shown to be mediated largely by the basic residue-rich (BRR) region of Ubr1p. However, mutations of the BRR domain that strongly decrease the interaction between Ubr1p and Ubc2p do not prevent the degradation of N-end rule substrates. In contrast, this degradation is completely dependent on the RING-H2 finger of Ubr1p adjacent to the BRR domain. Specifically, the first cysteine of RING-H2 is required for the ubiquitylation activity of the Ubr1p-Ubc2p complex, although this cysteine plays no detectable role in either the binding of N-end rule substrates by Ubr1p or the physical affinity between Ubr1p and Ubc2p. These results defined the topography of the Ubc2p-Ubr1p interaction and revealed the essential function of the RING-H2 finger, a domain that is present in many otherwise dissimilar E3 proteins of the ubiquitin system. (+info)