The transcription and processing of pre-mRNA in eukaryotic cells are regulated in part by reversible phosphorylation of the C-terminal domain of the largest RNA polymerase (RNAP) II subunit. The CTD phosphatase, FCP1, catalyzes the dephosphorylation of RNAP II and is thought to play a major role in polymerase recycling. This study describes a family of small CTD phosphatases (SCPs) that preferentially catalyze the dephosphorylation of Ser 5 within the consensus repeat. The preferred substrate for SCP1 is RNAP II phosphorylated by TFIIH. Like FCP1, the activity of SCP1 is enhanced by the RAP74 subunit of TFIIF. Expression of SCP1 inhibits activated transcription from a number of promoters, whereas a phosphatase-inactive mutant of SCP1 enhances transcription. Accordingly, SCP1 may play a role in the regulation of gene expression, possibly by controlling the transition from initiation/capping to processive transcript elongation.The largest subunit of RNA polymerase (RNAP) 1 II contains a C-terminal domain (CTD) composed of multiple repeats of the consensus sequence Tyr 1 -Ser 2 -Pro 3 -Thr 4 -Ser 5 -Pro 6 -Ser 7 . The progression of RNAP II through the transcription cycle is regulated by both the state of CTD phosphorylation and the specific site of phosphorylation within the consensus repeat (1, 2). The emerging overview of this process is that unphosphorylated RNAP II, designated RNAP IIA, enters the preinitiation complex, where phosphorylation of Ser 5 is catalyzed by TFIIH (which contains Cdk7/cyclin H subunits) concomitant with transcript initiation (3,4). This generates the phosphorylated form of RNAP II, designated RNAP IIO. Ser 5 phosphorylation facilitates the recruitment of the 7-methyl G capping enzyme complex (5-9). Phosphorylation of Ser 2 is catalyzed by the cyclin-dependent kinase P-TEFb (which contains Cdk9/cyclin T subunits) (10, 11 FCP1 is a class C phosphatase containing a BRCT domain that is required for interaction with RNAP II and dephosphorylation of the CTD (15, 16). FCP1 interacts with and is stimulated by RAP74, the larger subunit of TFIIF (16,17). Class C phosphatases are resistant to inhibitors that block other classes of Ser/Thr phosphatases and bind Mg 2ϩ or Mn 2ϩ in the binuclear metal center of the catalytic site (18, 19). The DX-DX(T/V) motif (where represents a hydrophobic residue) present in the FCP1 homology domain characterizes a subfamily of class C phosphatases, with both Asp residues being essential for activity (20,21).Synthetic lethality is observed between mutant FCP1 and reduced levels of RNAP II in Saccharomyces cerevisiae and Schizosaccharomyces pombe, indicating that FCP1 is an essential gene (21,22). It remains uncertain whether the activity of yeast FCP1 accounts for the dephosphorylation in vivo of both Ser 5 and Ser 2 and whether this is the sole activity that catalyzes CTD dephosphorylation. Mutations in FCP1 lead to increased phosphorylation of Ser 2 , suggesting that it functions in vivo in the dephosphorylation of Ser 2 (13). Yeast FCP1 appears more specific ...

We concluded that the endogenous NO synthase inhibitor ADMA is synthesized in human endothelial cells. Asymmetric dimethylarginine increases endothelial oxidative stress and potentiates monocyte binding. Asymmetric dimethylarginine may be an endogenous proatherogenic molecule.

The present study assessed whether impaired aerobic capacity previously observed in hypercholesterolemic mice is reversible by exercise training. Seventy-two 8-wk-old female C57BL/6J wild-type (+, n = 42) and apolipoprotein E-deficient (−, n = 30) mice were assigned to the following eight interventions: normal chow, sedentary (E+, n = 17; E−, n = 8) or exercised ([Formula: see text], n= 13; [Formula: see text], n = 7) and high-fat chow, sedentary ([Formula: see text], n = 6;[Formula: see text], n = 8) or exercised ([Formula: see text], n = 6;[Formula: see text], n = 7). Mice were trained on a treadmill 2 × 1 h/day, 6 days/wk, for 4 wk. Cholesterol levels correlated inversely with maximum oxygen uptake ( r = −0.35; P < 0.02), which was blunted in all hypercholesterolemic sedentary groups (all P < 0.05). Maximum oxygen uptake improved in all training groups but failed to match[Formula: see text] (all P< 0.05). Vascular reactivity and nitric oxide (NO) synthesis correlated with anaerobic threshold ( r = 0.36; P < 0.025) and maximal distance run ( r = 0.59; P < 0.007). We conclude that genetically induced hypercholesterolemia impairs aerobic capacity. This adverse impact of hypercholesterolemia on aerobic capacity may be related to its impairment of vascular NO synthesis and/or vascular smooth muscle sensitivity to nitrovasodilators. Aerobic capacity is improved to the same degree by exercise training in normal and genetically hypercholesterolemic mice, although there remains a persistent difference between these groups after training.

Damaged DNA binding proteins (DDBs) play a critical role in the initial recognition of UV-damaged DNA and mediate recruitment of nucleotide excision repair factors. Previous studies identified DDB2 as a target of the CUL-4A ubiquitin ligase. However, the biochemical mechanism governing DDB proteolysis and its underlying physiological function in the removal of UV-induced DNA damage are largely unknown. Here, we report that the c-Abl nonreceptor tyrosine kinase negatively regulates the repair of UV-induced photolesions on genomic DNA. Biochemical studies revealed that c-Abl promotes CUL-4A-mediated DDB ubiquitination and degradation in a manner that does not require its tyrosine kinase activity both under normal growth conditions and following UV irradiation. Moreover, c-Abl activates DDB degradation in part by alleviating the inhibitory effect of CAND1/TIP120A on CUL-4A. These results revealed a kinase-independent function of c-Abl in a ubiquitin-proteolytic pathway that regulates the damage recognition step of nucleotide excision repair.

The carboxyl-terminal domain (CTD) of the largest RNA polymerase (RNAP) II subunit undergoes reversible phosphorylation throughout the transcription cycle. The unphosphorylated form of RNAP II is referred to as IIA, whereas the hyperphosphorylated form is known as IIO. Phosphorylation occurs predominantly at serine 2 and serine 5 within the CTD heptapeptide repeat and has functional implications for RNAP II with respect to initiation, elongation, and transcription-coupled RNA processing. In an effort to determine the role of the major CTD phosphatase (FCP1) in regulating events in transcription that appear to be influenced by serine 2 and serine 5 phosphorylation, the specificity of FCP1 was examined. FCP1 is capable of dephosphorylating heterogeneous RNAP IIO populations of HeLa nuclear extracts. The extent of dephosphorylation at specific positions was assessed by immunoreactivity with monoclonal antibodies specific for phosphoserine 2 or phosphoserine 5. As an alternative method to assess FCP1 specificity, RNAP IIO isozymes were prepared in vitro by the phosphorylation of purified calf thymus RNAP IIA with specific CTD kinases and used as substrates for FCP1. FCP1 dephosphorylates serine 2 and serine 5 with comparable efficiency. Accordingly, the specificity of FCP1 is sufficiently broad to dephosphorylate RNAP IIO at any point in the transcription cycle irrespective of the site of serine phosphorylation within the consensus repeat. Reversible phosphorylation of the carboxyl-terminal domain (CTD)1 of the largest RNA polymerase (RNAP) II subunit plays an important role in the regulation of gene expression. The CTD of mammalian RNAP II is comprised of 52 repeats of the consensus sequence 1 YSPTSPS 7 (for a review, see Ref. 1). RNAP IIA, which contains an unmodified CTD, is actively recruited to the promoter as part of the preinitiation complex (2-5), whereas RNAP IIO, which contains a hyperphosphorylated CTD, is responsible for transcript elongation (6, 7). Therefore, protein kinases and phosphatases that alter the state of CTD phosphorylation can serve as transcriptional activators or repressors depending on the point in the transcription cycle at which they function.CTD phosphorylation occurs predominantly at serines 2 and 5 within the heptapeptide repeat. Genetic evidence indicates that the roles of serines in positions 2 and 5 are different. First, the partial substitution of serines in either position 2 or 5 have different effects on viability (8). Second, SRB (suppressors of RNA Polymerase IIB) mutations suppress the lethal effect of position 2 substitutions but not position 5 substitutions (9). Biochemical evidence has also confirmed differences between the two predominant serine positions. Serine 5 but not serine 2 phosphorylation recruits and activates the 5Ј-capping machinery (10, 11). Furthermore, nutritional stress and heat shock can independently alter the pattern of CTD phosphorylation, indicating that phosphoserine 2 and phosphoserine 5 are functionally different (12)(13)(14).A recent study using...