Heat shock of HeLa cells inactivates a nuclear protein phosphatase specific for dephosphorylation of the C-terminal domain of RNA polymerase II

The phosphorylation of the RNA polymerase II (RNAP II) carboxy-terminal domain (CTD) plays a key role in mRNA metabolism. The relative ratio of hyperphosphorylated RNAP II to hypophosphorylated RNAP II is determined by a dynamic equilibrium between CTD kinases and CTD phosphatase(s). The CTD is heavily phosphorylated in meiotic Xenopus laevis oocytes. In this report we show that the CTD undergoes fast and massive dephosphorylation upon fertilization. A cDNA was cloned and shown to code for a full-length xFCP1, the Xenopus orthologue of the FCP1 CTD phosphatases in humans and Saccharomyces cerevisiae. Two critical residues in the catalytic site were identified. CTD phosphatase activity was observed in extracts prepared from Xenopus eggs and cells and was shown to be entirely attributable to xFCP1. The CTD dephosphorylation triggered by fertilization was reproduced upon calcium activation of cytostatic factor-arrested egg extracts. Using immunodepleted extracts, we showed that this dephosphorylation is due to xFCP1. Although transcription does not occur at this stage, phosphorylation appears as a highly dynamic process involving the antagonist action of Xp42 mitogen-activated protein kinase and FCP1 phosphatase. This is the first report that free RNAP II is a substrate for FCP1 in vivo, independent from a transcription cycle.In many metazoans, early development is characterized by major changes in the global transcriptional activity of the zygote (13). After fertilization, development proceeds for a while in the absence of transcription and is dependent on the translation of maternal mRNAs stored during ovogenesis. Transcription resumes at a stage which is species specific and corresponds to the two-cell stage in mice. In Xenopus laevis, transcription resumes at the 12th cleavage division at a major developmental transition known as the midblastula transition. Important changes in RNA polymerase II (RNAP II) phosphorylation have been described to occur in worm (27), fly (20), mammalian (3), and amphibian (25) embryos during this period.RNAP II phosphorylation is a key event in the transcription cycle (11, 12). The largest subunit of RNAP II (Rpb1) can be extensively phosphorylated on its carboxy-terminal domain (CTD), which is comprised of up to 52 repeats of the consensus heptapeptide Tyr-Ser-Pro-Thr-Ser-Pro-Ser. Thus, in somatic cells, two forms of RNAP II coexist in a dynamic equilibrium (15). The hypophosphorylated IIA form assembles into preinitiation complexes, whereas the hyperphosphorylated IIO form catalyzes transcript elongation and facilitates the recruitment of the pre-mRNA processing machinery. CTD dephosphorylation is required to recycle the polymerase at the end of each round of transcription.Several CTD kinases have been identified (for reviews, see references 5, 21, and 28): the CDK8 subunit of the mediator complex in the RNAP II holoenzyme, the CDK7 subunit of the general transcription factor TFIIH which assembles in the preinitiation complex, the CDK9 subunit of P-TEFb (positive transcription el...

Section: Resultssupporting

confidence: 77%

“…An immunochemical analysis was employed to establish whether or not the CTD phosphatase activity detected in Xenopus extracts corresponded to xFCP1. An antiserum raised against hFCP1 reacted with an Ϸ150-kDa polypeptide in human cell extracts (1,14). The same antiserum reacted with a polypeptide of similar size in A6 cell extracts (Fig.…”

Section: Resultsmentioning

confidence: 77%

Transcription-Independent RNA Polymerase II Dephosphorylation by the FCP1 Carboxy-Terminal Domain Phosphatase in Xenopus laevis Early Embryos

Palancade

Dubois

Dahmus

et al. 2001

“…Extraction of inactive HSF2 required denaturing detergents capable of disrupting noncovalent interactions. These observations resemble the reported effects of heat shock on other intracellular proteins, including various endogenous mammalian proteins (6,7,8) as well as reporter enzymes (luciferase and ␤-galactosidase) transfected as heterologous genes into mammalian cells (29), which all undergo a heat shock-induced transition into a Triton X-100-insoluble state.…”

Section: Discussionsupporting

confidence: 80%

Stress-Specific Activation and Repression of Heat Shock Factors 1 and 2

Mathew

Mathur

Jolly

et al. 2001

113

Vertebrate cells express a family of heat shock transcription factors (HSF1 to HSF4) that coordinate the inducible regulation of heat shock genes in response to diverse signals. HSF1 is potent and activated rapidly though transiently by heat shock, whereas HSF2 is a less active transcriptional regulator but can retain its DNA binding properties for extended periods. Consequently, the differential activation of HSF1 and HSF2 by various stresses may be critical for cells to survive repeated and diverse stress challenges and to provide a mechanism for more precise regulation of heat shock gene expression. Here we show, using a novel DNA binding and detection assay, that HSF1 and HSF2 are coactivated to different levels in response to a range of conditions that cause cell stress. Above a low basal activity of both HSFs, heat shock preferentially activates HSF1, whereas the amino acid analogue azetidine or the proteasome inhibitor MG132 coactivates both HSFs to different levels and hemin preferentially induces HSF2. Unexpectedly, we also found that heat shock has dramatic adverse effects on HSF2 that lead to its reversible inactivation coincident with relocalization from the nucleus. The reversible inactivation of HSF2 is specific to heat shock and does not occur with other stressors or in cells expressing high levels of heat shock proteins. These results reveal that HSF2 activity is negatively regulated by heat and suggest a role for heat shock proteins in the positive regulation of HSF2.The heat shock response is a cellular defense against the deleterious effects of physiological and environmental stress that is mediated by heat shock transcription factors (HSFs). In vertebrates, four members of the HSF family have been identified (27,28,38,40,46), and of these, HSF1 and HSF2 are ubiquitously expressed and conserved (27,40). HSF1 and HSF2 are expressed as inert monomers and dimers, respectively, in unstressed cells. Upon activation, these HSFs trimerize and function as transcriptional activators that bind with similar, but not identical, specificities to the heat shock element (HSE), thus regulating heat shock gene transcription and hence the expression of diverse heat shock proteins and molecular chaperones (2,40,49,62).HSF1 and HSF2 differ in their pathways of activation and the nature of their transcriptional responses. Whereas HSF1 is activated upon exposure to a multitude of physiological and environmental stresses (41, 62), HSF2 activity appears to be more selective, being induced upon down-regulation of the ubiquitin-proteasome pathway (22), during differentiation (36,37,42,49), and in early development (9, 23, 37). Comparison of the HSF1 and HSF2 transactivation domains as chimeric proteins with a minimal heterologous DNA binding domain reveals that HSF1 is a more potent transcriptional activator than HSF2 (47, 59, 60). These observations are also supported by studies comparing hsp70 and hsp90 gene transcription rates in K562 cells in which endogenous HSF1 or HSF2 was activated (49). In those studies, HSF1 ...

“…Thus, it may be that in metazoans, or on some genes, the level of Ser5-P relative to Pol II is fairly constant along the gene. The possibility that the activity of a phosphatase may be system or gene specific is certainly plausible; for instance, heat shock of HeLa cells deactivates a CTD phosphatase (8).…”

Section: Discussionmentioning

confidence: 99%

Transcription Factor and Polymerase Recruitment, Modification, and Movement on dhsp70 In Vivo in the Minutes following Heat Shock

Boehm

Saunders

Werner

et al. 2003

207

225

The uninduced Drosophila hsp70 gene is poised for rapid activation. Here we examine the rapid changes upon heat shock in levels and location of heat shock factor (HSF), RNA polymerase II (Pol II) and its phosphorylated forms, and the Pol II kinase P-TEFb on hsp70 in vivo by using both real-time PCR assays of chromatin immunoprecipitates and polytene chromosome immunofluorescence. These studies capture Pol II recruitment and progression along hsp70 and reveal distinct spatial and temporal patterns of serine 2 and serine 5 phosphorylation: in uninduced cells, the promoter-paused Pol II shows Ser5 but not Ser2 phosphorylation, and in induced cells the relative level of Ser2-P Pol II is lower at the promoter than at regions downstream. An early time point of heat shock activation captures unphosphorylated Pol II recruited to the promoter prior to P-TEFb, and during the first wave of transcription Pol II and the P-TEFb kinase can be seen tracking together across hsp70 with indistinguishable kinetics. Pol II distributions on several other genes with paused Pol II show a pattern of Ser5 and Ser2 phosphorylation similar to that of hsp70. These studies of factor choreography set important limits in modeling transcription regulatory mechanisms.Recent studies of posttranslational modifications of, and factor interactions with, the C-terminal domain (CTD) of the largest subunit of RNA polymerase II (Pol II) have implicated this region of Pol II in productive transcriptional elongation and the coupling of transcription and pre-mRNA processing (for reviews see references 32 and 37). The CTD is highly conserved among eukaryotes, containing multiple repeats of a consensus heptad YSPTSPS; mammalian Pol II CTD consists of 52 repeats, yeast consists of 25 to 26 repeats, while Drosophila has 42 repeats (1, 6). This large flexible arm of Pol II appears to serve as a docking site for, or to stimulate the recruitment of, an orchestrated assembly of factors involved in pre-mRNA capping, splicing, and 3Ј polyadenylation at different stages in production of the nascent transcript (5,9,13,14,20,26). One way this coordination has been proposed to occur is through the known phosphorylation of serines 2 and 5 (Ser2-P and Ser5-P, respectively) of the heptad repeat (52, 56). Cdk7, the kinase subunit of general transcription factor TFIIH, has been shown to phosphorylate the CTD at Ser5, an event proposed to occur early in the transcription cycle (12,24,57). This in turn appears to influence the association and activity of the capping machinery (5,15,18,27,28,42,45). Positive transcription elongation factor b (P-TEFb) is able to phosphorylate the CTD at Ser2 and, under certain conditions, Ser5 (25, 38, 57). P-TEFb is a kinase composed of the proteins cyclin T (CycT) and cdk9 and is known to be recruited upon gene activation, overcoming the negative effects of factors like Spt5 and negative elongation factor (50) and aiding in the transition from transcription initiation to elongation (36). Cdk8, a component of the coactivator complex Mediator, h...